Michael Fischbach

All Publications

• Discovery and engineering of the antibody response to a prominent skin commensal. Nature Bousbaine, D., Bauman, K. D., Chen, Y. E., Lalgudi, P. V., Nguyen, T. T., Swenson, J. M., Yu, V. K., Tsang, E., Conlan, S., Li, D. B., Jbara, A., Zhao, A., Naziripour, A., Veinbachs, A., Lee, Y. E., Phung, J. L., Dimas, A., Jain, S., Meng, X., Pham, T. P., McLaughlin, M. I., Barkal, L. J., Gribonika, I., Van Rompay, K. K., Kong, H. H., Segre, J. A., Belkaid, Y., Barnes, C. O., Fischbach, M. A. 2024

  Abstract

  The ubiquitous skin colonist Staphylococcus epidermidis elicits a CD8+ T cell response pre-emptively, in the absence of an infection1. However, the scope and purpose of this anti-commensal immune program are not well defined, limiting our ability to harness it therapeutically. Here, we show that this colonist also induces a potent, durable, and specific antibody response that is conserved in humans and non-human primates. A series of S. epidermidis cell-wall mutants revealed that the cell surface protein Aap is a predominant target. By colonizing mice with a strain of S. epidermidis in which the parallel β-helix domain of Aap is replaced by tetanus toxin fragment C, we elicit a potent neutralizing antibody response that protects mice against a lethal challenge. A similar strain of S. epidermidis expressing an Aap-SpyCatcher chimera can be conjugated with recombinant immunogens; the resulting labeled commensal elicits high antibody titers under conditions of physiologic colonization, including a robust IgA response in the nasal and pulmonary mucosa. Thus, immunity to a common skin colonist involves a coordinated T and B cell response, the latter of which can be redirected against pathogens as a novel form of topical vaccination.

  View details for DOI 10.1038/s41586-024-08489-4

  View details for PubMedID 39662508

• Skin autonomous antibody production regulates host-microbiota interactions. Nature Gribonika, I., Band, V. I., Chi, L., Perez-Chaparro, P. J., Link, V. M., Ansaldo, E., Oguz, C., Bousbaine, D., Fischbach, M. A., Belkaid, Y. 2024

  Abstract

  The microbiota colonizes each barrier site and broadly controls host physiology1. However, when uncontrolled, microbial colonists can also promote inflammation and induce systemic infection2. The unique strategies employed at each barrier tissue to control the coexistence of the host with its microbiota remain largely elusive. Here we uncover that, within the skin, host-microbiota symbiosis depends on the remarkable ability of the skin to act as an autonomous lymphoid organ. Notably, an encounter with a new skin commensal promotes two parallel responses, both under the control of Langerhans cells. On one hand, skin commensals induce the formation of classical germinal centers within the lymph node associated with IgG1 and IgG3 antibody responses. On the other hand, microbial colonization also leads to the development of tertiary lymphoid organs within the skin that can locally sustain IgG2b and IgG2c responses. These phenomena are supported by the ability of regulatory T cells to convert into T follicular helper cells. Skin autonomous production of antibodies is sufficient to control local microbial biomass, as well as subsequent systemic infection with the same microbe. Collectively, these results reveal a striking compartmentalization of humoral responses to the microbiota allowing for control of both microbial symbiosis and potential pathogenesis.

  View details for DOI 10.1038/s41586-024-08376-y

  View details for PubMedID 39662506

• PTER is a N-acetyltaurine hydrolase that regulates feeding and obesity. Nature Wei, W., Lyu, X., Markhard, A. L., Fu, S., Mardjuki, R. E., Cavanagh, P. E., Zeng, X., Rajniak, J., Lu, N., Xiao, S., Zhao, M., Moya-Garzon, M. D., Truong, S. D., Chou, J. C., Wat, L. W., Chidambaranathan-Reghupaty, S., Coassolo, L., Xu, D., Shen, F., Huang, W., Ramirez, C. B., Jang, C., Li, L., Svensson, K. J., Fischbach, M. A., Long, J. Z. 2024

  Abstract

  Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in the biosynthesis of taurine from cysteine and in the downstream metabolism of secondary taurine metabolites4,5. One taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by stimuli that alter taurine or acetate flux, including endurance exercise7, dietary taurine supplementation8 and alcohol consumption6,9. So far, the identities of the enzymes involved in N-acetyltaurine metabolism, and the potential functions of N-acetyltaurine itself, have remained unknown. Here we show that the body mass index associated orphan enzyme phosphotriesterase-related (PTER)10 is a physiological N-acetyltaurine hydrolase. In vitro, PTER catalyses the hydrolysis of N-acetyltaurine to taurine and acetate. In mice, PTER is expressed in the kidney, liver and brainstem. Genetic ablation of Pter in mice results in complete loss of tissue N-acetyltaurine hydrolysis activity and a systemic increase in N-acetyltaurine levels. After stimuli that increase taurine levels, Pter knockout mice exhibit reduced food intake, resistance to diet-induced obesity and improved glucose homeostasis. Administration of N-acetyltaurine to obese wild-type mice also reduces food intake and body weight in a GFRAL-dependent manner. These data place PTER into a central enzymatic node of secondary taurine metabolism and uncover a role for PTER and N-acetyltaurine in body weight control and energy balance.

  View details for DOI 10.1038/s41586-024-07801-6

  View details for PubMedID 39112712

  View details for PubMedCentralID 3501277

• Gut microbe-generated phenylacetylglutamine is an endogenous allosteric modulator of β2-adrenergic receptors. Nature communications Saha, P. P., Gogonea, V., Sweet, W., Mohan, M. L., Singh, K. D., Anderson, J. T., Mallela, D., Witherow, C., Kar, N., Stenson, K., Harford, T., Fischbach, M. A., Brown, J. M., Karnik, S. S., Moravec, C. S., DiDonato, J. A., Naga Prasad, S. V., Hazen, S. L. 2024; 15 (1): 6696

  Abstract

  Allosteric modulation is a central mechanism for metabolic regulation but has yet to be described for a gut microbiota-host interaction. Phenylacetylglutamine (PAGln), a gut microbiota-derived metabolite, has previously been clinically associated with and mechanistically linked to cardiovascular disease (CVD) and heart failure (HF). Here, using cells expressing β1- versus β2-adrenergic receptors (β1AR and β2AR), PAGln is shown to act as a negative allosteric modulator (NAM) of β2AR, but not β1AR. In functional studies, PAGln is further shown to promote NAM effects in both isolated male mouse cardiomyocytes and failing human heart left ventricle muscle (contracting trabeculae). Finally, using in silico docking studies coupled with site-directed mutagenesis and functional analyses, we identified sites on β2AR (residues E122 and V206) that when mutated still confer responsiveness to canonical β2AR agonists but no longer show PAGln-elicited NAM activity. The present studies reveal the gut microbiota-obligate metabolite PAGln as an endogenous NAM of a host GPCR.

  View details for DOI 10.1038/s41467-024-50855-3

  View details for PubMedID 39107277

  View details for PubMedCentralID PMC11303761

• Discovery and characterization of dietary antigens in oral tolerance. bioRxiv : the preprint server for biology Blum, J. E., Kong, R., Schulman, E. A., Chen, F. M., Upadhyay, R., Romero-Meza, G., Littman, D. R., Fischbach, M. A., Nagashima, K., Sattely, E. S. 2024

  Abstract

  Food antigens elicit immune tolerance through the action of regulatory T cells (Tregs) in the intestine. Although antigens that trigger common food allergies are known, the epitopes that mediate tolerance to most foods have not been described. Here, we identified murine T cell receptors specific for maize, wheat, and soy, and used expression cloning to de-orphan their cognate epitopes. All of the epitopes derive from seed storage proteins that are resistant to degradation and abundant in the edible portion of the plant. Multiple unrelated T cell clones were specific for an epitope at the C-terminus of 19 kDa alpha-zein, a protein from maize kernel. An MHC tetramer loaded with this antigen revealed that zein-specific T cells are predominantly Tregs localized to the intestine. These cells, which develop concurrently with weaning, constitute up to 2% of the peripheral Treg pool. Bulk and single-cell RNA sequencing revealed that these cells express higher levels of immunosuppressive markers and chemokines compared to other Tregs. These data suggest that immune tolerance to plant-derived foods is focused on a specific class of antigens with common features, and they reveal the functional properties of naturally occurring food-specific Tregs.

  View details for DOI 10.1101/2024.05.26.593976

  View details for PubMedID 38853977

  View details for PubMedCentralID PMC11160622

• Production of deoxycholic acid by low-abundant microbial species is associated with impaired glucose metabolism. Nature communications Wahlström, A., Brumbaugh, A., Sjöland, W., Olsson, L., Wu, H., Henricsson, M., Lundqvist, A., Makki, K., Hazen, S. L., Bergström, G., Marschall, H. U., Fischbach, M. A., Bäckhed, F. 2024; 15 (1): 4276

  Abstract

  Alterations in gut microbiota composition are suggested to contribute to cardiometabolic diseases, in part by producing bioactive molecules. Some of the metabolites are produced by very low abundant bacterial taxa, which largely have been neglected due to limits of detection. However, the concentration of microbially produced metabolites from these taxa can still reach high levels and have substantial impact on host physiology. To explore this concept, we focused on the generation of secondary bile acids by 7α-dehydroxylating bacteria and demonstrated that addition of a very low abundant bacteria to a community can change the metabolic output dramatically. We show that Clostridium scindens converts cholic acid into the secondary bile acid deoxycholic acid (DCA) very efficiently even though the abundance of C. scindens is low, but still detectable by digital droplet PCR. We also show that colonization of germ-free female mice with a community containing C. scindens induces DCA production and affects host metabolism. Finally, we show that DCA correlates with impaired glucose metabolism and a worsened lipid profile in individuals with type 2 diabetes, which implies that this metabolic pathway may contribute to the development of cardiometabolic disease.

  View details for DOI 10.1038/s41467-024-48543-3

  View details for PubMedID 38769296

  View details for PubMedCentralID 8863577

• Comprehensive clinical and genetic analyses of circulating bile acids and their associations with diabetes and its indices. Diabetes Choucair, I., Mallela, D. P., Hilser, J. R., Hartiala, J. A., Nemet, I., Gogonea, V., Li, L., Lusis, A. J., Fischbach, M. A., Tang, W. H., Allayee, H., Hazen, S. L. 2024

  Abstract

  Bile acids (BAs) are cholesterol-derived compounds that regulate glucose, lipid, and energy metabolism. Despite their significance in glucose homeostasis, the association between specific BA molecular species and their synthetic pathways with diabetes mellitus (DM) is unclear. Here, we used a recently validated stable-isotope dilution highperformance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to quantify a panel of BAs in fasting plasma from subjects (n=2,145) and explored structural and genetic determinants of BAs linked to DM, insulin resistance and obesity. Multiple 12α-hydroxylated BAs were associated with DM [adjusted odds ratios (aORs):1.3-1.9 (all P<0.05)] and insulin resistance [aORs:1.3-2.2 (all P<0.05)]. Conversely, multiple 6a-hydroxylated BAs and isolithocholic acid (Iso-LCA) were inversely associated with DM and obesity [aORs:0.3-0.9 (all P<0.05)]. Genome-wide association studies (GWAS) revealed multiple genome-wide significant loci linked with nine of the 14 DM-associated BAs, including a locus for Iso-LCA (rs11866815). Mendelian randomization analyses showed genetically elevated DCA levels were causally associated with higher BMI, and Iso-LCA levels were causally associated with reduced BMI and DM risk. In conclusion, comprehensive large-scale quantitative mass spectrometry and genetics analyses show circulating levels of multiple structurally specific BAs, especially DCA and Iso-LCA, are clinically associated with and genetically linked to obesity and DM.

  View details for DOI 10.2337/db23-0676

  View details for PubMedID 38701355

• Problem choice and decision trees in science and engineering. Cell Fischbach, M. A. 2024; 187 (8): 1828-1833

  Abstract

  Scientists and engineers often spend days choosing a problem and years solving it. This imbalance limits impact. Here, we offer a framework for problem choice: prompts for ideation, guidelines for evaluating impact and likelihood of success, the importance of fixing one parameter at a time, and opportunities afforded by failure.

  View details for DOI 10.1016/j.cell.2024.03.012

  View details for PubMedID 38608651

• A PTER-dependent pathway of taurine metabolism linked to energy balance. bioRxiv : the preprint server for biology Wei, W., Lyu, X., Markhard, A. L., Fu, S., Mardjuki, R. E., Cavanagh, P. E., Zeng, X., Rajniak, J., Lu, N., Xiao, S., Zhao, M., Moya-Garzon, M. D., Truong, S. D., Chou, J. C., Wat, L. W., Chidambaranathan-Reghupaty, S., Coassolo, L., Xu, D., Shen, F., Huang, W., Ramirez, C. B., Jang, C., Svensson, K. J., Fischbach, M. A., Long, J. Z. 2024

  Abstract

  Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in biosynthesis of taurine from cysteine as well as the downstream derivatization of taurine into secondary taurine metabolites4,5. One such taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by diverse physiologic perturbations that alter taurine and/or acetate flux, including endurance exercise7, nutritional taurine supplementation8, and alcohol consumption6,9. While taurine N-acetyltransferase activity has been previously detected in mammalian cells6,7, the molecular identity of this enzyme, and the physiologic relevance of N-acetyltaurine, have remained unknown. Here we show that the orphan body mass index-associated enzyme PTER (phosphotriesterase-related)10 is the principal mammalian taurine N-acetyltransferase/hydrolase. In vitro, recombinant PTER catalyzes bidirectional taurine N-acetylation with free acetate as well as the reverse N-acetyltaurine hydrolysis reaction. Genetic ablation of PTER in mice results in complete loss of tissue taurine N-acetyltransferase/hydrolysis activities and systemic elevation of N-acetyltaurine levels. Upon stimuli that increase taurine levels, PTER-KO mice exhibit lower body weight, reduced adiposity, and improved glucose homeostasis. These phenotypes are recapitulated by administration of N-acetyltaurine to wild-type mice. Lastly, the anorexigenic and anti-obesity effects of N-acetyltaurine require functional GFRAL receptors. Together, these data uncover enzymatic control of a previously enigmatic pathway of secondary taurine metabolism linked to energy balance.

  View details for DOI 10.1101/2024.03.21.586194

  View details for PubMedID 38562797

  View details for PubMedCentralID PMC10983888

• Prognostic Value of Gut Microbe-Generated Metabolite Phenylacetylglutamine in Patients with Heart Failure. European journal of heart failure Tang, W. H., Nemet, I., Li, X. S., Wu, Y., Haghikia, A., Witkowski, M., Koeth, R. A., Demuth, I., König, M., Steinhagen-Thiessen, E., Bäckhed, F., Fischbach, M. A., Deb, A., Landmesser, U., Hazen, S. L. 2023

  Abstract

  Phenylacetylglutamine (PAGln) is a phenylalanine-derived metabolite produced by gut microbiota with mechanistic links to hear failure (HF)-relevant phenotypes. We sought to investigate the prognostic value of PAGln in patients with stable HF.Fasting plasma PAGln levels were measured by stable-isotope-dilution LC-MS/MS in patients with stable HF from two large cohorts. All-cause mortality was assessed at 5-year follow up in the Cleveland Cohort, and HF, hospitalization, or mortality were assessed at 3-year follow up in the Berlin Cohort. Fasting PAGln levels were measured by stable-isotope-dilution LC-MS/MS. Within the Cleveland cohort, median PAGln levels were 4.2[IQR 2.4-6.9] μM. Highest quartile of PAGln was associated with 3.09-fold increased mortality risk compared to lowest quartile. Following adjustments for traditional risk factors, as well as race, eGFR, NT-proBNP, hsCRP, LV ejection fraction, ischemic etiology, and heart failure drug treatment, elevated PAGln levels remained predictive of 5-year mortality in quartile comparisons (adjusted Hazard Ratio [95%CI] for Q4vQ1: 1.64 [1.07-2.53]). In the Berlin cohort, a similar distribution of PAGln levels was observed (median 3.2 μM [IQR 2.0-4.8] μM), and PAGln levels were associated with a 1.94-fold increase in 3-year HF hospitalization or all-cause mortality risk (adjusted HR [95%CI] for Q4vQ1: 1.94 [1.14-3.28]). Prognostic value of PAGln appears to be independent of trimethylamine N-oxide levels.High levels of PAGln are associated with adverse outcomes independent of traditional cardiac risk factors and cardio-renal risk markers. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/ejhf.3111

  View details for PubMedID 38124458

• Microbe-derived uremic solutes enhance thrombosis potential in the host. mBio Nemet, I., Funabashi, M., Li, X. S., Dwidar, M., Sangwan, N., Skye, S. M., Romano, K. A., Cajka, T., Needham, B. D., Mazmanian, S. K., Hajjar, A. M., Rey, F. E., Fiehn, O., Tang, W. H., Fischbach, M. A., Hazen, S. L. 2023: e0133123

  Abstract

  p-Cresol sulfate (pCS) and indoxyl sulfate (IS), gut microbiome-derived metabolites, are traditionally associated with cardiovascular disease (CVD) risks in the setting of impaired kidney function. While pharmacologic provision of pCS or IS can promote pro-thrombotic phenotypes, neither the microbial enzymes involved nor direct gut microbial production have been linked to CVD. Untargeted metabolomics was performed on a discovery cohort (n = 1,149) with relatively preserved kidney function, followed by stable isotope-dilution mass spectrometry quantification of pCS and IS in an independent validation cohort (n = 3,954). Genetic engineering of human commensals to produce p-cresol and indole gain-of-function and loss-of-function mutants, followed by colonization of germ-free mice, and studies on host thrombosis were performed. Systemic pCS and IS levels were independently associated with all-cause mortality. Both in vitro and within colonized germ-free mice p-cresol productions were recapitulated by collaboration of two organisms: a Bacteroides strain that converts tyrosine to 4-hydroxyphenylacetate, and a Clostridium strain that decarboxylates 4-hydroxyphenylacetate to p-cresol. We then engineered a single organism, Bacteroides thetaiotaomicron, to produce p-cresol, indole, or both metabolites. Colonizing germ-free mice with engineered strains, we show the gut microbial genes for p-cresol (hpdBCA) and indole (tryptophanase) are sufficient to confer a pro-thrombotic phenotype in vivo. Moreover, human fecal metagenomics analyses show that abundances of hpdBCA and tryptophanase are associated with CVD. These studies show that pCS and IS, two abundant microbiome-derived metabolites, play a broader potential role in CVD than was previously known. They also suggest that therapeutic targeting of gut microbial p-cresol- and indole-producing pathways represent rational targets for CVD.IMPORTANCEAlterations in gut microbial composition and function have been linked to numerous diseases. Identifying microbial pathways responsible for producing molecules that adversely impact the host is an important first step in the development of therapeutic interventions. Here, we first use large-scale clinical observations to link blood levels of defined microbial products to cardiovascular disease risks. Notably, the previously identified uremic toxins p-cresol sulfate and indoxyl sulfate were shown to predict 5-year mortality risks. After identifying the microbes and microbial enzymes involved in the generation of these uremic toxins, we used bioengineering technologies coupled with colonization of germ-free mice to show that the gut microbial genes that generate p-cresol and indole are sufficient to confer p-cresol sulfate and indoxyl sulfate formation, and a pro-thrombotic phenotype in vivo. The findings and tools developed serve as a critical step in both the study and targeting of these gut microbial pathways in vivo.

  View details for DOI 10.1128/mbio.01331-23

  View details for PubMedID 37947418

• Microbial triggering of myelin-specific immune cells in the gut drives central nervous system inflammation Siewert, L., Poessnecker, E., Dyckow, J., Baumann, R., Zulji, A., Schreiner, D., Sagan, S., Bosch, F., Wettig, A., Kogl, E., Modabber, M., Dhungel, B., Sabatino, J., Nagashima, K., Diard, M., Ganal, S., Fischbach, M., Zamvil, S., Schirmer, L., Baranzini, S. E., Proebstel, A. SAGE PUBLICATIONS LTD. 2023: 819-820

  View details for Web of Science ID 001091311305292

• Identification of human skin microbiome odorants that manipulate mosquito landing behavior. bioRxiv : the preprint server for biology Coutinho-Abreu, I. V., Jamshidi, O., Raban, R., Atabakhsh, K., Merriman, J. A., Fischbach, M. A., Akbari, O. S. 2023

  Abstract

  The resident human skin microbiome is responsible for the production of most of the human scents that are attractive to mosquitoes. Hence, engineering the human skin microbiome to synthesize less of mosquito attractants or produce repellents could potentially reduce bites and prevent the transmission of deadly mosquito-borne pathogens. In order to further characterize the human skin volatilome, we quantified the major volatiles of 39 strains of skin commensals (Staphylococci and Corynebacterium). Importantly, to validate the behavioral activity of these volatiles, we first assessed landing behavior triggered by human skin bacteria volatiles. We demonstrated that this behavioral step is gated by the presence of carbon dioxide and L-(+)-lactic acid, similar to the combinatorial coding triggering short range attraction. Repellency behavior to selected skin volatiles and the geraniol terpene was tested in the presence of carbon dioxide and L-(+)-lactic acid. In a 2-choice landing behavior context, the skin volatiles 2- and 3-methyl butyric acids reduced mosquito landing by 62.0-81.6% and 87.1-99.6%, respectively. Similarly, geraniol was capable of reducing mosquito landing behavior by 74.9%. We also tested the potential repellency effects of geraniol on mosquitoes at short-range using a 4-port olfactometer. In these assays, geraniol reduced mosquito attraction (69-78%) to a mixture of key human kairomones carbon dioxide, L-(+)-lactic acid, and ammonia. These findings demonstrate that carbon dioxide and L-(+)-lactic acid changes the valence of other skin volatiles towards mosquito landing behavior. Moreover, this study offers candidate odorants to be targeted in a novel strategy to reduce attractants or produce repellents by the human skin microbiota that may curtail mosquito bites, and subsequent mosquito-borne disease.

  View details for DOI 10.1101/2023.08.19.553996

  View details for PubMedID 37662338

• Mapping the T cell repertoire to a complex gut bacterial community. Nature Nagashima, K., Zhao, A., Atabakhsh, K., Bae, M., Blum, J. E., Weakley, A., Jain, S., Meng, X., Cheng, A. G., Wang, M., Higginbottom, S., Dimas, A., Murugkar, P., Sattely, E. S., Moon, J. J., Balskus, E. P., Fischbach, M. A. 2023

  Abstract

  Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.

  View details for DOI 10.1038/s41586-023-06431-8

  View details for PubMedID 37587342

  View details for PubMedCentralID 4128479

• Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome. Cell Wang, M., Osborn, L. J., Jain, S., Meng, X., Weakley, A., Yan, J., Massey, W. J., Varadharajan, V., Horak, A., Banerjee, R., Allende, D. S., Chan, E. R., Hajjar, A. M., Wang, Z., Dimas, A., Zhao, A., Nagashima, K., Cheng, A. G., Higginbottom, S., Hazen, S. L., Brown, J. M., Fischbach, M. A. 2023; 186 (13): 2839-2852.e21

  Abstract

  The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype.

  View details for DOI 10.1016/j.cell.2023.05.037

  View details for PubMedID 37352836

• Atlas of gut microbe-derived products from aromatic amino acids and risk of cardiovascular morbidity and mortality. European heart journal Nemet, I., Li, X. S., Haghikia, A., Li, L., Wilcox, J., Romano, K. A., Buffa, J. A., Witkowski, M., Demuth, I., König, M., Steinhagen-Thiessen, E., Bäckhed, F., Fischbach, M. A., Tang, W. H., Landmesser, U., Hazen, S. L. 2023

  Abstract

  Precision microbiome modulation as a novel treatment strategy is a rapidly evolving and sought goal. The aim of this study is to determine relationships among systemic gut microbial metabolite levels and incident cardiovascular disease risks to identify gut microbial pathways as possible targets for personalized therapeutic interventions.Stable isotope dilution mass spectrometry methods to quantitatively measure aromatic amino acids and their metabolites were used to examine sequential subjects undergoing elective diagnostic cardiac evaluation in two independent cohorts with longitudinal outcome data [US (n = 4000) and EU (n = 833) cohorts]. It was also used in plasma from humans and mice before vs. after a cocktail of poorly absorbed antibiotics to suppress gut microbiota. Multiple aromatic amino acid-derived metabolites that originate, at least in part, from gut bacteria are associated with incident (3-year) major adverse cardiovascular event (MACE) risks (myocardial infarction, stroke, or death) and all-cause mortality independent of traditional risk factors. Key gut microbiota-derived metabolites associated with incident MACE and poorer survival risks include: (i) phenylacetyl glutamine and phenylacetyl glycine (from phenylalanine); (ii) p-cresol (from tyrosine) yielding p-cresol sulfate and p-cresol glucuronide; (iii) 4-OH-phenyllactic acid (from tyrosine) yielding 4-OH-benzoic acid and 4-OH-hippuric acid; (iv) indole (from tryptophan) yielding indole glucuronide and indoxyl sulfate; (v) indole-3-pyruvic acid (from tryptophan) yielding indole-3-lactic acid and indole-3-acetyl-glutamine, and (vi) 5-OH-indole-3-acetic acid (from tryptophan).Key gut microbiota-generated metabolites derived from aromatic amino acids independently associated with incident adverse cardiovascular outcomes are identified, and thus will help focus future studies on gut-microbial metabolic outputs relevant to host cardiovascular health.

  View details for DOI 10.1093/eurheartj/ehad333

  View details for PubMedID 37342006

• Deciphering the crosstalk between skin commensals and the immune system. Bouladoux, N., Chen, Y., Fischbach, M. A., Belkaid, Y. AMER ASSOC IMMUNOLOGISTS. 2023

  View details for Web of Science ID 001106506502404

• Mapping the T cell repertoire to a complex defined gut bacterial community Nagashima, K., Fischbach, M. A. AMER ASSOC IMMUNOLOGISTS. 2023

  View details for Web of Science ID 001106506502265

• Engineered skin bacteria induce antitumor T cell responses against melanoma. Science (New York, N.Y.) Chen, Y. E., Bousbaine, D., Veinbachs, A., Atabakhsh, K., Dimas, A., Yu, V. K., Zhao, A., Enright, N. J., Nagashima, K., Belkaid, Y., Fischbach, M. A. 2023; 380 (6641): 203-210

  Abstract

  Certain bacterial colonists induce a highly specific T cell response. A hallmark of this encounter is that adaptive immunity develops preemptively, in the absence of an infection. However, the functional properties of colonist-induced T cells are not well defined, limiting our ability to understand anticommensal immunity and harness it therapeutically. We addressed both challenges by engineering the skin bacterium Staphylococcus epidermidis to express tumor antigens anchored to secreted or cell-surface proteins. Upon colonization, engineered S. epidermidis elicits tumor-specific T cells that circulate, infiltrate local and metastatic lesions, and exert cytotoxic activity. Thus, the immune response to a skin colonist can promote cellular immunity at a distal site and can be redirected against a target of therapeutic interest by expressing a target-derived antigen in a commensal.

  View details for DOI 10.1126/science.abp9563

  View details for PubMedID 37053311

• gutSMASH predicts specialized primary metabolic pathways from the human gut microbiota. Nature biotechnology Pascal Andreu, V., Augustijn, H. E., Chen, L., Zhernakova, A., Fu, J., Fischbach, M. A., Dodd, D., Medema, M. H. 2023

  Abstract

  The gut microbiota produce hundreds of small molecules, many of which modulate host physiology. Although efforts have been made to identify biosynthetic genes for secondary metabolites, the chemical output of the gut microbiome consists predominantly of primary metabolites. Here we introduce the gutSMASH algorithm for identification of primary metabolic gene clusters, and we used it to systematically profile gut microbiome metabolism, identifying 19,890 gene clusters in 4,240 high-quality microbial genomes. We found marked differences in pathway distribution among phyla, reflecting distinct strategies for energy capture. These data explain taxonomic differences in short-chain fatty acid production and suggest a characteristic metabolic niche for each taxon. Analysis of 1,135 individuals from a Dutch population-based cohort shows that the level of microbiome-derived metabolites in plasma and feces is almost completely uncorrelated with the metagenomic abundance of corresponding metabolic genes, indicating a crucial role for pathway-specific gene regulation and metabolite flux. This work is a starting point for understanding differences in how bacterial taxa contribute to the chemistry of the microbiome.

  View details for DOI 10.1038/s41587-023-01675-1

  View details for PubMedID 36782070

• Host-microbe co-metabolism via MCAD generates circulating metabolites including hippuric acid. Nature communications Pruss, K. M., Chen, H., Liu, Y., Van Treuren, W., Higginbottom, S. K., Jarman, J. B., Fischer, C. R., Mak, J., Wong, B., Cowan, T. M., Fischbach, M. A., Sonnenburg, J. L., Dodd, D. 2023; 14 (1): 512

  Abstract

  The human gut microbiota produces dozens of small molecules that circulate in blood, accumulate to comparable levels as pharmaceutical drugs, and influence host physiology. Despite the importance of these metabolites to human health and disease, the origin of most microbially-produced molecules and their fate in the host remains largely unknown. Here, we uncover a host-microbe co-metabolic pathway for generation of hippuric acid, one of the most abundant organic acids in mammalian urine. Combining stable isotope tracing with bacterial and host genetics, we demonstrate reduction of phenylalanine to phenylpropionic acid by gut bacteria; the host re-oxidizes phenylpropionic acid involving medium-chain acyl-CoA dehydrogenase (MCAD). Generation of germ-free male and female MCAD-/- mice enabled gnotobiotic colonization combined with untargeted metabolomics to identify additional microbial metabolites processed by MCAD in host circulation. Our findings uncover a host-microbe pathway for the abundant, non-toxic phenylalanine metabolite hippurate and identify β-oxidation via MCAD as a novel mechanism by which mammals metabolize microbiota-derived metabolites.

  View details for DOI 10.1038/s41467-023-36138-3

  View details for PubMedID 36720857

• Retroviral Infection and Commensal Bacteria Dependently Alter the Metabolomic Profile in a Sterile Organ. Viruses Spring, J., Beilinson, V., DeFelice, B. C., Sanchez, J. M., Fischbach, M., Chervonsky, A., Golovkina, T. 2023; 15 (2)

  Abstract

  Both viruses and bacteria produce "pathogen associated molecular patterns" that may affect microbial pathogenesis and anti-microbial responses. Additionally, bacteria produce metabolites, while viruses could change the metabolic profiles of the infected cells. Here, we used an unbiased metabolomics approach to profile metabolites in spleens and blood of murine leukemia virus-infected mice monocolonized with Lactobacillus murinus to show that viral infection significantly changes the metabolite profile of monocolonized mice. We hypothesize that these changes could contribute to viral pathogenesis or to the host response against the virus and thus open a new avenue for future investigations.

  View details for DOI 10.3390/v15020386

  View details for PubMedID 36851600

  View details for PubMedCentralID PMC9967258

• A Limited Effect of Chronic Renal Insufficiency on the Colon Microbiome. Journal of the American Society of Nephrology : JASN Guthrie, L., Sonnenburg, J. L., Fischbach, M. A., Meyer, T. W. 2023

  View details for DOI 10.1681/ASN.0000000000000064

  View details for PubMedID 36753629

• Two distinct gut microbial pathways contribute to meta-organismal production of phenylacetylglutamine with links to cardiovascular disease. Cell host & microbe Zhu, Y., Dwidar, M., Nemet, I., Buffa, J. A., Sangwan, N., Li, X. S., Anderson, J. T., Romano, K. A., Fu, X., Funabashi, M., Wang, Z., Keranahalli, P., Battle, S., Tittle, A. N., Hajjar, A. M., Gogonea, V., Fischbach, M. A., DiDonato, J. A., Hazen, S. L. 2022

  Abstract

  Recent studies show gut microbiota-dependent metabolism of dietary phenylalanine into phenylacetic acid (PAA) is critical in phenylacetylglutamine (PAGln) production, a metabolite linked to atherosclerotic cardiovascular disease (ASCVD). Accordingly, microbial enzymes involved in this transformation are of interest. Using genetic manipulation in selected microbes and monocolonization experiments in gnotobiotic mice, we identify two distinct gut microbial pathways for PAA formation; one is catalyzed by phenylpyruvate:ferredoxin oxidoreductase (PPFOR) and the other by phenylpyruvate decarboxylase (PPDC). PPFOR and PPDC play key roles in gut bacterial PAA production via oxidative and non-oxidative phenylpyruvate decarboxylation, respectively. Metagenomic analyses revealed a significantly higher abundance of both pathways in gut microbiomes of ASCVD patients compared with controls. The present studies show a role for these two divergent microbial catalytic strategies in the meta-organismal production of PAGln. Given the numerous links between PAGln and ASCVD, these findings will assist future efforts to therapeutically target PAGln formation in vivo.

  View details for DOI 10.1016/j.chom.2022.11.015

  View details for PubMedID 36549300

• Bacteroides thetaiotaomicron rough-type lipopolysaccharide: The chemical structure and the immunological activity CARBOHYDRATE POLYMERS Pither, M., Illiano, A., Pagliuca, C., Jacobson, A., Mantova, G., Stornaiuolo, A., Colicchio, R., Vitiello, M., Pinto, G., Silipo, A., Fischbach, M. A., Salvatore, P., Amoresano, A., Molinaro, A., Di Lorenzo, F. 2022; 297: 120040

  View details for DOI 10.1016/j.carbpol.2022.120040

  View details for Web of Science ID 000860478800003

  View details for PubMedID 36184180

• Gut commensal bacteria enhance pathogenesis of a tumorigenic murine retrovirus. Cell reports Spring, J., Khan, A. A., Lara, S., O'Grady, K., Wilks, J., Gurbuxani, S., Erickson, S., Fischbach, M., Jacobson, A., Chervonsky, A., Golovkina, T. 2022; 40 (11): 111341

  Abstract

  The influence of the microbiota on viral transmission and replication is well appreciated. However, its impact on retroviral pathogenesis outside of transmission/replication control remains unknown. Using murine leukemia virus (MuLV), we found that some commensal bacteria promoted the development of leukemia induced by this retrovirus. The promotion of leukemia development by commensals is due to suppression of the adaptive immune response through upregulation of several negative regulators of immunity. These negative regulators include Serpinb9b and Rnf128, which are associated with a poor prognosis of some spontaneous human cancers. Upregulation of Serpinb9b is mediated by sensing of bacteria by the NOD1/NOD2/RIPK2 pathway. This work describes a mechanism by which the microbiota enhances tumorigenesis within gut-distant organs and points at potential targets for cancer therapy.

  View details for DOI 10.1016/j.celrep.2022.111341

  View details for PubMedID 36103821

• Design, construction, and invivo augmentation of a complex gut microbiome. Cell Cheng, A. G., Ho, P., Aranda-Diaz, A., Jain, S., Yu, F. B., Meng, X., Wang, M., Iakiviak, M., Nagashima, K., Zhao, A., Murugkar, P., Patil, A., Atabakhsh, K., Weakley, A., Yan, J., Brumbaugh, A. R., Higginbottom, S., Dimas, A., Shiver, A. L., Deutschbauer, A., Neff, N., Sonnenburg, J. L., Huang, K. C., Fischbach, M. A. 2022

  Abstract

  Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex, but not both, limiting their utility. Here, we construct and characterize invitro a defined community of 104 bacterial species composed of the most common taxa from the human gut microbiota (hCom1). We then used an iterative experimental process to fill open niches: germ-free mice were colonized with hCom1 and then challenged with a human fecal sample. We identified new species that engrafted following fecal challenge and added them to hCom1, yielding hCom2. In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by either hCom2 or a human fecal community are phenotypically similar, suggesting that this consortium will enable a mechanistic interrogation of species and genes on microbiome-associated phenotypes.

  View details for DOI 10.1016/j.cell.2022.08.003

  View details for PubMedID 36070752

• Gut Microbe-derived Short-Chain Fatty Acids Regulate Arthritis and Myositis During Chikungunya Virus Infection Zhao, F. R., Winkler, E., Guo, C., Williams, R. B., Wang, L., Jung, A., Droit, L., Heath, L., Li, T., Mack, M., Baldridge, M. T., Stappenbeck, T. S., Thackray, L. B., Handley, S. A., Fischbach, M. A., Diamond, M. S. WILEY. 2022: 4325-4326

  View details for Web of Science ID 000877386506157

• Impact of a 7-day homogeneous diet on interpersonal variation in human gut microbiomes and metabolomes. Cell host & microbe Guthrie, L., Spencer, S. P., Perelman, D., Van Treuren, W., Han, S., Yu, F. B., Sonnenburg, E. D., Fischbach, M. A., Meyer, T. W., Sonnenburg, J. L. 2022

  Abstract

  Gut microbiota metabolism of dietary compounds generates a vast array of microbiome-dependent metabolites (MDMs), which are highly variable between individuals. The uremic MDMs (uMDMs) phenylacetylglutamine (PAG), p-cresol sulfate (PCS), and indoxyl sulfate (IS) accumulate during renal failure and are associated with poor outcomes. Targeted dietary interventions may reduce toxic MDM generation; however, it is unclear if inter-individual differences in diet or gut microbiome dominantly contribute to MDM variance. Here, we use a 7-day homogeneous average American diet to standardize dietary precursor availability in 21 healthy individuals. During dietary homogeneity, the coefficient of variation in PAG, PCS, and IS (primary outcome) did not decrease, nor did inter-individual variation in most identified metabolites; other microbiome metrics showed no or modest responses to the intervention. Host identity and age are dominant contributors to variability in MDMs. These results highlight the potential need to pair dietary modification with microbial therapies to control MDM profiles.

  View details for DOI 10.1016/j.chom.2022.05.003

  View details for PubMedID 35643079

• A gut-derived metabolite alters brain activity and anxiety behaviour in mice. Nature Needham, B. D., Funabashi, M., Adame, M. D., Wang, Z., Boktor, J. C., Haney, J., Wu, W., Rabut, C., Ladinsky, M. S., Hwang, S., Guo, Y., Zhu, Q., Griffiths, J. A., Knight, R., Bjorkman, P. J., Shapiro, M. G., Geschwind, D. H., Holschneider, D. P., Fischbach, M. A., Mazmanian, S. K. 2022

  Abstract

  Integration of sensory and molecular inputs from the environment shapes animal behaviour. A major site of exposure to environmental molecules is the gastrointestinal tract, in which dietary components are chemically transformed by the microbiota1 and gut-derived metabolites are disseminated to all organs, including the brain2. In mice, the gut microbiota impacts behaviour3, modulates neurotransmitter production in the gut and brain4,5, and influences brain development and myelination patterns6,7. The mechanisms that mediate the gut-brain interactions remain poorly defined, although they broadly involve humoral or neuronal connections. We previously reported that the levels of the microbial metabolite 4-ethylphenyl sulfate (4EPS) were increased in a mouse model of atypical neurodevelopment8. Here we identified biosynthetic genes from the gut microbiome that mediate the conversion of dietary tyrosine to 4-ethylphenol (4EP), and bioengineered gut bacteria to selectively produce 4EPS in mice. 4EPS entered the brain and was associated with changes in region-specific activity and functional connectivity. Gene expression signatures revealed altered oligodendrocyte function in the brain, and 4EPS impaired oligodendrocyte maturation in mice and decreased oligodendrocyte-neuron interactions in ex vivo brain cultures. Mice colonized with 4EP-producing bacteria exhibited reduced myelination of neuronal axons. Altered myelination dynamics in the brain have been associated with behavioural outcomes7,9-14. Accordingly, we observed that mice exposed to 4EPS displayed anxiety-like behaviours, and pharmacological treatments that promote oligodendrocyte differentiation prevented the behavioural effects of 4EPS. These findings reveal that a gut-derived molecule influences complex behaviours in mice through effects on oligodendrocyte function and myelin patterning in the brain.

  View details for DOI 10.1038/s41586-022-04396-8

  View details for PubMedID 35165440

• Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians. Nature Sato, Y., Atarashi, K., Plichta, D. R., Arai, Y., Sasajima, S., Kearney, S. M., Suda, W., Takeshita, K., Sasaki, T., Okamoto, S., Skelly, A. N., Okamura, Y., Vlamakis, H., Li, Y., Tanoue, T., Takei, H., Nittono, H., Narushima, S., Irie, J., Itoh, H., Moriya, K., Sugiura, Y., Suematsu, M., Moritoki, N., Shibata, S., Littman, D. R., Fischbach, M. A., Uwamino, Y., Inoue, T., Honda, A., Hattori, M., Murai, T., Xavier, R. J., Hirose, N., Honda, K. 2021

  Abstract

  Centenarians display decreased susceptibility to ageing-associated illness, chronic inflammation, and infectious disease1-3. Here we show that centenarians have a distinct gut microbiome enriched in microbes capable of generating unique secondary bile acids (BAs), including iso-, 3-oxo-, allo-, 3-oxoallo-, and isoallo-lithocholic acid (LCA). Among these BAs, the biosynthetic pathway for isoalloLCA had not been described previously. By screening 68 bacterial isolates from a centenarian's faecal microbiota, we identified Odoribacteraceae strains as effective producers of isoalloLCA both in vitro and in vivo. Furthermore, we found that the enzymes 5alpha-reductase (5AR) and 3beta-hydroxysteroid dehydrogenase (3betaHSDH) were responsible for isoalloLCA production. IsoalloLCA exerted potent antimicrobial effects against gram-positive (but not gram-negative) multidrug-resistant pathogens, including Clostridioides difficile and Enterococcus faecium. These findings suggest that specific bile acid metabolism may be involved in reducing the risk of pathobiont infection, thereby potentially contributing to the maintenance of intestinal homeostasis.

  View details for DOI 10.1038/s41586-021-03832-5

  View details for PubMedID 34325466

• Metagenomic compendium of 189,680 DNA viruses from the human gut microbiome. Nature microbiology Nayfach, S., Paez-Espino, D., Call, L., Low, S. J., Sberro, H., Ivanova, N. N., Proal, A. D., Fischbach, M. A., Bhatt, A. S., Hugenholtz, P., Kyrpides, N. C. 2021

  Abstract

  Bacteriophages have important roles in the ecology of the human gut microbiome but are under-represented in reference databases. To address this problem, we assembled the Metagenomic Gut Virus catalogue that comprises 189,680 viral genomes from 11,810 publicly available human stool metagenomes. Over 75% of genomes represent double-stranded DNA phages that infect members of the Bacteroidia and Clostridia classes. Based on sequence clustering we identified 54,118 candidate viral species, 92% of which were not found in existing databases. The Metagenomic Gut Virus catalogue improves detection of viruses in stool metagenomes and accounts for nearly 40% of CRISPR spacers found in human gut Bacteria and Archaea. We also produced a catalogue of 459,375 viral protein clusters to explore the functional potential of the gut virome. This revealed tens of thousands of diversity-generating retroelements, which use error-prone reverse transcription to mutate target genes and may be involved in the molecular arms race between phages and their bacterial hosts.

  View details for DOI 10.1038/s41564-021-00928-6

  View details for PubMedID 34168315

• Gut microbes impact stroke severity via the trimethylamine N-oxide pathway. Cell host & microbe Zhu, W., Romano, K. A., Li, L., Buffa, J. A., Sangwan, N., Prakash, P., Tittle, A. N., Li, X. S., Fu, X., Androjna, C., DiDonato, A. J., Brinson, K., Trapp, B. D., Fischbach, M. A., Rey, F. E., Hajjar, A. M., DiDonato, J. A., Hazen, S. L. 2021

  Abstract

  Clinical studies have demonstrated associations between circulating levels of the gut-microbiota-derived metabolite trimethylamine-N-oxide (TMAO) and stroke incident risk. However, a causal role of gut microbes in stroke has not yet been demonstrated. Herein we show that gut microbes, through dietary choline and TMAO generation, directly impact cerebral infarct size and adverse outcomes following stroke. Fecal microbial transplantation from low- versus high-TMAO-producing human subjects into germ-free mice shows that both TMAO generation and stroke severity are transmissible traits. Furthermore, employing multiple murine stroke models and transplantation of defined microbial communities with genetically engineered human commensals into germ-free mice, we demonstrate that the microbial cutC gene (an enzymatic source of choline-to-TMA transformation) is sufficient to transmit TMA/TMAO production, heighten cerebral infarct size, and lead to functional impairment. We thus reveal that gut microbiota in general, specifically the metaorganismal TMAO pathway, directly contributes to stroke severity.

  View details for DOI 10.1016/j.chom.2021.05.002

  View details for PubMedID 34139173

• The gutSMASH web server:automated identification of primary metabolic gene clusters from the gut microbiota. Nucleic acids research Pascal Andreu, V., Roel-Touris, J., Dodd, D., Fischbach, M. A., Medema, M. H. 2021

  Abstract

  Anaerobic bacteria from the human microbiome produce a wide array of molecules at high concentrations that can directly or indirectly affect the host. The production of these molecules, mostly derived from their primary metabolism, is frequently encoded in metabolic gene clusters (MGCs). However, despite the importance of microbiome-derived primary metabolites, no tool existed to predict the gene clusters responsible for their production. For this reason, we recently introduced gutSMASH. gutSMASH can predict 41 different known pathways, including MGCs involved in bioenergetics, but also putative ones that are candidates for novel pathway discovery. To make the tool more user-friendly and accessible, we here present the gutSMASH web server, hosted at https://gutsmash.bioinformatics.nl/. The user can either input the GenBank assembly accession or upload a genome file in FASTA or GenBank format. Optionally, the user can enable additional analyses to obtain further insights into the predicted MGCs. An interactive HTML output (viewable online or downloadable for offline use) provides a user-friendly way to browse functional gene annotations and sequence comparisons with reference gene clusters as well as gene clusters predicted in other genomes. Thus, this web server provides the community with a streamlined and user-friendly interface to analyze the metabolic potential of gut microbiomes.

  View details for DOI 10.1093/nar/gkab353

  View details for PubMedID 34019648

• CRISPR-based functional genomics in human dendritic cells. eLife Jost, M., Jacobson, A. N., Hussmann, J. A., Cirolia, G., Fischbach, M. A., Weissman, J. S. 2021; 10

  Abstract

  Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >94% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.

  View details for DOI 10.7554/eLife.65856

  View details for PubMedID 33904395

• A method for detection of SARS-CoV-2 RNA in healthy human stool: a validation study. The Lancet. Microbe Coryell, M. P., Iakiviak, M., Pereira, N., Murugkar, P. P., Rippe, J., Williams, D. B., Heald-Sargent, T., Sanchez-Pinto, L. N., Chavez, J., Hastie, J. L., Sava, R. L., Lien, C. Z., Wang, T. T., Muller, W. J., Fischbach, M. A., Carlson, P. E. 2021

  Abstract

  Background: Faecal shedding of SARS-CoV-2 has raised concerns about transmission through faecal microbiota transplantation procedures. Validation parameters of authorised tests for SARS-CoV-2 RNA detection in respiratory samples are described in product labelling, whereas the published methods for SARS-CoV-2 detection from faecal samples have not permitted a robust description of the assay parameters. We aimed to develop and validate a test specifically for detection of SARS-CoV-2 in human stool.Methods: In this validation study, we evaluated performance characteristics of a reverse transcriptase real-time PCR (RT-rtPCR) test for detection of SARS-CoV-2 in human stool specimens by spiking stool with inactivated SARS-CoV-2 material. A modified version of the US Centers for Disease Control and Prevention RT-rtPCR SARS-CoV-2 test was used for detection of viral RNA. Analytical sensitivity was evaluated in freshly spiked stool by testing two-fold dilutions in replicates of 20. Masked samples were tested by a second laboratory to evaluate interlaboratory reproducibility. Short-term (7-day) stability of viral RNA in stool samples was assessed with four different stool storage buffers (phosphate-buffered saline, Cary-Blair medium, Stool Transport and Recovery [STAR] buffer, and DNA/RNA Shield) kept at -80°C, 4°C, and ambient temperature (approximately 21°C). We also tested clinical stool and anal swab specimens from patients who were SARS-CoV-2 positive by nasopharyngeal testing.Findings: The lower limit of detection of the assay was found to be 3000 viral RNA copies per g of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Analytical sensitivity was diminished by approximately two times after a single freeze-thaw cycle at -80°C. At 100 times the limit of detection, spiked samples were generally stable in all four stool storage buffers tested for up to 7 days, with maximum changes in mean threshold cycle values observed at -80°C storage in Cary-Blair medium (from 29·4 [SD 0·27] at baseline to 30·8 [0·17] at day 7; p<0·0001), at 4°C storage in DNA/RNA Shield (from 28·5 [0·15] to 29·8 [0·09]; p=0·0019), and at ambient temperature in STAR buffer (from 30·4 [0·24] to 32·4 [0·62]; p=0·0083). 30 contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, SARS-CoV-2 RNA was detected using this assay in the stool and anal swab specimens of 11 of 23 individuals known to be positive for SARS-CoV-2.Interpretation: This is a sensitive and reproducible assay for detection of SARS-CoV-2 RNA in human stool, with potential uses in faecal microbiota transplantation donor screening, sewage monitoring, and further research into the effects of faecal shedding on the epidemiology of the COVID-19 pandemic.Funding: National Institute of Allergy and Infectious Diseases, US National Institutes of Health; Center for Biologics Evaluation and Research, US Food and Drug Administration.

  View details for DOI 10.1016/S2666-5247(21)00059-8

  View details for PubMedID 33821247

• Role of dietary fiber in the recovery of the human gut microbiome and its metabolome. Cell host & microbe Tanes, C., Bittinger, K., Gao, Y., Friedman, E. S., Nessel, L., Paladhi, U. R., Chau, L., Panfen, E., Fischbach, M. A., Braun, J., Xavier, R. J., Clish, C. B., Li, H., Bushman, F. D., Lewis, J. D., Wu, G. D. 2021

  Abstract

  Gut microbiota metabolites may be important for host health, yet few studies investigate the correlation between human gut microbiome and production of fecal metabolites and their impact on the plasma metabolome. Since gut microbiota metabolites are influenced by diet, we performed a longitudinal analysis of the impact of three divergent diets, vegan, omnivore, and a synthetic enteral nutrition (EEN) diet lacking fiber, on the human gut microbiome and its metabolome, including after a microbiota depletion intervention. Omnivore and vegan, but not EEN, diets altered fecal amino acid levels by supporting the growth of Firmicutes capable of amino acid metabolism. This correlated with relative abundance of a sizable number of fecal amino acid metabolites, some not previously associated with the gut microbiota. The effect on the plasma metabolome, in contrast, were modest. The impact of diet, particularly fiber, on the human microbiome influences broad classes of metabolites that may modify health.

  View details for DOI 10.1016/j.chom.2020.12.012

  View details for PubMedID 33440171

• A metabolomics pipeline for the mechanistic interrogation of the gut microbiome. Nature Han, S., Van Treuren, W., Fischer, C. R., Merrill, B. D., DeFelice, B. C., Sanchez, J. M., Higginbottom, S. K., Guthrie, L., Fall, L. A., Dodd, D., Fischbach, M. A., Sonnenburg, J. L. 2021; 595 (7867): 415-420

  Abstract

  Gut microorganisms modulate host phenotypes and are associated with numerous health effects in humans, ranging from host responses to cancer immunotherapy to metabolic disease and obesity. However, difficulty in accurate and high-throughput functional analysis of human gut microorganisms has hindered efforts to define mechanistic connections between individual microbial strains and host phenotypes. One key way in which the gut microbiome influences host physiology is through the production of small molecules1-3, yet progress in elucidating this chemical interplay has been hindered by limited tools calibrated to detect the products of anaerobic biochemistry in the gut. Here we construct a microbiome-focused, integrated mass-spectrometry pipeline to accelerate the identification of microbiota-dependent metabolites in diverse sample types. We report the metabolic profiles of 178 gut microorganism strains using our library of 833 metabolites. Using this metabolomics resource, we establish deviations in the relationships between phylogeny and metabolism, use machine learning to discover a previously undescribed type of metabolism in Bacteroides, and reveal candidate biochemical pathways using comparative genomics. Microbiota-dependent metabolites can be detected in diverse biological fluids from gnotobiotic and conventionally colonized mice and traced back to the corresponding metabolomic profiles of cultured bacteria. Collectively, our microbiome-focused metabolomics pipeline and interactive metabolomics profile explorer are a powerful tool for characterizing microorganisms and interactions between microorganisms and their host.

  View details for DOI 10.1038/s41586-021-03707-9

  View details for PubMedID 34262212

• Endogenous retroviruses promote homeostatic and inflammatory responses to the microbiota. Cell Lima-Junior, D. S., Krishnamurthy, S. R., Bouladoux, N., Collins, N., Han, S. J., Chen, E. Y., Constantinides, M. G., Link, V. M., Lim, A. I., Enamorado, M., Cataisson, C., Gil, L., Rao, I., Farley, T. K., Koroleva, G., Attig, J., Yuspa, S. H., Fischbach, M. A., Kassiotis, G., Belkaid, Y. 2021

  Abstract

  The microbiota plays a fundamental role in regulating host immunity. However, the processes involved in the initiation and regulation of immunity to the microbiota remain largely unknown. Here, we show that the skin microbiota promotes the discrete expression of defined endogenous retroviruses (ERVs). Keratinocyte-intrinsic responses to ERVs depended on cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING) signaling and promoted the induction of commensal-specific T cells. Inhibition of ERV reverse transcription significantly impacted these responses, resulting in impaired immunity to the microbiota and its associated tissue repair function. Conversely, a lipid-enriched diet primed the skin for heightened ERV- expression in response to commensal colonization, leading to increased immune responses and tissue inflammation. Together, our results support the idea that the host may have co-opted its endogenous virome as a means to communicate with the exogenous microbiota, resulting in a multi-kingdom dialog that controls both tissue homeostasis and inflammation.

  View details for DOI 10.1016/j.cell.2021.05.020

  View details for PubMedID 34166614

• BiG-MAP: an Automated Pipeline To Profile Metabolic Gene Cluster Abundance and Expression in Microbiomes. mSystems Pascal Andreu, V., Augustijn, H. E., van den Berg, K., van der Hooft, J. J., Fischbach, M. A., Medema, M. H. 2021: e0093721

  Abstract

  Microbial gene clusters encoding the biosynthesis of primary and secondary metabolites play key roles in shaping microbial ecosystems and driving microbiome-associated phenotypes. Although effective approaches exist to evaluate the metabolic potential of such bacteria through identification of these metabolic gene clusters in their genomes, no automated pipelines exist to profile the abundance and expression levels of such gene clusters in microbiome samples to generate hypotheses about their functional roles, and to find associations with phenotypes of interest. Here, we describe BiG-MAP, a bioinformatic tool to profile abundance and expression levels of gene clusters across metagenomic and metatranscriptomic data and evaluate their differential abundance and expression under different conditions. To illustrate its usefulness, we analyzed 96 metagenomic samples from healthy and caries-associated human oral microbiome samples and identified 252 gene clusters, including unreported ones, that were significantly more abundant in either phenotype. Among them, we found the muc operon, a gene cluster known to be associated with tooth decay. Additionally, we found a putative reuterin biosynthetic gene cluster from a Streptococcus strain to be enriched but not exclusively found in healthy samples; metabolomic data from the same samples showed masses with fragmentation patterns consistent with (poly)acrolein, which is known to spontaneously form from the products of the reuterin pathway and has been previously shown to inhibit pathogenic Streptococcus mutans strains. Thus, we show how BiG-MAP can be used to generate new hypotheses on potential drivers of microbiome-associated phenotypes and prioritize the experimental characterization of relevant gene clusters that may mediate them. IMPORTANCE Microbes play an increasingly recognized role in determining host-associated phenotypes by producing small molecules that interact with other microorganisms or host cells. The production of these molecules is often encoded in syntenic genomic regions, also known as gene clusters. With the increasing numbers of (multi)omics data sets that can help in understanding complex ecosystems at a much deeper level, there is a need to create tools that can automate the process of analyzing these gene clusters across omics data sets. This report presents a new software tool called BiG-MAP, which allows assessing gene cluster abundance and expression in microbiome samples using metagenomic and metatranscriptomic data. Here, we describe the tool and its functionalities, as well as its validation using a mock community. Finally, using an oral microbiome data set, we show how it can be used to generate hypotheses regarding the functional roles of gene clusters in mediating host phenotypes.

  View details for DOI 10.1128/mSystems.00937-21

  View details for PubMedID 34581602

• Bacterially Derived Tryptamine Increases Mucus Release by Activating a Host Receptor in a Mouse Model of Inflammatory Bowel Disease. iScience Bhattarai, Y., Jie, S., Linden, D. R., Ghatak, S., Mars, R. A., Williams, B. B., Pu, M., Sonnenburg, J. L., Fischbach, M. A., Farrugia, G., Sha, L., Kashyap, P. C. 2020; 23 (12): 101798

  Abstract

  Recent studies emphasize the role of microbial metabolites in regulating gastrointestinal (GI) physiology through activation of host receptors, highlighting the potential for inter-kingdom signaling in treating GI disorders. In this study, we show that tryptamine, a tryptophan-derived bacterial metabolite, stimulates mucus release from goblet cells via activation of G-protein-coupled receptor (GPCR) 5-HT4R. Germ-free mice colonized with engineered Bacteroides thetaiotaomicron optimized to produce tryptamine (Trp D+) exhibit decreased weight loss and increased mucus release following dextran sodium sulfate treatment when compared with mice colonized with control B.thetaiotaomicron (Trp D-). Additional beneficial effects in preventing barrier disruption and lower disease activity index were seen only in female mice, highlighting sex-specific effects of the bacterial metabolite. This study demonstrates potential for the precise modulation of mucus release by microbially produced 5-HT4 GPCR agonist as a therapeutic strategy to treat inflammatory conditions of the GI tract.

  View details for DOI 10.1016/j.isci.2020.101798

  View details for PubMedID 33299969

• A Cutibacterium acnes antibiotic modulates human skin microbiota composition in hair follicles. Science translational medicine Claesen, J., Spagnolo, J. B., Ramos, S. F., Kurita, K. L., Byrd, A. L., Aksenov, A. A., Melnik, A. V., Wong, W. R., Wang, S., Hernandez, R. D., Donia, M. S., Dorrestein, P. C., Kong, H. H., Segre, J. A., Linington, R. G., Fischbach, M. A., Lemon, K. P. 2020; 12 (570)

  Abstract

  The composition of the skin microbiota varies widely among individuals when sampled at the same body site. A key question is which molecular factors determine strain-level variability within sub-ecosystems of the skin microbiota. Here, we used a genomics-guided approach to identify an antibacterial biosynthetic gene cluster in Cutibacterium acnes (formerly Propionibacterium acnes), a human skin commensal bacterium that is widely distributed across individuals and skin sites. Experimental characterization of this biosynthetic gene cluster resulted in identification of a new thiopeptide antibiotic, cutimycin. Analysis of individual human skin hair follicles revealed that cutimycin contributed to the ecology of the skin hair follicle microbiota and helped to reduce colonization of skin hair follicles by Staphylococcus species.

  View details for DOI 10.1126/scitranslmed.aay5445

  View details for PubMedID 33208503

• Bifidobacterium alters the gut microbiota and modulates the functional metabolism of T regulatory cells in the context of immune checkpoint blockade. Proceedings of the National Academy of Sciences of the United States of America Sun, S., Luo, L., Liang, W., Yin, Q., Guo, J., Rush, A. M., Lv, Z., Liang, Q., Fischbach, M. A., Sonnenburg, J. L., Dodd, D., Davis, M. M., Wang, F. 2020

  Abstract

  Immune checkpoint-blocking antibodies that attenuate immune tolerance have been used to effectively treat cancer, but they can also trigger severe immune-related adverse events. Previously, we found that Bifidobacterium could mitigate intestinal immunopathology in the context of CTLA-4 blockade in mice. Here we examined the mechanism underlying this process. We found that Bifidobacterium altered the composition of the gut microbiota systematically in a regulatory T cell (Treg)-dependent manner. Moreover, this altered commensal community enhanced both the mitochondrial fitness and the IL-10-mediated suppressive functions of intestinal Tregs, contributing to the amelioration of colitis during immune checkpoint blockade.

  View details for DOI 10.1073/pnas.1921223117

  View details for PubMedID 33077598

• Computational genomic discovery of diverse gene clusters harbouring Fe-S flavoenzymes in anaerobic gut microbiota. Microbial genomics Pascal Andreu, V., Fischbach, M. A., Medema, M. H. 2020

  Abstract

  The gut contains an enormous diversity of simple as well as complex molecules from highly diverse food sources, together with host-secreted molecules. This presents a large metabolic opportunity for the gut microbiota, but little is known about how gut microbes are able to catabolize this large chemical diversity. Recently, Fe-S flavoenzymes were found to be key in the transformation of bile acids, catalysing the key step in the 7alpha-dehydroxylation pathway that allows gut bacteria to transform cholic acid into deoxycholic acid, an exclusively microbe-derived molecule with major implications for human health. While this enzyme family has also been implicated in a limited number of other catalytic transformations, little is known about the extent to which it is of more global importance in gut microbial metabolism. Here, we perform a large-scale computational genomic analysis to show that this enzyme superfamily has undergone a remarkable expansion in Clostridiales, and occurs throughout a diverse array of >1000 different families of putative metabolic gene clusters. Analysis of the enzyme content of these gene clusters suggests that they encode pathways with a wide range of predicted substrate classes, including saccharides, amino acids/peptides and lipids. Altogether, these results indicate a potentially important role of this protein superfamily in the human gut, and our dataset provides significant opportunities for the discovery of novel pathways that may have significant effects on human health.

  View details for DOI 10.1099/mgen.0.000373

  View details for PubMedID 32416747

• MAIT cells are imprinted by the microbiota in early life and promote tissue repair Constantinides, M. G., Link, V. M., Tamoutounour, S., Wong, A. C., Perez-Chaparro, P., Han, S., Chen, Y., Li, K., Farhat, S., Weckel, A., Krishnamurthy, S. R., Vujkovic-Cvijin, I., Linehan, J. L., Bouladoux, N., Merrill, E., Roy, S., Cua, D. J., Adams, E. J., Bhandoola, A., Scharschmidt, T. C., Aube, J., Fischbach, M. A., Belkaid, Y. AMER ASSOC IMMUNOLOGISTS. 2020

  View details for Web of Science ID 000589972400507

• Bile acid metabolites control Th17 and Treg cell differentiation Hang, S., Paik, D., Yao, L., Jamma, T., Lu, J., Ha, S., Nelson, B. N., Kelly, S. P., Wu, L., Zheng, Y., Longman, R. S., Rastinejad, F., Devlin, A., Krout, M. R., Fischbach, M. A., Littman, D. R., Huh, J. R. AMER ASSOC IMMUNOLOGISTS. 2020

  View details for Web of Science ID 000589972402506

• Dysbiosis-Induced Secondary Bile Acid Deficiency Promotes Intestinal Inflammation. Cell host & microbe Sinha, S. R., Haileselassie, Y., Nguyen, L. P., Tropini, C., Wang, M., Becker, L. S., Sim, D., Jarr, K., Spear, E. T., Singh, G., Namkoong, H., Bittinger, K., Fischbach, M. A., Sonnenburg, J. L., Habtezion, A. 2020

  Abstract

  Secondary bile acids (SBAs) are derived from primary bile acids (PBAs) in a process reliant on biosynthetic capabilities possessed by few microbes. To evaluate the role of BAs in intestinal inflammation, we performed metabolomic, microbiome, metagenomic, and transcriptomic profiling of stool from ileal pouches (surgically created resevoirs) in colectomy-treated patients with ulcerative colitis (UC) versus controls (familial adenomatous polyposis [FAP]). We show that relative to FAP, UC pouches have reduced levels of lithocholic acid and deoxycholic acid (normally the most abundant gut SBAs), genes required to convert PBAs to SBAs, and Ruminococcaceae (one of few taxa known to include SBA-producing bacteria). In three murine colitis models, SBA supplementation reduces intestinal inflammation. This anti-inflammatory effect is in part dependent on the TGR5 bile acid receptor. These data suggest that dysbiosis induces SBA deficiency in inflammatory-prone UC patients, which promotes a pro-inflammatory state within the intestine that may be treated by SBA restoration.

  View details for DOI 10.1016/j.chom.2020.01.021

  View details for PubMedID 32101703

• Michael Fischbach: Homing in on the molecules from microbes NATURE Scott, A., Fischbach, M. 2020; 577 (7792): S9

  View details for Web of Science ID 000510520700010

  View details for PubMedID 31996828

• Michael Fischbach: Homing in on the molecules from microbes NATURE Scott, A., Fischbach, M. 2020; 577 (7792): S9

  View details for Web of Science ID 000510520700011

• Bile acids profile, histopathological indices and genetic variants for non-alcoholic fatty liver disease progression. Metabolism: clinical and experimental Nimer, N. n., Choucair, I. n., Wang, Z. n., Nemet, I. n., Li, L. n., Gukasyan, J. n., Weeks, T. L., Alkhouri, N. n., Zein, N. n., Tang, W. H., Fischbach, M. A., Brown, J. M., Allayee, H. n., Dasarathy, S. n., Gogonea, V. n., Hazen, S. L. 2020: 154457

  Abstract

  Metabolomic studies suggest plasma levels of bile acids (BAs) are elevated amongst subjects with non-alcoholic fatty liver disease (NAFLD) compared to healthy controls. However, it remains unclear whether or not specific BAs are associated with the clinically relevant transition from nonalcoholic fatty liver (i.e. simple steatosis) to non-alcoholic steatohepatitis (NASH), or enhanced progression of hepatic fibrosis, or genetic determinants of NAFLD/NASH.Among sequential subjects (n=102) undergoing diagnostic liver biopsy, we examined the associations of a broad panel of BAs with distinct histopathological features of NAFLD, the presence of NASH, and their associations with genetic variants linked to NAFLD and NASH.Plasma BA alterations were observed through the entire spectrum of NAFLD, with several glycine conjugated forms of the BAs demonstrating significant associations with higher grades of inflammation and fibrosis. Plasma 7-Keto-DCA levels showed the strongest associations with advanced stages of hepatic fibrosis [odds ratio(95% confidence interval)], 4.2(1.2-16.4), NASH 24.5(4.1-473), and ballooning 18.7(4.8-91.9). Plasma 7-Keto-LCA levels were associated with NASH 9.4(1.5-185) and ballooning 5.9(1.4-28.8). Genetic variants at several NAFLD/NASH loci were nominally associated with increased levels of 7-Keto- and glycine-conjugated forms of BAs, and the NAFLD risk allele at the TRIB1 locus showed strong tendency toward increased plasma levels of GCA (p=0.02) and GUDCA (p=0.009).Circulating bile acid levels are associated with histopathological and genetic determinants of the transition from simple hepatic steatosis into NASH. Further studies exploring the potential involvement of bile acid metabolism in the development and/or progression of distinct histopathological features of NASH are warranted.

  View details for DOI 10.1016/j.metabol.2020.154457

  View details for PubMedID 33275980

• A Cardiovascular Disease-Linked Gut Microbial Metabolite Acts via Adrenergic Receptors. Cell Nemet, I. n., Saha, P. P., Gupta, N. n., Zhu, W. n., Romano, K. A., Skye, S. M., Cajka, T. n., Mohan, M. L., Li, L. n., Wu, Y. n., Funabashi, M. n., Ramer-Tait, A. E., Naga Prasad, S. V., Fiehn, O. n., Rey, F. E., Tang, W. H., Fischbach, M. A., DiDonato, J. A., Hazen, S. L. 2020; 180 (5): 862–77.e22

  Abstract

  Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and β2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.

  View details for DOI 10.1016/j.cell.2020.02.016

  View details for PubMedID 32142679

• Characterization of Serine Hydrolases Across Clinical Isolates of Commensal Skin Bacteria Staphylococcus epidermidis Using Activity-Based Protein Profiling. ACS infectious diseases Keller, L. J., Lentz, C. S., Chen, Y. E., Metivier, R. J., Weerapana, E. n., Fischbach, M. A., Bogyo, M. n. 2020

  Abstract

  The bacterial genus Staphylococcus comprises diverse species that colonize the skin as commensals but can also cause infection. Previous work identified a family of serine hydrolases termed fluorophoshonate-binding hydrolases (Fphs) in the pathogenic bacteria Staphylococcus aureus, one of which, FphB, functions as a virulence factor. Using a combination of bioinformatics and activity-based protein profiling (ABPP), we identify homologues of these enzymes in the related commensal bacteria Staphylococcus epidermidis. Two of the S. aureus Fph enzymes were not identified in S. epidermidis. Using ABPP, we identified several candidate hydrolases that were not previously identified in S. aureus that may be functionally related to the Fphs. Interestingly, the activity of the Fphs vary across clinical isolates of S. epidermidis. Biochemical characterization of the FphB homologue in S. epidermidis (SeFphB) suggests it is a functional homologue of FphB in S. aureus, but our preliminary studies suggest it may not have a role in colonization in vivo. This potential difference in biological function between the Fphs of closely related staphylococcal species may provide mechanisms for specific inhibition of S. aureus infection without perturbing commensal communities of related bacteria.

  View details for DOI 10.1021/acsinfecdis.0c00095

  View details for PubMedID 32298574

• Author Correction: Bile acid metabolites control TH17 and Treg cell differentiation. Nature Hang, S. n., Paik, D. n., Yao, L. n., Kim, E. n., Trinath, J. n., Lu, J. n., Ha, S. n., Nelson, B. N., Kelly, S. P., Wu, L. n., Zheng, Y. n., Longman, R. S., Rastinejad, F. n., Devlin, A. S., Krout, M. R., Fischbach, M. A., Littman, D. R., Huh, J. R. 2020

  Abstract

  An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

  View details for DOI 10.1038/s41586-020-2030-5

  View details for PubMedID 32094662

• Expansion of RiPP biosynthetic space through integration of pan-genomics and machine learning uncovers a novel class of lantibiotics. PLoS biology Kloosterman, A. M., Cimermancic, P. n., Elsayed, S. S., Du, C. n., Hadjithomas, M. n., Donia, M. S., Fischbach, M. A., van Wezel, G. P., Medema, M. H. 2020; 18 (12): e3001026

  Abstract

  Microbial natural products constitute a wide variety of chemical compounds, many which can have antibiotic, antiviral, or anticancer properties that make them interesting for clinical purposes. Natural product classes include polyketides (PKs), nonribosomal peptides (NRPs), and ribosomally synthesized and post-translationally modified peptides (RiPPs). While variants of biosynthetic gene clusters (BGCs) for known classes of natural products are easy to identify in genome sequences, BGCs for new compound classes escape attention. In particular, evidence is accumulating that for RiPPs, subclasses known thus far may only represent the tip of an iceberg. Here, we present decRiPPter (Data-driven Exploratory Class-independent RiPP TrackER), a RiPP genome mining algorithm aimed at the discovery of novel RiPP classes. DecRiPPter combines a Support Vector Machine (SVM) that identifies candidate RiPP precursors with pan-genomic analyses to identify which of these are encoded within operon-like structures that are part of the accessory genome of a genus. Subsequently, it prioritizes such regions based on the presence of new enzymology and based on patterns of gene cluster and precursor peptide conservation across species. We then applied decRiPPter to mine 1,295 Streptomyces genomes, which led to the identification of 42 new candidate RiPP families that could not be found by existing programs. One of these was studied further and elucidated as a representative of a novel subfamily of lanthipeptides, which we designate class V. The 2D structure of the new RiPP, which we name pristinin A3 (1), was solved using nuclear magnetic resonance (NMR), tandem mass spectrometry (MS/MS) data, and chemical labeling. Two previously unidentified modifying enzymes are proposed to create the hallmark lanthionine bridges. Taken together, our work highlights how novel natural product families can be discovered by methods going beyond sequence similarity searches to integrate multiple pathway discovery criteria.

  View details for DOI 10.1371/journal.pbio.3001026

  View details for PubMedID 33351797

• A metabolic pathway for bile acid dehydroxylation by the gut microbiome. Nature Funabashi, M. n., Grove, T. L., Wang, M. n., Varma, Y. n., McFadden, M. E., Brown, L. C., Guo, C. n., Higginbottom, S. n., Almo, S. C., Fischbach, M. A. 2020

  Abstract

  The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of Clostridium difficile1, promote hepatocellular carcinoma2 and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. 3). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids4; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.

  View details for DOI 10.1038/s41586-020-2396-4

  View details for PubMedID 32555455

• Depletion of microbiome-derived molecules in the host using Clostridium genetics. Science (New York, N.Y.) Guo, C., Allen, B. M., Hiam, K. J., Dodd, D., Van Treuren, W., Higginbottom, S., Nagashima, K., Fischer, C. R., Sonnenburg, J. L., Spitzer, M. H., Fischbach, M. A. 2019; 366 (6471)

  Abstract

  The gut microbiota produce hundreds of molecules that are present at high concentrations in the host circulation. Unraveling the contribution of each molecule to host biology remains difficult. We developed a system for constructing clean deletions in Clostridium spp., the source of many molecules from the gut microbiome. By applying this method to the model commensal organism Clostridium sporogenes, we knocked out genes for 10 C. sporogenes-derived molecules that accumulate in host tissues. In mice colonized by a C. sporogenes for which the production of branched short-chain fatty acids was knocked out, we discovered that these microbial products have immunoglobulin A-modulatory activity.

  View details for DOI 10.1126/science.aav1282

  View details for PubMedID 31831639

• Quantification of bile acids: A mass spectrometry platform for studying gut microbe connection to metabolic diseases. Journal of lipid research Choucair, I., Nemet, I., Li, L., Cole, M. A., Skye, S. M., Kirsop, J. D., Fischbach, M., Gogonea, V., Brown, J. M., Tang, W. H., Hazen, S. L. 2019

  Abstract

  Bile acids (BAs) serve multiple biological functions, ranging from absorption of lipids and fat-soluble vitamins, to serving as signaling molecules through the direct activation of dedicated cellular receptors.Synthesized by both host and microbial pathways, BAs are increasingly appreciated to participate in the regulation of numerous pathways relevant to metabolic diseases including lipid and glucose metabolism, energy expenditure and inflammation, pathways relevant to metabolic diseases. Quantitative analyses of BAs in biological matrices can be problematic due to their unusual and diverse physicochemical properties, making optimization of a method that shows good accuracy, precision, efficiency of extraction, and minimized matrix effects across structurally distinct human and murine BAs challenging. Herein we develop and clinically validate a stable isotope dilution liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the quantitative analysis of numerous primary and secondary BAs in both human and mouse biological matrices. We also utilize this tool to investigate gut microbiota participation in generation of structurally specific BAs in both humans and mice. We examine circulating levels of specific BAs and in a clinical case-control study of age- and gender-matched type 2 diabetics (T2DM) versus non-diabetics. BAs whose circulating levels are associated with T2DM include numerous 12alpha-hydroxyl BAs (taurocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, deoxycholic acid and 3-ketodeoxycholic acid), while taurohyodeoxycholic acid was negatively associated with diabetes. The LC/MS/MS based platform described should serve as a robust, high throughput investigative tool for studying the potential involvement of structurally specific BAs and the gut microbiome on both physiological and disease processes.

  View details for DOI 10.1194/jlr.RA119000311

  View details for PubMedID 31818878

• Bile acid metabolites control TH17 and Treg cell differentiation. Nature Hang, S. n., Paik, D. n., Yao, L. n., Kim, E. n., Jamma, T. n., Lu, J. n., Ha, S. n., Nelson, B. N., Kelly, S. P., Wu, L. n., Zheng, Y. n., Longman, R. S., Rastinejad, F. n., Devlin, A. S., Krout, M. R., Fischbach, M. A., Littman, D. R., Huh, J. R. 2019

  Abstract

  Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells.

  View details for DOI 10.1038/s41586-019-1785-z

  View details for PubMedID 31776512

• MAIT cells are imprinted by the microbiota in early life and promote tissue repair. Science (New York, N.Y.) Constantinides, M. G., Link, V. M., Tamoutounour, S., Wong, A. C., Perez-Chaparro, P. J., Han, S., Chen, Y. E., Li, K., Farhat, S., Weckel, A., Krishnamurthy, S. R., Vujkovic-Cvijin, I., Linehan, J. L., Bouladoux, N., Merrill, E. D., Roy, S., Cua, D. J., Adams, E. J., Bhandoola, A., Scharschmidt, T. C., Aube, J., Fischbach, M. A., Belkaid, Y. 2019; 366 (6464)

  Abstract

  How early-life colonization and subsequent exposure to the microbiota affect long-term tissue immunity remains poorly understood. Here, we show that the development of mucosal-associated invariant T (MAIT) cells relies on a specific temporal window, after which MAIT cell development is permanently impaired. This imprinting depends on early-life exposure to defined microbes that synthesize riboflavin-derived antigens. In adults, cutaneous MAIT cells are a dominant population of interleukin-17A (IL-17A)-producing lymphocytes, which display a distinct transcriptional signature and can subsequently respond to skin commensals in an IL-1-, IL-18-, and antigen-dependent manner. Consequently, local activation of cutaneous MAIT cells promotes wound healing. Together, our work uncovers a privileged interaction between defined members of the microbiota and MAIT cells, which sequentially controls both tissue-imprinting and subsequent responses to injury.

  View details for DOI 10.1126/science.aax6624

  View details for PubMedID 31649166

• Recovery of the Gut Microbiota after Antibiotics Depends on Host Diet, Community Context, and Environmental Reservoirs. Cell host & microbe Ng, K. M., Aranda-Díaz, A. n., Tropini, C. n., Frankel, M. R., Van Treuren, W. n., O'Laughlin, C. T., Merrill, B. D., Yu, F. B., Pruss, K. M., Oliveira, R. A., Higginbottom, S. K., Neff, N. F., Fischbach, M. A., Xavier, K. B., Sonnenburg, J. L., Huang, K. C. 2019; 26 (5): 650–65.e4

  Abstract

  Antibiotics alter microbiota composition and increase infection susceptibility. However, the generalizable effects of antibiotics on and the contribution of environmental variables to gut commensals remain unclear. To address this, we tracked microbiota dynamics with high temporal and taxonomic resolution during antibiotic treatment in a controlled murine system by isolating variables such as diet, treatment history, and housing co-inhabitants. Human microbiotas were remarkably resilient and recovered during antibiotic treatment, with transient dominance of resistant Bacteroides and taxa-asymmetric diversity reduction. In certain cases, in vitro sensitivities were not predictive of in vivo responses, underscoring the significance of host and community context. A fiber-deficient diet exacerbated microbiota collapse and delayed recovery. Species replacement through cross housing after ciprofloxacin treatment established resilience to a second treatment. Single housing drastically disrupted recovery, highlighting the importance of environmental reservoirs. Our findings highlight deterministic microbiota adaptations to perturbations and the translational potential for modulating diet, sanitation, and microbiota composition during antibiotics.

  View details for DOI 10.1016/j.chom.2019.10.011

  View details for PubMedID 31726029

• Microbial Transplantation With Human Gut Commensals Containing CutC Is Sufficient to Transmit Enhanced Platelet Reactivity and Thrombosis Potential. Circulation research Skye, S. M., Zhu, W., Romano, K. A., Guo, C., Wang, Z., Jia, X., Kirsop, J., Haag, B., Lang, J. M., DiDonato, J. A., Tang, W. H., Lusis, A. J., Rey, F. E., Fischbach, M. A., Hazen, S. L. 2018; 123 (10): 1164–76

  Abstract

  RATIONALE: Gut microbes influence cardiovascular disease and thrombosis risks through the production of trimethylamine N-oxide (TMAO). Microbiota-dependent generation of trimethylamine (TMA)-the precursor to TMAO-is rate limiting in the metaorganismal TMAO pathway in most humans and is catalyzed by several distinct microbial choline TMA-lyases, including the proteins encoded by the cutC/D (choline utilization C/D) genes in multiple human commensals.OBJECTIVE: Direct demonstration that the gut microbial cutC gene is sufficient to transmit enhanced platelet reactivity and thrombosis potential in a host via TMA/TMAO generation has not yet been reported.METHODS AND RESULTS: Herein, we use gnotobiotic mice and a series of microbial colonization studies to show that microbial cutC-dependent TMA/TMAO production is sufficient to transmit heightened platelet reactivity and thrombosis potential in a host. Specifically, we examine in vivo thrombosis potential employing germ-free mice colonized with either high TMA-producing stable human fecal polymcrobial communities or a defined CutC-deficient background microbial community coupled with a CutC-expressing human commensal±genetic disruption of its cutC gene (ie, Clostridium sporogenes Delta cutC).CONCLUSIONS: Collectively, these studies point to the microbial choline TMA-lyase pathway as a rational molecular target for the treatment of atherothrombotic heart disease.

  View details for PubMedID 30359185

• Microbial Transplantation With Human Gut Commensals Containing CutC Is Sufficient to Transmit Enhanced Platelet Reactivity and Thrombosis Potential CIRCULATION RESEARCH Skye, S. M., Zhu, W., Romano, K. A., Guo, C., Wang, Z., Jia, X., Kirsop, J., Haag, B., Lang, J. M., DiDonato, J. A., Tang, W., Lusis, A. J., Rey, F. E., Fischbach, M. A., Hazen, S. L. 2018; 123 (10): 1164–76

  View details for DOI 10.1161/CIRCRESAHA.118.313142

  View details for Web of Science ID 000451350700015

• Microbiome: Focus on Causation and Mechanism. Cell Fischbach, M. A. 2018; 174 (4): 785–90

  Abstract

  There is tremendous enthusiasm for the microbiome in academia and industry. This Perspective argues that in order to realize its potential, the field needs to focus on establishing causation and molecular mechanism with an emphasis on phenotypes that are large in magnitude, easy to measure, and unambiguously driven by the microbiota.

  View details for PubMedID 30096310

• Microbiome: Focus on Causation and Mechanism CELL Fischbach, M. A. 2018; 174 (4): 785–90

  View details for DOI 10.1016/j.cell.2018.07.038

  View details for Web of Science ID 000441145500006

• Mapping the Genetic Landscape of Human Cells CELL Horlbeck, M. A., Xu, A., Wang, M., Bennett, N. K., Park, C. Y., Bogdanoff, D., Adamson, B., Chow, E. D., Kampmann, M., Peterson, T. R., Nakamura, K., Fischbach, M. A., Weissman, J. S., Gilbert, L. A. 2018; 174 (4): 953-+

  Abstract

  Seminal yeast studies have established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Efforts in human cells using focused gene sets underscore the utility of this approach, but the feasibility of generating large-scale, diverse human GI maps remains unresolved. We developed a CRISPR interference platform for large-scale quantitative mapping of human GIs. We systematically perturbed 222,784 gene pairs in two cancer cell lines. The resultant maps cluster functionally related genes, assigning function to poorly characterized genes, including TMEM261, a new electron transport chain component. Individual GIs pinpoint unexpected relationships between pathways, exemplified by a specific cholesterol biosynthesis intermediate whose accumulation induces deoxynucleotide depletion, causing replicative DNA damage and a synthetic-lethal interaction with the ATR/9-1-1 DNA repair pathway. Our map provides a broad resource, establishes GI maps as a high-resolution tool for dissecting gene function, and serves as a blueprint for mapping the genetic landscape of human cells.

  View details for PubMedID 30033366

• Gut Microbiota-Produced Tryptamine Activates an Epithelial G-Protein-Coupled Receptor to Increase Colonic Secretion CELL HOST & MICROBE Bhattarai, Y., Williams, B. B., Battaglioli, E. J., Whitaker, W. R., Till, L., Grover, M., Linden, D. R., Akiba, Y., Kandimalla, K. K., Zachos, N. C., Kaunitz, J. D., Sonnenburg, J. L., Fischbach, M. A., Farrugia, G., Kashyap, P. C. 2018; 23 (6): 775-+

  Abstract

  Tryptamine, a tryptophan-derived monoamine similar to 5-hydroxytryptamine (5-HT), is produced by gut bacteria and is abundant in human and rodent feces. However, the physiologic effect of tryptamine in the gastrointestinal (GI) tract remains unknown. Here, we show that the biological effects of tryptamine are mediated through the 5-HT4 receptor (5-HT4R), a G-protein-coupled receptor (GPCR) uniquely expressed in the colonic epithelium. Tryptamine increases both ionic flux across the colonic epithelium and fluid secretion in colonoids from germ-free (GF) and humanized (ex-GF colonized with human stool) mice, consistent with increased intestinal secretion. The secretory effect of tryptamine is dependent on 5-HT4R activation and is blocked by 5-HT4R antagonist and absent in 5-HT4R-/- mice. GF mice colonized by Bacteroides thetaiotaomicron engineered to produce tryptamine exhibit accelerated GI transit. Our study demonstrates an aspect of host physiology under control of a bacterial metabolite that can be exploited as a therapeutic modality. VIDEO ABSTRACT.

  View details for PubMedID 29902441

  View details for PubMedCentralID PMC6055526

• Contextual control of skin immunity and inflammation byCorynebacterium. The Journal of experimental medicine Ridaura, V. K., Bouladoux, N. n., Claesen, J. n., Chen, Y. E., Byrd, A. L., Constantinides, M. G., Merrill, E. D., Tamoutounour, S. n., Fischbach, M. A., Belkaid, Y. n. 2018; 215 (3): 785–99

  Abstract

  How defined microbes influence the skin immune system remains poorly understood. Here we demonstrate thatCorynebacteria, dominant members of the skin microbiota, promote a dramatic increase in the number and activation of a defined subset of γδ T cells. This effect is long-lasting, occurs independently of other microbes, and is, in part, mediated by interleukin (IL)-23. Under steady-state conditions, the impact ofCorynebacteriumis discrete and noninflammatory. However, when applied to the skin of a host fed a high-fat diet,Corynebacterium accolensalone promotes inflammation in an IL-23-dependent manner. Such effect is highly conserved among species ofCorynebacteriumand dependent on the expression of a dominant component of the cell envelope, mycolic acid. Our data uncover a mode of communication between the immune system and a dominant genus of the skin microbiota and reveal that the functional impact of canonical skin microbial determinants is contextually controlled by the inflammatory and metabolic state of the host.

  View details for PubMedID 29382696

  View details for PubMedCentralID PMC5839758

• A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology. Journal of the American Chemical Society Casini, A. n., Chang, F. Y., Eluere, R. n., King, A. M., Young, E. M., Dudley, Q. M., Karim, A. n., Pratt, K. n., Bristol, C. n., Forget, A. n., Ghodasara, A. n., Warden-Rothman, R. n., Gan, R. n., Cristofaro, A. n., Borujeni, A. E., Ryu, M. H., Li, J. n., Kwon, Y. C., Wang, H. n., Tatsis, E. n., Rodriguez-Lopez, C. n., O'Connor, S. n., Mdema, M. H., Fischbach, M. A., Jewett, M. C., Voigt, C. n., Gordon, D. B. 2018

  Abstract

  Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

  View details for PubMedID 29480720

• The Biosynthesis of Lipooligosaccharide fromBacteroides thetaiotaomicron. mBio Jacobson, A. N., Choudhury, B. P., Fischbach, M. A. 2018; 9 (2)

  Abstract

  Lipopolysaccharide (LPS), a cell-associated glycolipid that makes up the outer leaflet of the outer membrane of Gram-negative bacteria, is a canonical mediator of microbe-host interactions. The most prevalent Gram-negative gut bacterial taxon,Bacteroides, makes up around 50% of the cells in a typical Western gut; these cells harbor ~300 mg of LPS, making it one of the highest-abundance molecules in the intestine. As a starting point for understanding the biological function ofBacteroidesLPS, we have identified genes inBacteroides thetaiotaomicronVPI 5482 involved in the biosynthesis of its lipid A core and glycan, generated mutants that elaborate altered forms of LPS, and used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to interrogate the molecular features of these variants. We demonstrate,inter alia, that the glycan does not appear to have a repeating unit, and so this strain produces lipooligosaccharide (LOS) rather than LPS. This result contrasts withBacteroides vulgatusATCC 8482, which by SDS-PAGE analysis appears to produce LPS with a repeating unit. Additionally, our identification of theB. thetaiotaomicronLOS oligosaccharide gene cluster allowed us to identify similar clusters in otherBacteroidesspecies. Our work lays the foundation for developing a structure-function relationship forBacteroidesLPS/LOS in the context of host colonization.IMPORTANCEMuch is known about the bacterial species and genes that make up the human microbiome, but remarkably little is known about the molecular mechanisms through which the microbiota influences host biology. A well-known mechanism by which bacteria influence the host centers around lipopolysaccharide (LPS), a component of the Gram-negative bacterial outer membrane. Pathogen-derived LPS is a potent ligand for host receptor Toll-like receptor 4, which plays an important role in sensing bacteria as part of the innate immune response. Puzzlingly, the most common genus of human gut bacteria,Bacteroides, produces LPS but does not elicit a potent proinflammatory response. Previous work showing thatBacteroidesLPS differs structurally from pathogen-derived LPS suggested the outlines of an explanation. Here, we take the next step, elucidating the biosynthetic pathway forBacteroidesLPS and generating mutants in the process that will be of great use in understanding how this molecule modulates the host immune response.

  View details for DOI 10.1128/mBio.02289-17

  View details for PubMedID 29535205

• Skin microbiota-host interactions. Nature Chen, Y. E., Fischbach, M. A., Belkaid, Y. n. 2018; 553 (7689): 427–36

  Abstract

  The skin is a complex and dynamic ecosystem that is inhabited by bacteria, archaea, fungi and viruses. These microbes-collectively referred to as the skin microbiota-are fundamental to skin physiology and immunity. Interactions between skin microbes and the host can fall anywhere along the continuum between mutualism and pathogenicity. In this Review, we highlight how host-microbe interactions depend heavily on context, including the state of immune activation, host genetic predisposition, barrier status, microbe localization, and microbe-microbe interactions. We focus on how context shapes the complex dialogue between skin microbes and the host, and the consequences of this dialogue for health and disease.

  View details for PubMedID 29364286

• Discovery of Reactive Microbiota-Derived Metabolites that Inhibit Host Proteases CELL Guo, C., Chang, F., Wyche, T. P., Backus, K. M., Acker, T. M., Funabashi, M., Taketani, M., Donia, M. S., Nayfach, S., Pollard, K. S., Craik, C. S., Cravatt, B. F., Clardy, J., Voigt, C. A., Fischbach, M. A. 2017; 168 (3): 517-?

  Abstract

  The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals.

  View details for DOI 10.1016/j.cell.2016.12.021

  View details for Web of Science ID 000396249600017

  View details for PubMedID 28111075

  View details for PubMedCentralID PMC5302092

• A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites. Nature Dodd, D. n., Spitzer, M. H., Van Treuren, W. n., Merrill, B. D., Hryckowian, A. J., Higginbottom, S. K., Le, A. n., Cowan, T. M., Nolan, G. P., Fischbach, M. A., Sonnenburg, J. L. 2017; 551 (7682): 648–52

  Abstract

  The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.

  View details for PubMedID 29168502

• Modulation of a Circulating Uremic Solute via Rational Genetic Manipulation of the Gut Microbiota CELL HOST & MICROBE Devlin, A. S., Marcobal, A., Dodd, D., Nayfach, S., Plummer, N., Meyer, T., Pollard, K. S., Sonnenburg, J. L., Fischbach, M. A. 2016; 20 (6): 709-715

  Abstract

  Renal disease is growing in prevalence and has striking co-morbidities with metabolic and cardiovascular disease. Indoxyl sulfate (IS) is a toxin that accumulates in plasma when kidney function declines and contributes to the progression of chronic kidney disease. IS derives exclusively from the gut microbiota. Bacterial tryptophanases convert tryptophan to indole, which is absorbed and modified by the host to produce IS. Here, we identify a widely distributed family of tryptophanases in the gut commensal Bacteroides and find that deleting this gene eliminates the production of indole in vitro. By altering the status or abundance of the Bacteroides tryptophanase, we can modulate IS levels in gnotobiotic mice and in the background of a conventional murine gut community. Our results demonstrate that it is possible to control host IS levels by targeting the microbiota and suggest a possible strategy for treating renal disease.

  View details for DOI 10.1016/j.chom.2016.10.021

  View details for Web of Science ID 000392843500008

  View details for PubMedID 27916477

  View details for PubMedCentralID PMC5159218

• Signaling in Host-Associated Microbial Communities CELL Fischbach, M. A., Segre, J. A. 2016; 164 (6): 1288-1300

  Abstract

  Human-associated microbiota form and stabilize communities based on interspecies interactions. We review how these microbe-microbe and microbe-host interactions are communicated to shape communities over a human's lifespan, including periods of health and disease. Modeling and dissecting signaling in host-associated communities is crucial to understand their function and will open the door to therapies that prevent or correct microbial community dysfunction to promote health and treat disease.

  View details for DOI 10.1016/j.cell.2016.02.037

  View details for Web of Science ID 000372784900025

  View details for PubMedID 26967294

  View details for PubMedCentralID PMC4801507

• Synthetic biology to access and expand nature's chemical diversity NATURE REVIEWS MICROBIOLOGY Smanski, M. J., Zhou, H., Claesen, J., Shen, B., Fischbach, M. A., Voigt, C. A. 2016; 14 (3): 135-149

  Abstract

  Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Accessing these natural products promises to reinvigorate drug discovery pipelines and provide novel routes to synthesize complex chemicals. The pathways leading to the production of these molecules often comprise dozens of genes spanning large areas of the genome and are controlled by complex regulatory networks with some of the most interesting molecules being produced by non-model organisms. In this Review, we discuss how advances in synthetic biology--including novel DNA construction technologies, the use of genetic parts for the precise control of expression and for synthetic regulatory circuits--and multiplexed genome engineering can be used to optimize the design and synthesis of pathways that produce natural products.

  View details for DOI 10.1038/nrmicro.2015.24

  View details for Web of Science ID 000370469800008

  View details for PubMedID 26876034

  View details for PubMedCentralID PMC5048682

• A Wave of Regulatory T Cells into Neonatal Skin Mediates Tolerance to Commensal Microbes IMMUNITY Scharschmidt, T. C., Vasquez, K. S., Truong, H., Gearty, S. V., Pauli, M. L., Nosbaum, A., Gratz, I. K., Otto, M., Moon, J. J., Liese, J., Abbas, A. K., Fischbach, M. A., Rosenblum, M. D. 2015; 43 (5): 1011-1021

  Abstract

  The skin is a site of constant dialog between the immune system and commensal bacteria. However, the molecular mechanisms that allow us to tolerate the presence of skin commensals without eliciting destructive inflammation are unknown. Using a model system to study the antigen-specific response to S. epidermidis, we demonstrated that skin colonization during a defined period of neonatal life was required for establishing immune tolerance to commensal microbes. This crucial window was characterized by an abrupt influx of highly activated regulatory T (Treg) cells into neonatal skin. Selective inhibition of this Treg cell wave completely abrogated tolerance. Thus, the host-commensal relationship in the skin relied on a unique Treg cell population that mediated tolerance to bacterial antigens during a defined developmental window. This suggests that the cutaneous microbiome composition in neonatal life is crucial in shaping adaptive immune responses to commensals, and disrupting these interactions might have enduring health implications.

  View details for DOI 10.1016/j.immuni.2015.10.016

  View details for Web of Science ID 000366846000020

  View details for PubMedID 26588783

  View details for PubMedCentralID PMC4654993

• MetaQuery: a web server for rapid annotation and quantitative analysis of specific genes in the human gut microbiome BIOINFORMATICS Nayfach, S., Fischbach, M. A., Pollard, K. S. 2015; 31 (20): 3368-3370

  Abstract

  Microbiome researchers frequently want to know how abundant a particular microbial gene or pathway is across different human hosts, including its association with disease and its co-occurrence with other genes or microbial taxa. With thousands of publicly available metagenomes, these questions should be easy to answer. However, computational barriers prevent most researchers from conducting such analyses. We address this problem with MetaQuery, a web application for rapid and quantitative analysis of specific genes in the human gut microbiome. The user inputs one or more query genes, and our software returns the estimated abundance of these genes across 1267 publicly available fecal metagenomes from American, European and Chinese individuals. In addition, our application performs downstream statistical analyses to identify features that are associated with gene variation, including other query genes (i.e. gene co-variation), taxa, clinical variables (e.g. inflammatory bowel disease and diabetes) and average genome size. The speed and accessibility of MetaQuery are a step toward democratizing metagenomics research, which should allow many researchers to query the abundance and variation of specific genes in the human gut microbiome.http://metaquery.docpollard.org.snayfach@gmail.comS UPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  View details for DOI 10.1093/bioinformatics/btv382

  View details for Web of Science ID 000362846600022

  View details for PubMedID 26104745

  View details for PubMedCentralID PMC4595903

• Mammalian Lipopolysaccharide Receptors Incorporated into the Retroviral Envelope Augment Virus Transmission CELL HOST & MICROBE Wilks, J., Lien, E., Jacobson, A. N., Fischbach, M. A., Qureshi, N., Chervonsky, A. V., Golovkina, T. V. 2015; 18 (4): 456-462

  Abstract

  The orally transmitted retrovirus mouse mammary tumor virus (MMTV) requires the intestinal microbiota for persistence. Virion-associated lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4), stimulating production of the immunosuppressive cytokine IL-10 and MMTV evasion of host immunity. However, the mechanisms by which MMTV associates with LPS remain unknown. We find that the viral envelope contains the mammalian LPS-binding factors CD14, TLR4, and MD-2, which, in conjunction with LPS-binding protein (LBP), bind LPS to the virus and augment transmission. MMTV isolated from infected mice lacking these LBPs cannot engage LPS or stimulate TLR4 and have a transmission defect. Furthermore, MMTV incorporation of a weak agonist LPS from Bacteroides, a prevalent LPS source in the gut, significantly enhances the ability of this LPS to stimulate TLR4, suggesting that MMTV intensifies these immunostimulatory properties. Thus, an orally transmitted retrovirus can capture, modify, and exploit mammalian receptors for bacterial ligands to ensure successful transmission.

  View details for DOI 10.1016/j.chom.2015.09.005

  View details for Web of Science ID 000365111600013

  View details for PubMedID 26468748

  View details for PubMedCentralID PMC4795803

• Genome-Directed Lead Discovery: Biosynthesis, Structure Elucidation, and Biological Evaluation of Two Families of Polyene Macrolactams against Trypanosoma brucei ACS CHEMICAL BIOLOGY Schulze, C. J., Donia, M. S., Siqueira-Neto, J. L., Ray, D., Raskatov, J. A., Green, R. E., McKerrow, J. H., Fischbach, M. A., Linington, R. G. 2015; 10 (10): 2373-2381

  Abstract

  Marine natural products are an important source of lead compounds against many pathogenic targets. Herein, we report the discovery of lobosamides A-C from a marine actinobacterium, Micromonospora sp., representing three new members of a small but growing family of bacterially produced polyene macrolactams. The lobosamides display growth inhibitory activity against the protozoan parasite Trypanosoma brucei (lobosamide A IC50 = 0.8 μM), the causative agent of human African trypanosomiasis (HAT). The biosynthetic gene cluster of the lobosamides was sequenced and suggests a conserved cluster organization among the 26-membered macrolactams. While determination of the relative and absolute configurations of many members of this family is lacking, the absolute configurations of the lobosamides were deduced using a combination of chemical modification, detailed spectroscopic analysis, and bioinformatics. We implemented a "molecules-to-genes-to-molecules" approach to determine the prevalence of similar clusters in other bacteria, which led to the discovery of two additional macrolactams, mirilactams A and B from Actinosynnema mirum. These additional analogs have allowed us to identify specific structure-activity relationships that contribute to the antitrypanosomal activity of this class. This approach illustrates the power of combining chemical analysis and genomics in the discovery and characterization of natural products as new lead compounds for neglected disease targets.

  View details for DOI 10.1021/acschembio.5b00308

  View details for Web of Science ID 000363225100022

  View details for PubMedID 26270237

• Computational approaches to natural product discovery NATURE CHEMICAL BIOLOGY Medema, M. H., Fischbach, M. A. 2015; 11 (9): 639-648

  Abstract

  Starting with the earliest Streptomyces genome sequences, the promise of natural product genome mining has been captivating: genomics and bioinformatics would transform compound discovery from an ad hoc pursuit to a high-throughput endeavor. Until recently, however, genome mining has advanced natural product discovery only modestly. Here, we argue that the development of algorithms to mine the continuously increasing amounts of (meta)genomic data will enable the promise of genome mining to be realized. We review computational strategies that have been developed to identify biosynthetic gene clusters in genome sequences and predict the chemical structures of their products. We then discuss networking strategies that can systematize large volumes of genetic and chemical data and connect genomic information to metabolomic and phenotypic data. Finally, we provide a vision of what natural product discovery might look like in the future, specifically considering longstanding questions in microbial ecology regarding the roles of metabolites in interspecies interactions.

  View details for DOI 10.1038/NCHEMBIO.1884

  View details for Web of Science ID 000359954700007

  View details for PubMedID 26284671

  View details for PubMedCentralID PMC5024737

• A biosynthetic pathway for a prominent class of microbiota-derived bile acids NATURE CHEMICAL BIOLOGY Devlin, A. S., Fischbach, M. A. 2015; 11 (9): 685-?

  Abstract

  The gut bile acid pool is millimolar in concentration, varies widely in composition among individuals and is linked to metabolic disease and cancer. Although these molecules are derived almost exclusively from the microbiota, remarkably little is known about which bacterial species and genes are responsible for their biosynthesis. Here we report a biosynthetic pathway for the second most abundant class in the gut, 3β-hydroxy(iso)-bile acids, whose levels exceed 300 μM in some humans and are absent in others. We show, for the first time, that iso-bile acids are produced by Ruminococcus gnavus, a far more abundant commensal than previously known producers, and that the iso-bile acid pathway detoxifies deoxycholic acid and thus favors the growth of the keystone genus Bacteroides. By revealing the biosynthetic genes for an abundant class of bile acids, our work sets the stage for predicting and rationally altering the composition of the bile acid pool.

  View details for DOI 10.1038/NCHEMBIO.1864

  View details for Web of Science ID 000359954700012

  View details for PubMedID 26192599

  View details for PubMedCentralID PMC4543561

• A Phase-Variable Surface Layer from the Gut Symbiont Bacteroides thetaiotaomicron MBIO Taketani, M., Donia, M. S., Jacobson, A. N., Lambris, J. D., Fischbach, M. A. 2015; 6 (5)

  Abstract

  The capsule from Bacteroides, a common gut symbiont, has long been a model system for studying the molecular mechanisms of host-symbiont interactions. The Bacteroides capsule is thought to consist of an array of phase-variable polysaccharides that give rise to subpopulations with distinct cell surface structures. Here, we report the serendipitous discovery of a previously unknown surface structure in Bacteroides thetaiotaomicron: a surface layer composed of a protein of unknown function, BT1927. BT1927, which is expressed in a phase-variable manner by ~1:1,000 cells in a wild-type culture, forms a hexagonally tessellated surface layer. The BT1927-expressing subpopulation is profoundly resistant to complement-mediated killing, due in part to the BT1927-mediated blockade of C3b deposition. Our results show that the Bacteroides surface structure is capable of a far greater degree of structural variation than previously known, and they suggest that structural variation within a Bacteroides species is important for productive gut colonization.Many bacterial species elaborate a capsule, a structure that resides outside the cell wall and mediates microbe-microbe and microbe-host interactions. Species of Bacteroides, the most abundant genus in the human gut, produce a capsule that consists of an array of polysaccharides, some of which are known to mediate interactions with the host immune system. Here, we report the discovery of a previously unknown surface structure in Bacteroides thetaiotaomicron. We show that this protein-based structure is expressed by a subset of cells in a population and protects Bacteroides from killing by complement, a component of the innate immune system. This novel surface layer protein is conserved across many species of the genus Bacteroides, suggesting an important role in colonization and host immune modulation.

  View details for DOI 10.1128/mBio.01339-15

  View details for Web of Science ID 000364523100038

  View details for PubMedID 26419879

  View details for PubMedCentralID PMC4611039

• Minimum Information about a Biosynthetic Gene cluster NATURE CHEMICAL BIOLOGY Medema, M. H., Kottmann, R., Yilmaz, P., Cummings, M., Biggins, J. B., Blin, K., de Bruijn, I., Chooi, Y. H., Claesen, J., Coates, R. C., Cruz-Morales, P., Duddela, S., Duesterhus, S., Edwards, D. J., Fewer, D. P., Garg, N., Geiger, C., Gomez-Escribano, J. P., Greule, A., Hadjithomas, M., Haines, A. S., Helfrich, E. J., Hillwig, M. L., Ishida, K., Jones, A. C., Jones, C. S., Jungmann, K., Kegler, C., Kim, H. U., Koetter, P., Krug, D., Masschelein, J., Melnik, A. V., Mantovani, S. M., Monroe, E. A., Moore, M., Moss, N., Nuetzmann, H., Pan, G., Pati, A., Petras, D., Reen, F. J., Rosconi, F., Rui, Z., Tian, Z., Tobias, N. J., Tsunematsu, Y., Wiemann, P., Wyckoff, E., Yan, X., Yim, G., Yu, F., Xie, Y., Aigle, B., Apel, A. K., Balibar, C. J., Balskus, E. P., Barona-Gomez, F., Bechthold, A., Bode, H. B., Borriss, R., Brady, S. F., Brakhage, A. A., Caffrey, P., Cheng, Y., Clardy, J., Cox, R. J., De Mot, R., Donadio, S., Donia, M. S., van der Donk, W. A., Dorrestein, P. C., Doyle, S., Driessen, A. J., Ehling-Schulz, M., Entian, K., Fischbach, M. A., Gerwick, L., Gerwick, W. H., Gross, H., Gust, B., Hertweck, C., Hofte, M., Jensen, S. E., Ju, J., Katz, L., Kaysser, L., Klassen, J. L., Keller, N. P., Kormanec, J., Kuipers, O. P., Kuzuyama, T., Kyrpides, N. C., Kwon, H., Lautru, S., Lavigne, R., Lee, C. Y., Linquan, B., Liu, X., Liu, W., Luzhetskyy, A., Mahmud, T., Mast, Y., Mendez, C., Metsa-Ketela, M., Micklefield, J., Mitchell, D. A., Moore, B. S., Moreira, L. M., Mueller, R., Neilan, B. A., Nett, M., Nielsen, J., O'Gara, F., Oikawa, H., Osbourn, A., Osburne, M. S., Ostash, B., Payne, S. M., Pernodet, J., Petricek, M., Piel, J., Ploux, O., Raaijmakers, J. M., Salas, J. A., Schmitt, E. K., Scott, B., Seipke, R. F., Shen, B., Sherman, D. H., Sivonen, K., Smanski, M. J., Sosio, M., Stegmann, E., Suessmuth, R. D., Tahlan, K., Thomas, C. M., Tang, Y., Truman, A. W., Viaud, M., Walton, J. D., Walsh, C. T., Weber, T., van Wezel, G. P., Wilkinson, B., Willey, J. M., Wohlleben, W., Wright, G. D., Ziemert, N., Zhang, C., Zotchev, S. B., Breitling, R., Takano, E., Gloeckner, F. O. 2015; 11 (9): 625-631

  View details for PubMedID 26284661

• HUMAN MICROBIOTA. Small molecules from the human microbiota. Science Donia, M. S., Fischbach, M. A. 2015; 349 (6246)

  Abstract

  Developments in the use of genomics to guide natural product discovery and a recent emphasis on understanding the molecular mechanisms of microbiota-host interactions have converged on the discovery of small molecules from the human microbiome. Here, we review what is known about small molecules produced by the human microbiota. Numerous molecules representing each of the major metabolite classes have been found that have a variety of biological activities, including immune modulation and antibiosis. We discuss technologies that will affect how microbiota-derived molecules are discovered in the future and consider the challenges inherent in finding specific molecules that are critical for driving microbe-host and microbe-microbe interactions and understanding their biological relevance.

  View details for DOI 10.1126/science.1254766

  View details for PubMedID 26206939

  View details for PubMedCentralID PMC4641445

• IMG-ABC: A Knowledge Base To Fuel Discovery of Biosynthetic Gene Clusters and Novel Secondary Metabolites MBIO Hadjithomas, M., Chen, I. A., Chu, K., Ratner, A., Palaniappan, K., Szeto, E., Huang, J., Reddy, T. B., Cimermancic, P., Fischbach, M. A., Ivanova, N. N., Markowitz, V. M., Kyrpides, N. C., Pati, A. 2015; 6 (4)

  Abstract

  In the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of "big" genomic data for discovering small molecules. IMG-ABC relies on IMG's comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC's focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time in Alphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules.IMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG's extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to expand, with the goal of becoming an essential component of any bioinformatic exploration of the secondary metabolism world.

  View details for DOI 10.1128/mBio.00932-15

  View details for Web of Science ID 000360839400025

  View details for PubMedID 26173699

  View details for PubMedCentralID PMC4502231

• antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters NUCLEIC ACIDS RESEARCH Weber, T., Blin, K., Duddela, S., Krug, D., Kim, H. U., Bruccoleri, R., Lee, S. Y., Fischbach, M. A., Mueller, R., Wohlleben, W., Breitling, R., Takano, E., Medema, M. H. 2015; 43 (W1): W237-W243

  Abstract

  Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.

  View details for DOI 10.1093/nar/gkv437

  View details for Web of Science ID 000359772700037

  View details for PubMedID 25948579

  View details for PubMedCentralID PMC4489286

• Synthetic Microbes As Drug Delivery Systems ACS SYNTHETIC BIOLOGY Claesen, J., Fischbach, M. A. 2015; 4 (4): 358-364

  Abstract

  Synthetic cell therapy is a field that has broad potential for future applications in human disease treatment. Next generation therapies will consist of engineered bacterial strains capable of diagnosing disease, producing and delivering therapeutics, and controlling their numbers to meet containment and safety concerns. A thorough understanding of the microbial ecology of the human body and the interaction of the microbes with the immune system will benefit the choice of an appropriate chassis that engrafts stably and interacts productively with the resident community in specific body niches.

  View details for DOI 10.1021/sb500258b

  View details for Web of Science ID 000353313600001

  View details for PubMedID 25079685

  View details for PubMedCentralID PMC4410909

• A Systematic Computational Analysis of Biosynthetic Gene Cluster Evolution: Lessons for Engineering Biosynthesis PLOS COMPUTATIONAL BIOLOGY Medema, M. H., Cimermancic, P., Sali, A., Takano, E., Fischbach, M. A. 2014; 10 (12)

  Abstract

  Bacterial secondary metabolites are widely used as antibiotics, anticancer drugs, insecticides and food additives. Attempts to engineer their biosynthetic gene clusters (BGCs) to produce unnatural metabolites with improved properties are often frustrated by the unpredictability and complexity of the enzymes that synthesize these molecules, suggesting that genetic changes within BGCs are limited by specific constraints. Here, by performing a systematic computational analysis of BGC evolution, we derive evidence for three findings that shed light on the ways in which, despite these constraints, nature successfully invents new molecules: 1) BGCs for complex molecules often evolve through the successive merger of smaller sub-clusters, which function as independent evolutionary entities. 2) An important subset of polyketide synthases and nonribosomal peptide synthetases evolve by concerted evolution, which generates sets of sequence-homogenized domains that may hold promise for engineering efforts since they exhibit a high degree of functional interoperability, 3) Individual BGC families evolve in distinct ways, suggesting that design strategies should take into account family-specific functional constraints. These findings suggest novel strategies for using synthetic biology to rationally engineer biosynthetic pathways.

  View details for DOI 10.1371/journal.pcbi.1004016

  View details for Web of Science ID 000346656700050

  View details for PubMedID 25474254

  View details for PubMedCentralID PMC4256081

• Discovery and Characterization of Gut Microbiota Decarboxylases that Can Produce the Neurotransmitter Tryptamine CELL HOST & MICROBE Williams, B. B., Van Benschoten, A. H., Cimermancic, P., Donia, M. S., Zimmermann, M., Taketani, M., Ishihara, A., Kashyap, P. C., Fraser, J. S., Fischbach, M. A. 2014; 16 (4): 495-503

  Abstract

  Several recent studies describe the influence of the gut microbiota on host brain and behavior. However, the mechanisms responsible for microbiota-nervous system interactions are largely unknown. Using a combination of genetics, biochemistry, and crystallography, we identify and characterize two phylogenetically distinct enzymes found in the human microbiome that decarboxylate tryptophan to form the β-arylamine neurotransmitter tryptamine. Although this enzymatic activity is exceedingly rare among bacteria more broadly, analysis of the Human Microbiome Project data demonstrate that at least 10% of the human population harbors at least one bacterium encoding a tryptophan decarboxylase in their gut community. Our results uncover a previously unrecognized enzymatic activity that can give rise to host-modulatory compounds and suggests a potential direct mechanism by which gut microbiota can influence host physiology, including behavior.

  View details for DOI 10.1016/j.chom.2014.09.001

  View details for Web of Science ID 000343826100013

  View details for PubMedID 25263219

  View details for PubMedCentralID PMC4260654

• A Systematic Analysis of Biosynthetic Gene Clusters in the Human Microbiome Reveals a Common Family of Antibiotics CELL Donia, M. S., Cimermancic, P., Schulze, C. J., Brown, L. C., Martin, J., Mitreva, M., Clardy, J., Linington, R. G., Fischbach, M. A. 2014; 158 (6): 1402-1414

  Abstract

  In complex biological systems, small molecules often mediate microbe-microbe and microbe-host interactions. Using a systematic approach, we identified 3,118 small-molecule biosynthetic gene clusters (BGCs) in genomes of human-associated bacteria and studied their representation in 752 metagenomic samples from the NIH Human Microbiome Project. Remarkably, we discovered that BGCs for a class of antibiotics in clinical trials, thiopeptides, are widely distributed in genomes and metagenomes of the human microbiota. We purified and solved the structure of a thiopeptide antibiotic, lactocillin, from a prominent member of the vaginal microbiota. We demonstrate that lactocillin has potent antibacterial activity against a range of Gram-positive vaginal pathogens, and we show that lactocillin and other thiopeptide BGCs are expressed in vivo by analyzing human metatranscriptomic sequencing data. Our findings illustrate the widespread distribution of small-molecule-encoding BGCs in the human microbiome, and they demonstrate the bacterial production of drug-like molecules in humans. PAPERCLIP:

  View details for DOI 10.1016/j.cell.2014.08.032

  View details for Web of Science ID 000343094200020

  View details for PubMedID 25215495

  View details for PubMedCentralID PMC4164201

• Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters. Cell Cimermancic, P., Medema, M. H., Claesen, J., Kurita, K., Wieland Brown, L. C., Mavrommatis, K., Pati, A., Godfrey, P. A., Koehrsen, M., Clardy, J., Birren, B. W., Takano, E., Sali, A., Linington, R. G., Fischbach, M. A. 2014; 158 (2): 412-421

  Abstract

  Although biosynthetic gene clusters (BGCs) have been discovered for hundreds of bacterial metabolites, our knowledge of their diversity remains limited. Here, we used a novel algorithm to systematically identify BGCs in the extensive extant microbial sequencing data. Network analysis of the predicted BGCs revealed large gene cluster families, the vast majority uncharacterized. We experimentally characterized the most prominent family, consisting of two subfamilies of hundreds of BGCs distributed throughout the Proteobacteria; their products are aryl polyenes, lipids with an aryl head group conjugated to a polyene tail. We identified a distant relationship to a third subfamily of aryl polyene BGCs, and together the three subfamilies represent the largest known family of biosynthetic gene clusters, with more than 1,000 members. Although these clusters are widely divergent in sequence, their small molecule products are remarkably conserved, indicating for the first time the important roles these compounds play in Gram-negative cell biology.

  View details for DOI 10.1016/j.cell.2014.06.034

  View details for PubMedID 25036635

  View details for PubMedCentralID PMC4123684

• Propionibacterium-Produced Coproporphyrin III Induces Staphylococcus aureus Aggregation and Biofilm Formation MBIO Wollenberg, M. S., Claesen, J., Escapa, I. F., Aldridge, K. L., Fischbach, M. A., Lemon, K. P. 2014; 5 (4)

  Abstract

  The majority of bacteria detected in the nostril microbiota of most healthy adults belong to three genera: Propionibacterium, Corynebacterium, and Staphylococcus. Among these staphylococci is the medically important bacterium Staphylococcus aureus. Almost nothing is known about interspecies interactions among bacteria in the nostrils. We observed that crude extracts of cell-free conditioned medium from Propionibacterium spp. induce S. aureus aggregation in culture. Bioassay-guided fractionation implicated coproporphyrin III (CIII), the most abundant extracellular porphyrin produced by human-associated Propionibacterium spp., as a cause of S. aureus aggregation. This aggregation response depended on the CIII dose and occurred during early stationary-phase growth, and a low pH (~4 to 6) was necessary but was not sufficient for its induction. Additionally, CIII induced plasma-independent S. aureus biofilm development on an abiotic surface in multiple S. aureus strains. In strain UAMS-1, CIII stimulation of biofilm depended on sarA, a key biofilm regulator. This study is one of the first demonstrations of a small-molecule-mediated interaction among medically relevant members of the nostril microbiota and the first description of a role for CIII in bacterial interspecies interactions. Our results indicate that CIII may be an important mediator of S. aureus aggregation and/or biofilm formation in the nostril or other sites inhabited by Propionibacterium spp. and S. aureus. Importance: Very little is known about interspecies interactions among the bacteria that inhabit the adult nostril, including Staphylococcus aureus, a potential pathogen that colonizes about a quarter of adults. We demonstrated that coproporphyrin III (CIII), a diffusible small molecule excreted by nostril- and skin-associated Propionibacterium spp., induces S. aureus aggregation in a manner dependent on dose, growth phase, and pH. CIII also induces S. aureus to form a plasma-independent surface-attached biofilm. This report is the first description of a role for CIII in bacterial interspecies interactions at any human body site and a novel demonstration that nostril microbiota physiology is influenced by small-molecule-mediated interactions.

  View details for DOI 10.1128/mBio.01286-14

  View details for Web of Science ID 000341588100067

  View details for PubMedID 25053784

  View details for PubMedCentralID PMC4120196

• Key applications of plant metabolic engineering. PLoS biology Lau, W., Fischbach, M. A., Osbourn, A., Sattely, E. S. 2014; 12 (6): e1001879

  Abstract

  Great strides have been made in plant metabolic engineering over the last two decades, with notable success stories including Golden rice. Here, we discuss the field's progress in addressing four long-standing challenges: creating plants that satisfy their own nitrogen requirement, so reducing or eliminating the need for nitrogen fertilizer; enhancing the nutrient content of crop plants; engineering biofuel feed stocks that harbor easy-to-access fermentable saccharides by incorporating self-destructing lignin; and increasing photosynthetic efficiency. We also look to the future at emerging areas of research in this field.

  View details for DOI 10.1371/journal.pbio.1001879

  View details for PubMedID 24915445

  View details for PubMedCentralID PMC4051588

• Key applications of plant metabolic engineering. PLoS biology Lau, W., Fischbach, M. A., Osbourn, A., Sattely, E. S. 2014; 12 (6)

  Abstract

  Great strides have been made in plant metabolic engineering over the last two decades, with notable success stories including Golden rice. Here, we discuss the field's progress in addressing four long-standing challenges: creating plants that satisfy their own nitrogen requirement, so reducing or eliminating the need for nitrogen fertilizer; enhancing the nutrient content of crop plants; engineering biofuel feed stocks that harbor easy-to-access fermentable saccharides by incorporating self-destructing lignin; and increasing photosynthetic efficiency. We also look to the future at emerging areas of research in this field.

  View details for DOI 10.1371/journal.pbio.1001879

  View details for PubMedID 24915445

  View details for PubMedCentralID PMC4051588

• The Prevalence of Species and Strains in the Human Microbiome: A Resource for Experimental Efforts PLOS ONE Kraal, L., Abubucker, S., Kota, K., Fischbach, M. A., Mitreva, M. 2014; 9 (5)

  Abstract

  Experimental efforts to characterize the human microbiota often use bacterial strains that were chosen for historical rather than biological reasons. Here, we report an analysis of 380 whole-genome shotgun samples from 100 subjects from the NIH Human Microbiome Project. By mapping their reads to 1,751 reference genome sequences and analyzing the resulting relative strain abundance in each sample we present metrics and visualizations that can help identify strains of interest for experimentalists. We also show that approximately 14 strains of 10 species account for 80% of the mapped reads from a typical stool sample, indicating that the function of a community may not be irreducibly complex. Some of these strains account for >20% of the sequence reads in a subset of samples but are absent in others, a dichotomy that could underlie biological differences among subjects. These data should serve as an important strain selection resource for the community of researchers who take experimental approaches to studying the human microbiota.

  View details for DOI 10.1371/journal.pone.0097279

  View details for Web of Science ID 000336857400072

  View details for PubMedID 24827833

  View details for PubMedCentralID PMC4020798

• Diet rapidly and reproducibly alters the human gut microbiome NATURE David, L. A., Maurice, C. F., Carmody, R. N., Gootenberg, D. B., Button, J. E., Wolfe, B. E., Ling, A. V., Devlin, A. S., Varma, Y., Fischbach, M. A., Biddinger, S. B., Dutton, R. J., Turnbaugh, P. J. 2014; 505 (7484): 559-?

  Abstract

  Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.

  View details for DOI 10.1038/nature12820

  View details for Web of Science ID 000329995000042

  View details for PubMedID 24336217

  View details for PubMedCentralID PMC3957428

• What Lives On Our Skin: Ecology, Genomics and Therapeutic Opportunities Of the Skin Microbiome. Drug discovery today. Disease mechanisms Scharschmidt, T. C., Fischbach, M. A. 2013; 10 (3-4)

  Abstract

  Our skin is home to a rich community of microorganisms. Recent advances in sequencing technology have allowed more accurate enumeration of these human-associated microbiota and investigation of their genomic content.Staphylococcus,CorynebacteriumandPropionibacteriumrepresent the dominant bacterial genera on skin and illustrate how bacteria adapt to life in this harsh environment and also provide us with unique benefits. In healthy states, our skin peacefully co-exists with commensal bacteria while fending off potentially dangerous invaders. Disruption of this equilibrium, termed "dysbiosis", can result from changes in the composition of our skin bacteria, an altered immune response to them, or both and may be a driving factor in certain types of inflammatory skin disease. Engineering topical therapeutics to favourably influence the composition of our skin flora and optimize interactions with them represents a real therapeutic opportunity for the field of dermatology and warrants additional investigation into skin microbial ecology and disease mechanisms related to host-microbe dysbiosis.

  View details for PubMedID 24273587

  View details for PubMedCentralID PMC3833721

• A metabolomic view of how the human gut microbiota impacts the host metabolome using humanized and gnotobiotic mice. ISME journal Marcobal, A., Kashyap, P. C., Nelson, T. A., Aronov, P. A., Donia, M. S., Spormann, A., Fischbach, M. A., Sonnenburg, J. L. 2013; 7 (10): 1933-1943

  Abstract

  Defining the functional status of host-associated microbial ecosystems has proven challenging owing to the vast number of predicted genes within the microbiome and relatively poor understanding of community dynamics and community-host interaction. Metabolomic approaches, in which a large number of small molecule metabolites can be defined in a biological sample, offer a promising avenue to 'fingerprint' microbiota functional status. Here, we examined the effects of the human gut microbiota on the fecal and urinary metabolome of a humanized (HUM) mouse using an optimized ultra performance liquid chromatography-mass spectrometry-based method. Differences between HUM and conventional mouse urine and fecal metabolomic profiles support host-specific aspects of the microbiota's metabolomic contribution, consistent with distinct microbial compositions. Comparison of microbiota composition and metabolome of mice humanized with different human donors revealed that the vast majority of metabolomic features observed in donor samples are produced in the corresponding HUM mice, and individual-specific features suggest 'personalized' aspects of functionality can be reconstituted in mice. Feeding the mice a defined, custom diet resulted in modification of the metabolite signatures, illustrating that host diet provides an avenue for altering gut microbiota functionality, which in turn can be monitored via metabolomics. Using a defined model microbiota consisting of one or two species, we show that simplified communities can drive major changes in the host metabolomic profile. Our results demonstrate that metabolomics constitutes a powerful avenue for functional characterization of the intestinal microbiota and its interaction with the host.The ISME Journal advance online publication, 6 June 2013; doi:10.1038/ismej.2013.89.

  View details for DOI 10.1038/ismej.2013.89

  View details for PubMedID 23739052

• Production of alpha-Galactosylceramide by a Prominent Member of the Human Gut Microbiota PLOS BIOLOGY Brown, L. C., Penaranda, C., Kashyap, P. C., Williams, B. B., Clardy, J., Kronenberg, M., Sonnenburg, J. L., Comstock, L. E., Bluestone, J. A., Fischbach, M. A. 2013; 11 (7)

  Abstract

  While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCer(Bf)), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCer(Bf) has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.

  View details for DOI 10.1371/journal.pbio.1001610

  View details for Web of Science ID 000322592700014

  View details for PubMedCentralID PMC3712910

• antiSMASH 2.0--a versatile platform for genome mining of secondary metabolite producers. Nucleic acids research Blin, K., Medema, M. H., Kazempour, D., Fischbach, M. A., Breitling, R., Takano, E., Weber, T. 2013; 41 (Web Server issue): W204-12

  Abstract

  Microbial secondary metabolites are a potent source of antibiotics and other pharmaceuticals. Genome mining of their biosynthetic gene clusters has become a key method to accelerate their identification and characterization. In 2011, we developed antiSMASH, a web-based analysis platform that automates this process. Here, we present the highly improved antiSMASH 2.0 release, available at http://antismash.secondarymetabolites.org/. For the new version, antiSMASH was entirely re-designed using a plug-and-play concept that allows easy integration of novel predictor or output modules. antiSMASH 2.0 now supports input of multiple related sequences simultaneously (multi-FASTA/GenBank/EMBL), which allows the analysis of draft genomes comprising multiple contigs. Moreover, direct analysis of protein sequences is now possible. antiSMASH 2.0 has also been equipped with the capacity to detect additional classes of secondary metabolites, including oligosaccharide antibiotics, phenazines, thiopeptides, homo-serine lactones, phosphonates and furans. The algorithm for predicting the core structure of the cluster end product is now also covering lantipeptides, in addition to polyketides and non-ribosomal peptides. The antiSMASH ClusterBlast functionality has been extended to identify sub-clusters involved in the biosynthesis of specific chemical building blocks. The new features currently make antiSMASH 2.0 the most comprehensive resource for identifying and analyzing novel secondary metabolite biosynthetic pathways in microorganisms.

  View details for DOI 10.1093/nar/gkt449

  View details for PubMedID 23737449

  View details for PubMedCentralID PMC3692088

• Production of a-galactosylceramide by a prominent member of the human gut microbiota. PLoS biology Wieland Brown, L. C., Penaranda, C., Kashyap, P. C., Williams, B. B., Clardy, J., Kronenberg, M., Sonnenburg, J. L., Comstock, L. E., Bluestone, J. A., Fischbach, M. A. 2013; 11 (7)

  Abstract

  While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCer(Bf)), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCer(Bf) has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.

  View details for DOI 10.1371/journal.pbio.1001610

  View details for PubMedID 23874157

• Cell-Based Therapeutics: The Next Pillar of Medicine SCIENCE TRANSLATIONAL MEDICINE Fischbach, M. A., Bluestone, J. A., Lim, W. A. 2013; 5 (179)

  Abstract

  Two decades ago, the pharmaceutical industry-long dominated by small-molecule drugs-was revolutionized by the the advent of biologics. Today, biomedicine sits on the cusp of a new revolution: the use of microbial and human cells as versatile therapeutic engines. Here, we discuss the promise of this "third pillar" of therapeutics in the context of current scientific, regulatory, economic, and perceptual challenges. History suggests that the advent of cellular medicines will require the development of a foundational cellular engineering science that provides a systematic framework for safely and predictably altering and regulating cellular behaviors.

  View details for DOI 10.1126/scitranslmed.3005568

  View details for Web of Science ID 000317037000004

  View details for PubMedID 23552369

  View details for PubMedCentralID PMC3772767

• Trehalose Biosynthesis Promotes Pseudomonas aeruginosa Pathogenicity in Plants PLOS PATHOGENS Djonovic, S., Urbach, J. M., Drenkard, E., Bush, J., Feinbaum, R., Ausubel, J. L., Traficante, D., Risech, M., Kocks, C., Fischbach, M. A., Priebe, G. P., Ausubel, F. M. 2013; 9 (3)

  Abstract

  Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ΔtreYZΔtreS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ΔtreYZΔtreS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ΔtreYZΔtreS in trans. Surprisingly, the growth defect of the double ΔtreYZΔtreS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved "house-keeping" anabolic pathway (trehalose biosynthesis) as a potent virulence factor that allows it to replicate in the intercellular environment of a leaf.

  View details for DOI 10.1371/journal.ppat.1003217

  View details for Web of Science ID 000316953800022

  View details for PubMedID 23505373

  View details for PubMedCentralID PMC3591346

• Dyeing to Learn More about the Gut Microbiota CELL HOST & MICROBE Donia, M. S., Fischbach, M. A. 2013; 13 (2): 119-120

  Abstract

  The switch from culture-based enumeration to deep sequencing has enabled microbial community composition to be profiled en masse. In a new article, Maurice et al. (2013) report the use of fluorescence-activated cell sorting (FACS) to perform a high-throughput analysis of gut microbiota community function.

  View details for DOI 10.1016/j.chom.2013.01.011

  View details for Web of Science ID 000330850200001

  View details for PubMedID 23414750

  View details for PubMedCentralID PMC3690940

• Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Natural product reports Arnison, P. G., Bibb, M. J., Bierbaum, G., Bowers, A. A., Bugni, T. S., Bulaj, G., Camarero, J. A., Campopiano, D. J., Challis, G. L., Clardy, J., Cotter, P. D., Craik, D. J., Dawson, M., Dittmann, E., Donadio, S., Dorrestein, P. C., Entian, K., Fischbach, M. A., Garavelli, J. S., Göransson, U., Gruber, C. W., Haft, D. H., Hemscheidt, T. K., Hertweck, C., Hill, C., Horswill, A. R., Jaspars, M., Kelly, W. L., Klinman, J. P., Kuipers, O. P., Link, A. J., Liu, W., Marahiel, M. A., Mitchell, D. A., Moll, G. N., Moore, B. S., Müller, R., Nair, S. K., Nes, I. F., Norris, G. E., Olivera, B. M., Onaka, H., Patchett, M. L., Piel, J., Reaney, M. J., Rebuffat, S., Ross, R. P., Sahl, H., Schmidt, E. W., Selsted, M. E., Severinov, K., Shen, B., Sivonen, K., Smith, L., Stein, T., Süssmuth, R. D., Tagg, J. R., Tang, G., Truman, A. W., Vederas, J. C., Walsh, C. T., Walton, J. D., Wenzel, S. C., Willey, J. M., van der Donk, W. A. 2013; 30 (1): 108-160

  Abstract

  This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

  View details for DOI 10.1039/c2np20085f

  View details for PubMedID 23165928

  View details for PubMedCentralID PMC3954855

• Molecular Analysis of Model Gut Microbiotas by Imaging Mass Spectrometry and Nanodesorption Electrospray Ionization Reveals Dietary Metabolite Transformations ANALYTICAL CHEMISTRY Rath, C. M., Alexandrov, T., Higginbottom, S. K., Song, J., Milla, M. E., Fischbach, M. A., Sonnenburg, J. L., Dorrestein, P. C. 2012; 84 (21): 9259-9267

  Abstract

  The communities constituting our microbiotas are emerging as mediators of the health-disease continuum. However, deciphering the functional impact of microbial communities on host pathophysiology represents a formidable challenge, due to the heterogeneous distribution of chemical and microbial species within the gastrointestinal (GI) tract. Herein, we apply imaging mass spectrometry (IMS) to localize metabolites from the interaction between the host and colonizing microbiota. This approach complements other molecular imaging methodologies in that analytes need not be known a priori, offering the possibility of untargeted analysis. Localized molecules within the GI tract were then identified in situ by surface sampling with nanodesorption electrospray ionization Fourier transform ion cyclotron resonance-mass spectrometry (nanoDESI FTICR-MS). Products from diverse structural classes were identified including cholesterol-derived lipids, glycans, and polar metabolites. Specific chemical transformations performed by the microbiota were validated with bacteria in culture. This study illustrates how untargeted spatial characterization of metabolites can be applied to the molecular dissection of complex biology in situ.

  View details for DOI 10.1021/ac302039u

  View details for Web of Science ID 000310664600055

  View details for PubMedID 23009651

  View details for PubMedCentralID PMC3711173

• Molecular Insights into the Biosynthesis of Guadinomine: A Type III Secretion System Inhibitor JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Holmes, T. C., May, A. E., Zaleta-Riyera, K., Ruby, J. G., Skewes-Cox, P., Fischbach, M. A., DeRisi, J. L., Iwatsuki, M., Omura, S., Khosla, C. 2012; 134 (42): 17797-17806

  Abstract

  Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509 with a novel mode of action. With an IC(50) of 14 nM, guadinomine B is the most potent known inhibitor of the type III secretion system (TTSS) of Gram-negative bacteria. TTSS activity is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli , Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. The guadinomine (gdn) biosynthetic gene cluster has been cloned and sequenced and includes 26 open reading frames spanning 51.2 kb. It encodes a chimeric multimodular polyketide synthase, a nonribosomal peptide synthetase, along with enzymes responsible for the biosynthesis of the unusual aminomalonyl-acyl carrier protein extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by targeted disruption of the gene cluster as well as by heterologous expression and analysis of key enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these scarce but potentially important natural products.

  View details for DOI 10.1021/ja308622d

  View details for Web of Science ID 000310103800078

  View details for PubMedID 23030602

  View details for PubMedCentralID PMC3483642

• Microbiota-Targeted Therapies: An Ecological Perspective SCIENCE TRANSLATIONAL MEDICINE Lemon, K. P., Armitage, G. C., Relman, D. A., Fischbach, M. A. 2012; 4 (137)

  Abstract

  The connection between disease and the disruption of homeostatic interactions between the host and its microbiota is now well established. Drug developers and clinicians are starting to rely more heavily on therapies that directly target the microbiota and on the ecology of the microbiota to understand the outcomes of these treatments. The effects of those microbiota-targeted therapies that alter community composition range in scale from eliminating individual strains of a single species (for example, with antibacterial conjugate vaccines) to replacing the entire community with a new intact microbiota (for example, by fecal transplantation). Secondary infections linked to antibiotic use provide a cautionary tale of the unintended consequences of perturbing a microbial species network and highlight the need for new narrow-spectrum antibiotics with rapid companion diagnostics. Insights into microbial ecology will also benefit the development of probiotics, whose therapeutic prospects will depend on rigorous clinical testing. Future probiotics may take the form of a consortium of long-term community residents: "a fecal transplant in a capsule." The efficacy of microbiota-targeted therapies will need to be assessed using new diagnostic tools that measure community function rather than composition, including the temporal response of a microbial community to a defined perturbation such as an antibiotic or probiotic.

  View details for DOI 10.1126/scitranslmed.3004183

  View details for Web of Science ID 000305075700012

  View details for PubMedID 22674555

• Production of an NKT cell stimulatory glycosphingolipid by a prominent member of the human gut microbiota Penaranda, C., Brown, L., Kashyap, P., Clardy, J., Kronenberg, M., Sonnenburg, J., Comstock, L., Bluestone, J., Fischbach, M. AMER ASSOC IMMUNOLOGISTS. 2012

  View details for Web of Science ID 000304659700365

• A mass spectrometry-guided genome mining approach for natural product peptidogenomics NATURE CHEMICAL BIOLOGY Kersten, R. D., Yang, Y., Xu, Y., Cimermancic, P., Nam, S., Fenical, W., Fischbach, M. A., Moore, B. S., Dorrestein, P. C. 2011; 7 (11): 794-802

  Abstract

  Peptide natural products show broad biological properties and are commonly produced by orthogonal ribosomal and nonribosomal pathways in prokaryotes and eukaryotes. To harvest this large and diverse resource of bioactive molecules, we introduce here natural product peptidogenomics (NPP), a new MS-guided genome-mining method that connects the chemotypes of peptide natural products to their biosynthetic gene clusters by iteratively matching de novo tandem MS (MS(n)) structures to genomics-based structures following biosynthetic logic. In this study, we show that NPP enabled the rapid characterization of over ten chemically diverse ribosomal and nonribosomal peptide natural products of previously unidentified composition from Streptomycete bacteria as a proof of concept to begin automating the genome-mining process. We show the identification of lantipeptides, lasso peptides, linardins, formylated peptides and lipopeptides, many of which are from well-characterized model Streptomycetes, highlighting the power of NPP in the discovery of new peptide natural products from even intensely studied organisms.

  View details for DOI 10.1038/nchembio.684

  View details for Web of Science ID 000296381600009

  View details for PubMedID 21983601

  View details for PubMedCentralID PMC3258187

• Eating For Two: How Metabolism Establishes lnterspecies Interactions in the Gut CELL HOST & MICROBE Fischbach, M. A., Sonnenburg, J. L. 2011; 10 (4): 336-347

  Abstract

  In bacterial communities, "tight economic times" are the norm. Of the many challenges bacteria face in making a living, perhaps none are more important than generating energy, maintaining redox balance, and acquiring carbon and nitrogen to synthesize primary metabolites. The ability of bacteria to meet these challenges depends heavily on the rest of their community. Indeed, the most fundamental way in which bacteria communicate is by importing the substrates for metabolism and exporting metabolic end products. As an illustration of this principle, we will travel down a carbohydrate catabolic pathway common to many species of Bacteroides, highlighting the interspecies interactions established (often inevitably) at its key steps. We also discuss the metabolic considerations in maintaining the stability of host-associated microbial communities.

  View details for DOI 10.1016/j.chom.2011.10.002

  View details for Web of Science ID 000296600700009

  View details for PubMedID 22018234

  View details for PubMedCentralID PMC3225337

• Combination therapies for combating antimicrobial resistance CURRENT OPINION IN MICROBIOLOGY Fischbach, M. A. 2011; 14 (5): 519-523

  Abstract

  New drug development strategies are needed to combat antimicrobial resistance. The object of this perspective is to highlight one such strategy: treating infections with sets of drugs rather than individual drugs. We will highlight three categories of combination therapy: those that inhibit targets in different pathways; those that inhibit distinct nodes in the same pathway; and those that inhibit the very same target in different ways. We will then consider examples of naturally occurring combination therapies produced by micro-organisms, and conclude by discussing key opportunities and challenges for making more widespread use of drug combinations.

  View details for DOI 10.1016/j.mib.2011.08.003

  View details for Web of Science ID 000296945800003

  View details for PubMedID 21900036

  View details for PubMedCentralID PMC3196371

• antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences NUCLEIC ACIDS RESEARCH Medema, M. H., Blin, K., Cimermancic, P., de Jager, V., Zakrzewski, P., Fischbach, M. A., Weber, T., Takano, E., Breitling, R. 2011; 39: W339-W346

  Abstract

  Bacterial and fungal secondary metabolism is a rich source of novel bioactive compounds with potential pharmaceutical applications as antibiotics, anti-tumor drugs or cholesterol-lowering drugs. To find new drug candidates, microbiologists are increasingly relying on sequencing genomes of a wide variety of microbes. However, rapidly and reliably pinpointing all the potential gene clusters for secondary metabolites in dozens of newly sequenced genomes has been extremely challenging, due to their biochemical heterogeneity, the presence of unknown enzymes and the dispersed nature of the necessary specialized bioinformatics tools and resources. Here, we present antiSMASH (antibiotics & Secondary Metabolite Analysis Shell), the first comprehensive pipeline capable of identifying biosynthetic loci covering the whole range of known secondary metabolite compound classes (polyketides, non-ribosomal peptides, terpenes, aminoglycosides, aminocoumarins, indolocarbazoles, lantibiotics, bacteriocins, nucleosides, beta-lactams, butyrolactones, siderophores, melanins and others). It aligns the identified regions at the gene cluster level to their nearest relatives from a database containing all other known gene clusters, and integrates or cross-links all previously available secondary-metabolite specific gene analysis methods in one interactive view. antiSMASH is available at http://antismash.secondarymetabolites.org.

  View details for DOI 10.1093/nar/gkr466

  View details for Web of Science ID 000292325300055

  View details for PubMedID 21672958

  View details for PubMedCentralID PMC3125804

• Community Health Care: Therapeutic Opportunities in the Human Microbiome SCIENCE TRANSLATIONAL MEDICINE Sonnenburg, J. L., Fischbach, M. A. 2011; 3 (78)

  Abstract

  We are never alone. Humans coexist with diverse microbial species that live within and upon us--our so-called microbiota. It is now clear that this microbial community is essentially another organ that plays a fundamental role in human physiology and disease. Basic and translational research efforts have begun to focus on deciphering mechanisms of microbiome function--and learning how to manipulate it to benefit human health. In this Perspective, we discuss therapeutic opportunities in the human microbiome.

  View details for DOI 10.1126/scitranslmed.3001626

  View details for Web of Science ID 000292976400002

  View details for PubMedID 21490274

  View details for PubMedCentralID PMC3287364

• Draft Genome Sequence of Streptomyces clavuligerus NRRL 3585, a Producer of Diverse Secondary Metabolites JOURNAL OF BACTERIOLOGY Song, J. Y., Jeong, H., Yu, D. S., Fischbach, M. A., Park, H., Kim, J. J., Seo, J., Jensen, S. E., Oh, T. K., Lee, K. J., Kim, J. F. 2010; 192 (23): 6317-6318

  Abstract

  Streptomyces clavuligerus is an important industrial strain that produces a number of antibiotics, including clavulanic acid and cephamycin C. A high-quality draft genome sequence of the S. clavuligerus NRRL 3585 strain was produced by employing a hybrid approach that involved Sanger sequencing, Roche/454 pyrosequencing, optical mapping, and partial finishing. Its genome, comprising four linear replicons, one chromosome, and four plasmids, carries numerous sets of genes involved in the biosynthesis of secondary metabolites, including a variety of antibiotics.

  View details for DOI 10.1128/JB.00859-10

  View details for Web of Science ID 000283994300028

  View details for PubMedID 20889745

  View details for PubMedCentralID PMC2981214

• A Family of Pyrazinone Natural Products from a Conserved Nonribosomal Peptide Synthetase in Staphylococcus aureus CHEMISTRY & BIOLOGY Zimmermann, M., Fischbach, M. A. 2010; 17 (9): 925-930

  Abstract

  Each year in the United States, infections by methicillin-resistant Staphylococcus aureus (MRSA) are responsible for ∼19,000 deaths and result in $3-$4 billion of health care costs. Because skin colonization is a major risk factor for S. aureus infection, identifying novel small molecules produced by S. aureus can lead to new molecular insights into its ability to colonize and infect the host and new targets for antibacterial intervention. Here, we report that a nonribosomal peptide synthetase conserved across S. aureus and other skin-associated staphylococci encodes a family of three pyrazinone natural products. These molecules likely result from the synthesis and release of a dipeptide aldehyde, its spontaneous cyclization to a dihydropyrazinone, and subsequent oxidation to a pyrazinone. As an unexpected family of small molecule natural products from the pathogen S. aureus, the pyrazinones may open a new window into the interspecies interactions that underlie the poorly understood process of skin colonization.

  View details for DOI 10.1016/j.chembiol.2010.08.006

  View details for Web of Science ID 000283283200006

  View details for PubMedID 20851341

• A Chromatin-Mediated Reversible Drug-Tolerant State in Cancer Cell Subpopulations CELL Sharma, S. V., Lee, D. Y., Li, B., Quinlan, M. P., Takahashi, F., Maheswaran, S., McDermott, U., Azizian, N., Zou, L., Fischbach, M. A., Wong, K., Brandstetter, K., Wittner, B., Ramaswamy, S., Classon, M., Settleman, J. 2010; 141 (1): 69-80

  Abstract

  Accumulating evidence implicates heterogeneity within cancer cell populations in the response to stressful exposures, including drug treatments. While modeling the acute response to various anticancer agents in drug-sensitive human tumor cell lines, we consistently detected a small subpopulation of reversibly "drug-tolerant" cells. These cells demonstrate >100-fold reduced drug sensitivity and maintain viability via engagement of IGF-1 receptor signaling and an altered chromatin state that requires the histone demethylase RBP2/KDM5A/Jarid1A. This drug-tolerant phenotype is transiently acquired and relinquished at low frequency by individual cells within the population, implicating the dynamic regulation of phenotypic heterogeneity in drug tolerance. The drug-tolerant subpopulation can be selectively ablated by treatment with IGF-1 receptor inhibitors or chromatin-modifying agents, potentially yielding a therapeutic opportunity. Together, these findings suggest that cancer cell populations employ a dynamic survival strategy in which individual cells transiently assume a reversibly drug-tolerant state to protect the population from eradication by potentially lethal exposures.

  View details for DOI 10.1016/j.cell.2010.02.027

  View details for Web of Science ID 000276211100013

  View details for PubMedID 20371346

  View details for PubMedCentralID PMC2851638

• Natural Products Version 2.0: Connecting Genes to Molecules JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Walsh, C. T., Fischbach, M. A. 2010; 132 (8): 2469-2493

  Abstract

  Natural products have played a prominent role in the history of organic chemistry, and they continue to be important as drugs, biological probes, and targets of study for synthetic and analytical chemists. In this Perspective, we explore how connecting Nature's small molecules to the genes that encode them has sparked a renaissance in natural product research, focusing primarily on the biosynthesis of polyketides and non-ribosomal peptides. We survey monomer biogenesis, coupling chemistries from templated and non-templated pathways, and the broad set of tailoring reactions and hybrid pathways that give rise to the diverse scaffolds and functionalization patterns of natural products. We conclude by considering two questions: What would it take to find all natural product scaffolds? What kind of scientists will be studying natural products in the future?

  View details for DOI 10.1021/ja909118a

  View details for Web of Science ID 000275117900001

  View details for PubMedID 20121095

  View details for PubMedCentralID PMC2828520

• Dapdiamides, Tripeptide Antibiotics Formed by Unconventional Amide Ligases JOURNAL OF NATURAL PRODUCTS Dawlaty, J., Zhang, X., Fischbach, M. A., Clardy, J. 2010; 73 (3): 441-446

  Abstract

  Construction of a genomic DNA library from Pantoea agglomerans strain CU0119 and screening against the plant pathogen Erwinia amylovora yielded a new family of antibiotics, dapdiamides A-E (1-5). The structures were established through 2D-NMR experiments and mass spectrometry, as well as the synthesis of dapdiamide A (1). Transposon mutagenesis of the active cosmid allowed identification of the biosynthetic gene cluster. The dapdiamide family's promiscuous biosynthetic pathway contains two unconventional amide ligases that are predicted to couple its constituent monomers.

  View details for DOI 10.1021/np900685z

  View details for Web of Science ID 000275885000026

  View details for PubMedID 20041689

  View details for PubMedCentralID PMC2846032

• The next frontier of systems biology: higher-order and interspecies interactions GENOME BIOLOGY Fischbach, M. A., Krogan, N. J. 2010; 11 (5)

  Abstract

  Systems approaches are not so different in essence from classical genetic and biochemical approaches, and in the future may become adopted so widely that the term 'systems biology' itself will become obsolete.

  View details for DOI 10.1186/gb-2010-11-5-208

  View details for Web of Science ID 000279631000007

  View details for PubMedID 20441613

  View details for PubMedCentralID PMC2898071

• Antibiotics from microbes: converging to kill CURRENT OPINION IN MICROBIOLOGY Fischbach, M. A. 2009; 12 (5): 520-527

  Abstract

  As genetically encoded small molecules, antibiotics are phenotypes that have resulted from mutation and natural selection. Advances in genetics, biochemistry, and bioinformatics have connected hundreds of antibiotics to the gene clusters that encode them, allowing these molecules to be analyzed using the tools of evolutionary biology. This review surveys examples of convergent evolution from microbially produced antibiotics, including the convergence of distinct gene clusters on similar phenotypes and the merger of distinct gene clusters into a single functional unit. Examining antibiotics through an evolutionary lens highlights the versatility of biosynthetic pathways, reveals lessons for combating antibiotic resistance, and provides an entry point for studying the natural roles of these natural products.

  View details for DOI 10.1016/j.mib.2009.07.002

  View details for Web of Science ID 000271563200008

  View details for PubMedID 19695947

  View details for PubMedCentralID PMC3176294

• Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans NATURE Haas, B. J., Kamoun, S., Zody, M. C., Jiang, R. H., Handsaker, R. E., Cano, L. M., Grabherr, M., Kodira, C. D., Raffaele, S., Torto-Alalibo, T., Bozkurt, T. O., Ah-Fong, A. M., Alvarado, L., Anderson, V. L., Armstrong, M. R., Avrova, A., Baxter, L., Beynon, J., Boevink, P. C., Bollmann, S. R., Bos, J. I., Bulone, V., Cai, G., Cakir, C., Carrington, J. C., Chawner, M., Conti, L., Costanzo, S., Ewan, R., Fahlgren, N., Fischbach, M. A., Fugelstad, J., Gilroy, E. M., Gnerre, S., Green, P. J., Grenville-Briggs, L. J., Griffith, J., Gruenwald, N. J., Horn, K., Horner, N. R., Hu, C., Huitema, E., Jeong, D., Jones, A. M., Jones, J. D., Jones, R. W., Karlsson, E. K., Kunjeti, S. G., Lamour, K., Liu, Z., Ma, L., MacLean, D., Chibucos, M. C., McDonald, H., McWalters, J., Meijer, H. J., Morgan, W., Morris, P. F., Munro, C. A., O'Neill, K., Ospina-Giraldo, M., Pinzon, A., Pritchard, L., Ramsahoye, B., Ren, Q., Restrepo, S., Roy, S., Sadanandom, A., Savidor, A., Schornack, S., Schwartz, D. C., Schumann, U. D., Schwessinger, B., Seyer, L., Sharpe, T., Silvar, C., Song, J., Studholme, D. J., Sykes, S., Thines, M., van de Vondervoort, P. J., Phuntumart, V., Wawra, S., Weide, R., Win, J., Young, C., Zhou, S., Fry, W., Meyers, B. C., van West, P., Ristaino, J., Govers, F., Birch, P. R., Whisson, S. C., Judelson, H. S., Nusbaum, C. 2009; 461 (7262): 393-398

  Abstract

  Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

  View details for DOI 10.1038/nature08358

  View details for Web of Science ID 000269828100039

  View details for PubMedID 19741609

• Antibiotics for Emerging Pathogens SCIENCE Fischbach, M. A., Walsh, C. T. 2009; 325 (5944): 1089-1093

  Abstract

  Antibiotic-resistant strains of pathogenic bacteria are increasingly prevalent in hospitals and the community. New antibiotics are needed to combat these bacterial pathogens, but progress in developing them has been slow. Historically, most antibiotics have come from a small set of molecular scaffolds whose functional lifetimes have been extended by generations of synthetic tailoring. The emergence of multidrug resistance among the latest generation of pathogens suggests that the discovery of new scaffolds should be a priority. Promising approaches to scaffold discovery are emerging; they include mining underexplored microbial niches for natural products, designing screens that avoid rediscovering old scaffolds, and repurposing libraries of synthetic molecules for use as antibiotics.

  View details for DOI 10.1126/science.1176667

  View details for Web of Science ID 000269382300030

  View details for PubMedID 19713519

  View details for PubMedCentralID PMC2802854

• New Ways to Squash Superbugs SCIENTIFIC AMERICAN Walsh, C. T., Fischbach, M. A. 2009; 301 (1): 44-51

  View details for Web of Science ID 000267198900032

  View details for PubMedID 19555023

• The natural history of antibiotics CURRENT BIOLOGY Clardy, J., Fischbach, M. A., Currie, C. R. 2009; 19 (11): R437-R441

  View details for Web of Science ID 000266891900009

  View details for PubMedID 19515346

  View details for PubMedCentralID PMC2731226

• Thirteen posttranslational modifications convert a 14-residue peptide into the antibiotic thiocillin PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brown, L. C., Acker, M. G., Clardy, J., Walsh, C. T., Fischbach, M. A. 2009; 106 (8): 2549-2553

  Abstract

  The thiazolylpeptides are a family of >50 bactericidal antibiotics that block the initial steps of bacterial protein synthesis. Here, we report a biosynthetic gene cluster for thiocillin and establish that it, and by extension the whole class, is ribosomally synthesized. Remarkably, the C-terminal 14 residues of a 52-residue peptide precursor undergo 13 posttranslational modifications to give rise to thiocillin, making this antibiotic the most heavily posttranslationally-modified peptide known to date.

  View details for DOI 10.1073/pnas.0900008106

  View details for Web of Science ID 000263652900017

  View details for PubMedID 19196969

  View details for PubMedCentralID PMC2650375

• Repurposing libraries of eukaryotic protein kinase inhibitors for antibiotic discovery PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Walsh, C. T., Fischbach, M. A. 2009; 106 (6): 1689-1690

  View details for DOI 10.1073/pnas.0813405106

  View details for Web of Science ID 000263252500004

  View details for PubMedID 19193851

  View details for PubMedCentralID PMC2644097

• Structurally diverse natural products that cause potassium leakage trigger multicellularity in Bacillus subtilis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lopez, D., Fischbach, M. A., Chu, F., Losick, R., Kolter, R. 2009; 106 (1): 280-285

  Abstract

  We report a previously undescribed quorum-sensing mechanism for triggering multicellularity in Bacillus subtilis. B. subtilis forms communities of cells known as biofilms in response to an unknown signal. We discovered that biofilm formation is stimulated by a variety of small molecules produced by bacteria--including the B. subtilis nonribosomal peptide surfactin--that share the ability to induce potassium leakage. Natural products that do not cause potassium leakage failed to induce multicellularity. Small-molecule-induced multicellularity was prevented by the addition of potassium, but not sodium or lithium. Evidence is presented that potassium leakage stimulates the activity of a membrane protein kinase, KinC, which governs the expression of genes involved in biofilm formation. We propose that KinC responds to lowered intracellular potassium concentration and that this is a quorum-sensing mechanism that enables B. subtilis to respond to related and unrelated bacteria.

  View details for DOI 10.1073/pnas.0810940106

  View details for Web of Science ID 000262263900052

  View details for PubMedID 19114652

  View details for PubMedCentralID PMC2629187

• Total biosynthesis: in vitro reconstitution of polyketide and nonribosomal peptide pathways NATURAL PRODUCT REPORTS Sattely, E. S., Fischbach, M. A., Walsh, C. T. 2008; 25 (4): 757-793

  Abstract

  This review surveys efforts to reconstitute key steps in polyketide and nonribosomal peptide biosynthetic pathways with purified enzymes and substrates; 344 references are cited.

  View details for DOI 10.1039/b801747f

  View details for Web of Science ID 000258506200007

  View details for PubMedID 18663394

• Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis GENOME RESEARCH Stinear, T. P., Seemann, T., Harrison, P. F., Jenkin, G. A., Davies, J. K., Johnson, P. D., Abdellah, Z., Arrowsmith, C., Chillingworth, T., Churcher, C., Clarke, K., Cronin, A., Davis, P., Goodhead, I., Holroyd, N., Jagels, K., Lord, A., Moule, S., Mungall, K., Norbertczak, H., Quail, M. A., Rabbinowitsch, E., Walker, D., White, B., Whitehead, S., Small, P. L., Brosch, R., Ramakrishnan, L., Fischbach, M. A., Parkhill, J., Cole, S. T. 2008; 18 (5): 729-741

  Abstract

  Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis.

  View details for DOI 10.1101/gr.075069.107

  View details for Web of Science ID 000255504600005

  View details for PubMedID 18403782

  View details for PubMedCentralID PMC2336800

• The evolution of gene collectives: How natural selection drives chemical innovation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fischbach, M. A., Walsh, C. T., Clardy, J. 2008; 105 (12): 4601-4608

  Abstract

  DNA sequencing has become central to the study of evolution. Comparing the sequences of individual genes from a variety of organisms has revolutionized our understanding of how single genes evolve, but the challenge of analyzing polygenic phenotypes has complicated efforts to study how genes evolve when they are part of a group that functions collectively. We suggest that biosynthetic gene clusters from microbes are ideal candidates for the evolutionary study of gene collectives; these selfish genetic elements evolve rapidly, they usually comprise a complete pathway, and they have a phenotype-a small molecule-that is easy to identify and assay. Because these elements are transferred horizontally as well as vertically, they also provide an opportunity to study the effects of horizontal transmission on gene evolution. We discuss known examples to begin addressing two fundamental questions about the evolution of biosynthetic gene clusters: How do they propagate by horizontal transfer? How do they change to create new molecules?

  View details for Web of Science ID 000254772700015

  View details for PubMedID 18216259

  View details for PubMedCentralID PMC2290807

• Inhibitors of sterol biosynthesis as Staphylococcus aureus antibiotics ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Walsh, C. T., Fischbach, M. A. 2008; 47 (31): 5700-5702

  View details for DOI 10.1002/anie.200801801

  View details for Web of Science ID 000257963900001

  View details for PubMedID 18576462

  View details for PubMedCentralID PMC2603329

• Biosynthetic tailoring of microcin e492m: Post-translational modification affords an antibacterial siderophore-peptide conjugate JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Nolan, E. M., Fischbach, M. A., Koglin, A., Walsh, C. T. 2007; 129 (46): 14336-14347

  Abstract

  The present work reveals that four proteins, MceCDIJ, encoded by the MccE492 gene cluster are responsible for the remarkable post-translational tailoring of microcin E492 (MccE492), an 84-residue protein toxin secreted by Klebsiella pneumonaie RYC492 that targets neighboring Gram-negative species. This modification results in attachment of a linearized and monoglycosylated derivative of enterobactin, a nonribosomal peptide and iron scavenger (siderophore), to the MccE492m C-terminus. MceC and MceD derivatize enterobactin by C-glycosylation at the C5 position of a N-(2,3-dihydroxybenzoyl)serine (DHB-Ser) moiety and regiospecific hydrolysis of an ester linkage in the trilactone scaffold, respectively. MceI and MceJ form a protein complex that attaches C-glycosylated enterobactins to the C-terminal serine residue of both a C10 model peptide and full-length MccE492. In the enzymatic product, the C-terminal serine residue is covalently attached to the C4' oxygen of the glucose moiety. Nonenzymatic and base-catalyzed migration of the peptide to the C6' position affords the C6' glycosyl ester linkage observed in the mature toxin, MccE492m, isolated from bacterial cultures.

  View details for DOI 10.1021/ja074650f

  View details for Web of Science ID 000251182000059

  View details for PubMedID 17973380

  View details for PubMedCentralID PMC2522288

• Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fischbach, M. A., Lai, J. R., Roche, E. D., Walsh, C. T., Liu, D. R. 2007; 104 (29): 11951-11956

  Abstract

  Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced with a domain from a foreign NRPS to create a chimeric assembly line that produces a new variant of a natural NRP. However, such chimeric NRPS modules are often heavily impaired, impeding efforts to create novel NRP variants by swapping domains from different modules or organisms. Here we show that impaired chimeric NRPSs can be functionally restored by directed evolution. Using rounds of mutagenesis coupled with in vivo screens for NRP production, we rapidly isolated variants of two different chimeric NRPSs with approximately 10-fold improvements in enzyme activity and product yield, including one that produces new derivatives of the potent NRP/polyketide antibiotic andrimid. Because functional restoration in these examples required only modest library sizes (10(3) to 10(4) clones) and three or fewer rounds of screening, our approach may be widely applicable even for NRPSs from genetically challenging hosts.

  View details for DOI 10.1073/pnas.0705348104

  View details for Web of Science ID 000248199200018

  View details for PubMedID 17620609

  View details for PubMedCentralID PMC1924594

• One pathway, many products NATURE CHEMICAL BIOLOGY Fischbach, M. A., Clardy, J. 2007; 3 (7): 353-355

  Abstract

  Biosynthetic pathways for secondary metabolites usually make many products, not just one. In this Commentary, we consider why molecular promiscuity might be an evolutionarily advantageous feature of these pathways.

  View details for DOI 10.1038/nchembio0707-353

  View details for Web of Science ID 000247462800003

  View details for PubMedID 17576415

• The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Butcher, R. A., Schroeder, F. C., Fischbach, M. A., Straightt, P. D., Kolter, R., Walsh, C. T., Clardy, J. 2007; 104 (5): 1506-1509

  Abstract

  The approximately 80-kb pksX gene cluster in Bacillus subtilis encodes an unusual hybrid polyketide/nonribosomal peptide synthase that has been linked to the production of the uncharacterized antibiotic bacillaene. Multiple copies of this synthase, each similar in size to the ribosome, assemble into a single organelle-like complex with a mass of tens to hundreds of megadaltons. The resource requirements of the assembled megacomplex suggest that bacillaene has an important biological role. By coupling a differential NMR spectroscopic technique with genetically manipulated strains of B. subtilis, we were able to characterize the structure of this unusual secondary metabolite, which could not be predicted by using bioinformatic analysis. We report that bacillaene is a linear molecule with two amide bonds: the first links an alpha-hydroxy carboxylic acid to a omega-amino carboxylic acid containing a conjugated hexaene, and the second links the hexaene-containing carboxylic acid to an (omega-1) amino carboxylic acid containing a conjugated triene. Knowledge of bacillaene's structure has enabled us to annotate the pksX gene cluster and should facilitate the study of bacillaene's biosynthesis as well as its biological role in B. subtilis.

  View details for DOI 10.1073/pnas.0610503104

  View details for Web of Science ID 000244081000014

  View details for PubMedID 17234808

  View details for PubMedCentralID PMC1785240

• A singular enzymatic megacomplex from Bacillus subtilis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Straight, P. D., Fischbach, M. A., Walsh, C. T., Rudner, D. Z., Kolter, R. 2007; 104 (1): 305-310

  Abstract

  Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

  View details for DOI 10.1073/pnas.0609073103

  View details for Web of Science ID 000243456300056

  View details for PubMedID 17190806

  View details for PubMedCentralID PMC1765455

• New antibiotics from bacterial natural products NATURE BIOTECHNOLOGY Clardy, J., Fischbach, M. A., Walsh, C. T. 2006; 24 (12): 1541-1550

  Abstract

  For the past five decades, the need for new antibiotics has been met largely by semisynthetic tailoring of natural product scaffolds discovered in the middle of the 20(th) century. More recently, however, advances in technology have sparked a resurgence in the discovery of natural product antibiotics from bacterial sources. In particular, efforts have refocused on finding new antibiotics from old sources (for example, streptomycetes) and new sources (for example, other actinomycetes, cyanobacteria and uncultured bacteria). This has resulted in several newly discovered antibiotics with unique scaffolds and/or novel mechanisms of action, with the potential to form a basis for new antibiotic classes addressing bacterial targets that are currently underexploited.

  View details for DOI 10.1038/nbt1266

  View details for Web of Science ID 000242795800032

  View details for PubMedID 17160060

• The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fischbach, M. A., Lin, H., Zhou, L., Yu, Y., Abergel, R. J., Liu, D. R., Raymond, K. N., Wanner, B. L., Strong, R. K., Walsh, C. T., Aderem, A., Smith, K. D. 2006; 103 (44): 16502-16507

  Abstract

  Numerous bacteria cope with the scarcity of iron in their microenvironment by synthesizing small iron-scavenging molecules known as siderophores. Mammals have evolved countermeasures to block siderophore-mediated iron acquisition as part of their innate immune response. Secreted lipocalin 2 (Lcn2) sequesters the Escherichia coli siderophore enterobactin (Ent), preventing E. coli from acquiring iron and protecting mammals from infection by E. coli. Here, we show that the iroA gene cluster, found in many pathogenic strains of Gram-negative enteric bacteria, including E. coli, Salmonella spp., and Klebsiella pneumoniae, allows bacteria to evade sequestration of Ent by Lcn2. We demonstrate that C-glucosylated derivatives of Ent produced by iroA-encoded enzymes do not bind purified Lcn2, and an iroA-harboring strain of E. coli is insensitive to the growth inhibitory effects of Lcn2 in vitro. Furthermore, we show that mice rapidly succumb to infection by an iroA-harboring strain of E. coli but not its wild-type counterpart, and that this increased virulence depends on evasion of host Lcn2. Our findings indicate that the iroA gene cluster allows bacteria to evade this component of the innate immune system, rejuvenating their Ent-mediated iron-acquisition pathway and playing an important role in their virulence.

  View details for DOI 10.1073/pnas.0604636103

  View details for Web of Science ID 000241879500078

  View details for PubMedID 17060628

  View details for PubMedCentralID PMC1637611

• Biochemistry - Directing biosynthesis SCIENCE Fischbach, M. A., Walsh, C. T. 2006; 314 (5799): 603-605

  View details for DOI 10.1126/science.1132692

  View details for Web of Science ID 000241557800031

  View details for PubMedID 17068249

• Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lai, J. R., Fischbach, M. A., Liu, D. R., Walsh, C. T. 2006; 128 (34): 11002-11003

  Abstract

  Carrier proteins are 80- to 100-residue way stations that are central to polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. Because the biosynthetic intermediates for catalytic operations are presented on carrier proteins as covalently attached thioesters (via a 4'-phosphopantetheine prosthetic group), the specific protein-protein interactions between carrier proteins and other NRPS/PKS domains are critical for high-fidelity conversion to the final product. Here we show by combinatorial mutagenesis and selection that the aryl carrier protein of EntB (EntB-ArCP) contains localized protein interaction surfaces. Our strategy involved random mutagenesis of N-terminal regions of EntB-ArCP, then selection for clones that produce enterobactin by plating onto iron-deficient media. We identified several residues that were highly conserved from our selection, two of which (G242 and D244) constitute an interaction surface on EntB-ArCP for the phosphopantetheinyl transferases (PPTases) EntD and Sfp. This PPTase interface is distinct from a previously characterized interface on EntB-ArCP for the downstream elongation module, EntF. These results suggest that different protein components recognize different faces of EntB-ArCP in the enterobactin synthetase and that the majority of EntB-ArCP surface residues are not involved in these interactions. Therefore, designing noncognate carrier protein interactions in PKS and NRPS systems should be possible with very few mutations on a particular carrier protein.

  View details for DOI 10.1021/ja063238h

  View details for Web of Science ID 000239932500010

  View details for PubMedID 16925399

• Structural characterization of enterobactin hydrolase IroE BIOCHEMISTRY Larsen, N. A., Lin, H., Wei, R., Fischbach, M. A., Walsh, C. T. 2006; 45 (34): 10184-10190

  Abstract

  The proliferation of many pathogenic bacteria is limited by the scarcity of soluble iron in their environment. Many of these bacteria scavenge iron by synthesizing and exporting small molecule siderophores that chelate iron. Iron-bound siderophores are subsequently imported for metabolic processing. Three related serine hydrolases have been characterized biochemically in this pathway: Fes, IroD, and IroE. Here, we report the crystal structure of IroE from uropathogenic Escherichia coli CFT073. The native structure and a complex with diisopropyl fluorophosphonate (DFP, a potent serine hydrolase inhibitor) were determined at 2.3 and 1.4 A resolution, respectively. IroE has the typical alpha/beta-hydrolase fold with an atypical catalytic dyad composed of Ser 189 and His 287. Mutation of either residue was detrimental to catalysis. In addition, rather than the typical oxyanion hole composed of backbone amides, IroE employs the atypical guanidinium moiety of Arg 130. Asp 90 anchors Arg 130 in the active site, and mutation of either residue was likewise detrimental to catalysis. We also compare the structure of IroE to the structure of Fes from Shigella flexneri (PDB entry 2B20). Both enzymes have similar active sites, but Fes has an additional amino-terminal lid domain. These lid domains are proposed to confer specificity to these related hydrolases.

  View details for DOI 10.1021/bi060950i

  View details for Web of Science ID 000239922200003

  View details for PubMedID 16922493

• A biosynthetic gene cluster for the acetyl-CoA carboxylase inhibitor andrimid JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Jin, M., Fischbach, M. A., Clardy, J. 2006; 128 (33): 10660-10661

  Abstract

  Increasing bacterial resistance to antibiotics with conventional targets has focused attention on antibiotics with unconventional targets. One promising candidate, the acetyl-CoA carboxylase (ACC) inhibitor andrimid, is a potent, broad-spectrum antibiotic with high selectivity for prokaryotic ACC. Here, we report the use of a DNA-based approach to clone the andrimid biosynthetic gene cluster from Pantoea agglomerans, yielding a cosmid that confers robust andrimid production on Escherichia coli. This gene cluster encodes a hybrid nonribosomal peptide/polyketide (NRP/PK) synthase with several unusual features, including three enzymes that form and insert beta-phenylalanine, two transglutaminase-like enzymes that likely serve as condensation catalysts, and four densely hybrid modules that form the succinimide precursor. Unlike most type I NRPSs and PKSs, the andrimid gene cluster is a dissociated system comprised of small proteins. Therefore, future efforts can exploit the genetic manipulability of E. coli to engineer the andrimid synthase with the goal of producing a diverse set of andrimid analogues for clinical evaluation.

  View details for DOI 10.1021/ja063194c

  View details for Web of Science ID 000239791100013

  View details for PubMedID 16910643

  View details for PubMedCentralID PMC2529255

• Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: Logic, machinery, and mechanisms CHEMICAL REVIEWS Fischbach, M. A., Walsh, C. T. 2006; 106 (8): 3468-3496

  View details for DOI 10.1021/cr0503097

  View details for Web of Science ID 000239624000023

  View details for PubMedID 16895337

• Bromoenterobactins as potent inhibitors of a pathogen-associated, siderophore-modifying C-glycosyltransferase JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lin, H., Fischbach, M. A., Gatto, G. J., Liu, D. R., Walsh, C. T. 2006; 128 (29): 9324-9325

  Abstract

  IroB is a C-glycosyltransferase encoded in the iroA cluster. C-Glucosylation of the bacterial siderophore enterobactin by IroB is a strategy some pathogenic bacteria use to evade the host's innate immunity mediated by lipocalin 2 (Lcn2). Without this modification, enterobactin can be tightly bound by host Lcn2, rendering it ineffective as a siderophore. Therefore, IroB inhibitors could be potential antibiotics against iroA-harboring pathogenic bacteria. We used enterobactin analogues to probe the properties of the active site of IroB and found that enterobactin analogues brominated at the C5 positions of the 2,3-dihydroxybenzoyl rings are potent inhibitors of IroB. This finding could lead to the discovery of effective antibiotics targeting iroA-containing bacteria.

  View details for DOI 10.1021/ja063236x

  View details for Web of Science ID 000239120700033

  View details for PubMedID 16848455

• "Oncogenic shock": Explaining oncogene addiction through differential signal attenuation CLINICAL CANCER RESEARCH Sharma, S. V., Fischbach, M. A., Haber, D. A., Settleman, J. 2006; 12 (14): 4392S-4395S

  Abstract

  "Oncogene addiction" describes the curious acquired dependence of tumor cells on an activated oncogene for their survival and/or proliferation, a phenomenon that has important implications for the success of targeted cancer therapies. However, the mechanisms explaining oncogene addiction remain elusive. We propose that "addiction" may be an illusion generated as a consequence of differential attenuation rates of prosurvival and proapoptotic signals emanating from an oncoprotein acutely following its inactivation. According to this model, which we call "oncogenic shock," prosurvival signals dissipate quickly on oncoprotein inactivation whereas proapoptotic signals linger sufficiently long to commit the cell to an apoptotic death. This mechanism may contribute to the rapid and dramatic clinical responses observed in some cancer patients treated with selective tyrosine kinase inhibitors and could yield additional drug targets that determine the balance of signaling outputs from activated oncoproteins.

  View details for DOI 10.1158/1078-0432.CCR-06-0096

  View details for Web of Science ID 000239437800005

  View details for PubMedID 16857816

• A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lai, J. R., Fischbach, M. A., Liu, D. R., Walsh, C. T. 2006; 103 (14): 5314-5319

  Abstract

  Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via covalent attachment to carrier proteins, by catalytic domains is critical for NRPS and polyketide synthase function. We report the use of combinatorial mutagenesis coupled with in vivo selection for the production of the Escherichia coli NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF. Two libraries spanning the predicted helix 2 and loop 2/helix 3 of EntB-ArCP were generated by shotgun alanine scanning and selected for their ability to support enterobactin production. From the surviving pools, we identified several hydrophobic residues (M249, F264, and A268) that were highly conserved. These residues cluster near the phosphopantetheinylated serine in a structural model, and two of these positions are in the predicted helix 3 region. Subsequent in vitro studies are consistent with the hypothesis that these residues form a surface on EntB required for interaction with EntF. These results suggest that helix 3 is a major recognition element in EntB-ArCP and demonstrate the utility of selection-based approaches for studying NRPS biosynthesis.

  View details for DOI 10.1073/pnas.0601038103

  View details for Web of Science ID 000236636400016

  View details for PubMedID 16567620

  View details for PubMedCentralID PMC1459352

• How pathogenic bacteria evade mammalian sabotage in the battle for iron NATURE CHEMICAL BIOLOGY Fischbach, M. A., Lin, H. N., Liu, D. R., Walsh, C. T. 2006; 2 (3): 132-138

  Abstract

  Many bacteria, including numerous human pathogens, synthesize small molecules known as siderophores to scavenge iron. Enterobactin, a siderophore produced by enteric bacteria, is surprisingly ineffective as an iron-scavenging agent for bacteria growing in animals because of its hydrophobicity and its sequestration by the mammalian protein siderocalin, a component of the innate immune system. However, pathogenic strains of Escherichia coli and Salmonella use enzymes encoded by the iroA gene cluster to tailor enterobactin by glycosylation and linearization. The resulting modified forms of enterobactin, known as salmochelins, can evade siderocalin and are less hydrophobic than enterobactin, restoring this siderophore's iron-scavenging ability in mammals.

  View details for Web of Science ID 000235424100009

  View details for PubMedID 16485005

• Activity screening of carrier domains within nonribosomal peptide synthetases using complex substrate mixtures and large molecule mass spectrometry BIOCHEMISTRY Dorrestein, P. C., Blackhall, J., Straight, P. D., Fischbach, M. A., Garneau-Tsodikova, S., Edwards, D. J., McLaughlin, S., Lin, M., Gerwick, W. H., Kolter, R., Walsh, C. T., Kelleher, N. L. 2006; 45 (6): 1537-1546

  Abstract

  For screening a pool of potential substrates that load carrier domains found in nonribosomal peptide synthetases, large molecule mass spectrometry is shown to be a new, unbiased assay. Combining the high resolving power of Fourier transform mass spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A1, yersiniabactin, pyochelin, and enterobactin biosynthetic pathways as proof of principle, preferred substrates are readily identified from substrate pools. Furthermore, this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the pksN and pksJ genes that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate.

  View details for DOI 10.1021/bi052333k

  View details for Web of Science ID 000235631400001

  View details for PubMedID 16460000

  View details for PubMedCentralID PMC2565507

• Mixing with bubbles: a practical technology for use with portable microfluidic devices LAB ON A CHIP Garstecki, P., Fuerstman, M. J., Fischbach, M. A., Sia, S. K., Whitesides, G. M. 2006; 6 (2): 207-212

  Abstract

  This paper demonstrates a methodology for micromixing that is sufficiently simple that it can be used in portable microfluidic devices. It illustrates the use of the micromixer by incorporating it into an elementary, portable microfluidic system that includes sample introduction, sample filtration, and valving. This system has the following characteristics: (i) it is powered with a single hand-operated source of vacuum, (ii) it allows samples to be loaded easily by depositing them into prefabricated wells, (iii) the samples are filtered in situ to prevent clogging of the microchannels, (iv) the structure of the channels ensure mixing of the laminar streams by interaction with bubbles of gas introduced into the channels, (v) the device is prepared in a single-step soft-lithographic process, and (vi) the device can be prepared to be resistant to the adsorption of proteins, and can be used with or without surface-active agents.

  View details for DOI 10.1039/b510843h

  View details for Web of Science ID 000235684900012

  View details for PubMedID 16450029

• Enzymatic tailoring of enterobactin alters membrane partitioning and iron acquisition ACS CHEMICAL BIOLOGY Luo, M., Lin, H., Fischbach, M. A., Liu, D. R., Walsh, C. T., Groves, J. T. 2006; 1 (1): 29-32

  Abstract

  Enterobactin (Ent), a prototypic bacterial siderophore, is modified by both the C-glucosyltransferase IroB and the macrolactone hydrolase IroE in pathogenic bacteria that contain the iroA cluster. To investigate the possible effects of glucosylation and macrolactone hydrolysis on the physical properties of Ent, the membrane affinities and iron acquisition rates of Ent and Ent-derived siderophores were measured. The data obtained indicate that Ent has a high membrane affinity (K(x) = 1.5 x 10(4)) similar to that of ferric acinetoferrin, an amphiphile containing two eight-carbon hydrophobic chains. Glucosylation and macrolactone hydrolysis decrease the membrane affinity of Ent by 5-25-fold. Furthermore, in the presence of phospholipid vesicles, the iron acquisition rate is significantly increased by glucosylation and macrolactone hydrolysis, due to the resultant decrease in membrane sequestration of the siderophore. These results suggest that IroB and IroE enhance the ability of Ent-producing pathogens to acquire iron in membrane-rich microenvironments.

  View details for DOI 10.1021/cb0500034

  View details for Web of Science ID 000240419700014

  View details for PubMedID 17163637

• Regulation of the nucleotide state of oncogenic Ras proteins by nucleoside diphosphate kinase REGULATORS AND EFFECTORS OF SMALL GTPASES: RAS FAMILY Fischbach, M. A., Settleman, J. 2006; 407: 33-45

  Abstract

  Oncogenic forms of the Ras GTPase exhibit defective GTP hydrolase activity and are insensitive to the stimulatory activity of GTPase activating proteins. It has been suggested that a potential therapeutic strategy to inactivate such mutant forms of Ras could involve small molecules that restore GTP hydrolase activity to mutant Ras proteins; however, thus far, such molecules have not been developed. While characterizing the biochemical properties of several commonly detected K-Ras mutants, we made the unexpected observation that an activity in crude bacterial cell extracts was capable of stimulating the conversion of the oncogenic K-RasG13D mutant from a GTP-bound, active form to a GDP-bound, inactive form. The activity was purified, and the protein, nucleoside diphosphate kinase (NDK), was identified as being responsible for the Ras regulating activity. NDK is closely related to the human metastasis suppressor, NM23, which has previously been implicated in regulating the nucleotide state of small GTPases of the Ras family. Although the physiological relevance of such regulation has been controversial, our biochemical findings in in vitro assays indicate that it may be feasible to develop a therapeutic strategy to achieve the selective biochemical inactivation of oncogenic Ras proteins.

  View details for DOI 10.1016/S0076-6879(05)07004-7

  View details for Web of Science ID 000237082800004

  View details for PubMedID 16757312

• In vitro characterization of salmochelin and enterobactin trilactone hydrolases IroD, IroE, and Fes JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lin, H., Fiscchbach, M. A., Liu, D. R., Walsh, C. T. 2005; 127 (31): 11075-11084

  Abstract

  The iroA locus encodes five genes (iroB, iroC, iroD, iroE, iroN) that are found in pathogenic Salmonella and Escherichia coli strains. We recently reported that IroB is an enterobactin (Ent) C-glucosyltransferase, converting the siderophore into mono-, di-, and triglucosyl enterobactins (MGE, DGE, and TGE, respectively). Here, we report the characterization of IroD and IroE as esterases for the apo and Fe(3+)-bound forms of Ent, MGE, DGE, and TGE, and we compare their activities with those of Fes, the previously characterized enterobactin esterase. IroD hydrolyzes both apo and Fe(3+)-bound siderophores distributively to generate DHB-Ser and/or Glc-DHB-Ser, with higher catalytic efficiencies (k(cat)/K(m)) on Fe(3+)-bound forms, suggesting that IroD is the ferric MGE/DGE esterase responsible for cytoplasmic iron release. Similarly, Fes hydrolyzes ferric Ent more efficiently than apo Ent, confirming Fes is the ferric Ent esterase responsible for Fe(3+) release from ferric Ent. Although each enzyme exhibits lower k(cat)'s processing ferric siderophores, dramatic decreases in K(m)'s for ferric siderophores result in increased catalytic efficiencies. The inability of Fes to efficiently hydrolyze ferric MGE, ferric DGE, or ferric TGE explains the requirement for IroD in the iroA cluster. IroE, in contrast, prefers apo siderophores as substrates and tends to hydrolyze the trilactone just once to produce linearized trimers. These data and the periplasmic location of IroE suggest that it hydrolyzes apo enterobactins while they are being exported. IroD hydrolyzes apo MGE (and DGE) regioselectively to give a single linear trimer product and a single linear dimer product as determined by NMR.

  View details for DOI 10.1021/ja0522027

  View details for Web of Science ID 000231052100065

  View details for PubMedID 16076215

  View details for PubMedCentralID PMC2536649

• In vitro characterization of IroB, a pathogen-associated C-glycosyltransferase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fischbach, M. A., Lin, H. N., Liu, D. R., Walsh, C. T. 2005; 102 (3): 571-576

  Abstract

  Pathogenic strains of Escherichia coli and Salmonella enterica modify the tricatecholic siderophore enterobactin (Ent) by glucosylation of three aryl carbon atoms, a process controlled by the iroA locus [Hantke, K., Nicholson, G., Rabsch, W. & Winkelmann, G. (2003) Proc. Natl. Acad. Sci. USA 100, 3677-3682]. Here, we report the purification of the IroB protein and its characterization as the Ent C-glucosyltransferase. IroB transfers glucosyl groups from uridine-5'-diphosphoglucose to C5 of one, two, or three of the 2,3-dihydroxybenzoyl units of Ent to yield monoglucosyl-C-Ent (MGE), diglucosyl-C-Ent (DGE), and triglucosyl-C-Ent (TGE). DGE, also known as salmochelin S4, and macrolactone-opened derivatives have been isolated from the culture broths of S. enterica and uropathogenic E. coli [Bister, B., Bischoff, D., Nicholson, G. J., Valdebenito, M., Schneider, K., Winkelmann, G., Hantke, K. & Sussmuth, R. D. (2004) Biometals 17, 471-481], but MGE and TGE have not been reported previously. IroB has a k(cat) of approximately 10 min(-1) for the first C-glucosylation and is distributive, with sequential conversion and buildup of MGE and then DGE. The C5 to C1' regio-selectivity of the 2,3-dihydroxybenzoyl-glucose linkage at all three rings of TGE suggests a C5 carbanion, para to the C2 phenolate oxygen, as the carbon nucleophile in this novel enzymatic C-glucosylation.

  View details for Web of Science ID 000226436000011

  View details for PubMedID 15598734

  View details for PubMedCentralID PMC545562

• Dynamic control of liquid-core/liquid-cladding optical waveguides PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wolfe, D. B., Conroy, R. S., Garstecki, P., Mayers, B. T., Fischbach, M. A., Paul, K. E., Prentiss, M., Whitesides, G. M. 2004; 101 (34): 12434-12438

  Abstract

  This report describes the manipulation of light in waveguides that comprise a liquid core and a liquid cladding (liq/liq waveguide). These waveguides are dynamic: Their structure and function depend on a continuous, laminar flow of the core and cladding liquids. Because they are dynamic, they can be reconfigured and adapted continuously in ways that are not possible with solid-state waveguides. The liquids are introduced into the channels of a microfluidic network designed to sandwich the flowing core liquid between flowing slabs of the cladding fluid. At low and moderate Reynolds numbers, flow is laminar, and the liq/liq interfaces are optically smooth. Small irregularities in the solid walls of the channels do not propagate into these interfaces, and liq/liq waveguides therefore exhibit low optical loss because of scattering. Manipulating the rate of flow and the composition of the liquids tunes the characteristics of these optical systems.

  View details for DOI 10.1073/pnas.0404423101

  View details for Web of Science ID 000223596200009

  View details for PubMedID 15314232

  View details for PubMedCentralID PMC515079

• Specific biochemical inactivation of oncogenic Ras proteins by nucleoside diphosphate kinase CANCER RESEARCH Fischbach, M. A., Settleman, J. 2003; 63 (14): 4089-4094

  Abstract

  Activating mutations of Ras have been implicated in approximately 30% of human cancers. In every case, the biochemical consequence of such mutations is to disrupt the GTPase activity of Ras and to render Ras resistant to the actions of GTPase activating proteins. Consequently, oncogenic Ras mutants are "locked" in a GTP-bound active state. We detected a potent activity in Escherichia coli extract that can efficiently convert mutationally activated GTP-bound Ras to the inactive GDP-bound form. Purification of the protein responsible for this activity led to the identification of the enzyme nucleoside diphosphate kinase (Ndk). The human orthologue of Ndk is the NM23 metastasis suppressor, which we found to exhibit a similar activity. Purified Ndk effectively inactivates several of the oncogenic forms of Ras that are seen frequently in human cancers, including RasD12, the most commonly detected Ras mutation. Significantly, Ndk does not detectably affect wild-type Ras or an activated form of the Ras-related Rho GTPase. These results demonstrate that it is possible, through biochemical means, to specifically inactivate oncogenic Ras as a potential therapeutic approach to tumors that harbor Ras mutations. Moreover, the results suggest that the loss of NM23 expression that is commonly observed during tumor progression could lead to increased potency of oncogenic Ras proteins.

  View details for Web of Science ID 000184379800041

  View details for PubMedID 12874011