Benjamin Wang

All Publications

• High-throughput fitness experiments reveal specific vulnerabilities of human-adapted Salmonella during stress and infection. Nature genetics Wang, B. X., Leshchiner, D., Luo, L., Tuncel, M., Hokamp, K., Hinton, J. C., Monack, D. M. 2024

  Abstract

  Salmonella enterica is comprised of genetically distinct 'serovars' that together provide an intriguing model for exploring the genetic basis of pathogen evolution. Although the genomes of numerous Salmonella isolates with broad variations in host range and human disease manifestations have been sequenced, the functional links between genetic and phenotypic differences among these serovars remain poorly understood. Here, we conduct high-throughput functional genomics on both generalist (Typhimurium) and human-restricted (Typhi and Paratyphi A) Salmonella at unprecedented scale in the study of this enteric pathogen. Using a comprehensive systems biology approach, we identify gene networks with serovar-specific fitness effects across 25 host-associated stresses encountered at key stages of human infection. By experimentally perturbing these networks, we characterize previously undescribed pseudogenes in human-adapted Salmonella. Overall, this work highlights specific vulnerabilities encoded within human-restricted Salmonella that are linked to the degradation of their genomes, shedding light into the evolution of this enteric pathogen.

  View details for DOI 10.1038/s41588-024-01779-7

  View details for PubMedID 38831009

  View details for PubMedCentralID 10023398

• One species, different diseases: the unique molecular mechanisms that underlie the pathogenesis of typhoidal Salmonella infections. Current opinion in microbiology Wang, B. X., Butler, D. S., Hamblin, M., Monack, D. M. 2023; 72: 102262

  Abstract

  Salmonella enterica is one of the most widespread bacterial pathogens found worldwide, resulting in approximately 100 million infections and over 200 000 deaths per year. Salmonella isolates, termed 'serovars', can largely be classified as either nontyphoidal or typhoidal Salmonella, which differ in regard to disease manifestation and host tropism. Nontyphoidal Salmonella causes gastroenteritis in many hosts, while typhoidal Salmonella is human-restricted and causes typhoid fever, a systemic disease with a mortality rate of up to 30% without treatment. There has been considerable interest in understanding how different Salmonella serovars cause different diseases, but the molecular details that underlie these infections have not yet been fully characterized, especially in the case of typhoidal Salmonella. In this review, we highlight the current state of research into understanding the pathogenesis of both nontyphoidal and typhoidal Salmonella, with a specific interest in serovar-specific traits that allow human-adapted strains of Salmonella to cause enteric fever. Overall, a more detailed molecular understanding of how different Salmonella isolates infect humans will provide critical insights into how we can eradicate these dangerous enteric pathogens.

  View details for DOI 10.1016/j.mib.2022.102262

  View details for PubMedID 36640585

• Host-derived O-glycans inhibit toxigenic conversion by a virulence-encoding phage in Vibrio cholerae. The EMBO journal Wang, B. X., Takagi, J., McShane, A., Park, J. H., Aoki, K., Griffin, C., Teschler, J., Kitts, G., Minzer, G., Tiemeyer, M., Hevey, R., Yildiz, F., Ribbeck, K. 2022: e111562

  Abstract

  Pandemic and endemic strains of Vibrio cholerae arise from toxigenic conversion by the CTXphi bacteriophage, a process by which CTXphi infects nontoxigenic strains of V.cholerae. CTXphi encodes the cholera toxin, an enterotoxin responsible for the watery diarrhea associated with cholera infections. Despite the critical role of CTXphi during infections, signals that affect CTXphi-driven toxigenic conversion or expression of the CTXphi-encoded cholera toxin remain poorly characterized, particularly in the context of the gut mucosa. Here, we identify mucin polymers as potent regulators of CTXphi-driven pathogenicity in V.cholerae. Our results indicate that mucin-associated O-glycans block toxigenic conversion by CTXphi and suppress the expression of CTXphi-related virulence factors, including the toxin co-regulated pilus and cholera toxin, by interfering with the TcpP/ToxR/ToxT virulence pathway. By synthesizing individual mucin glycan structures de novo, we identify the Core 2 motif as the critical structure governing this virulence attenuation. Overall, our results highlight a novel mechanism by which mucins and their associated O-glycan structures affect CTXphi-mediated evolution and pathogenicity of V.cholerae, underscoring the potential regulatory power housed within mucus.

  View details for DOI 10.15252/embj.2022111562

  View details for PubMedID 36504455

• Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles NATURE COMMUNICATIONS Jones, R. D., Qian, Y., Ilia, K., Wang, B., Laub, M. T., Del Vecchio, D., Weiss, R. 2022; 13 (1): 1720

  Abstract

  Engineered signaling networks can impart cells with new functionalities useful for directing differentiation and actuating cellular therapies. For such applications, the engineered networks must be tunable, precisely regulate target gene expression, and be robust to perturbations within the complex context of mammalian cells. Here, we use bacterial two-component signaling proteins to develop synthetic phosphoregulation devices that exhibit these properties in mammalian cells. First, we engineer a synthetic covalent modification cycle based on kinase and phosphatase proteins derived from the bifunctional histidine kinase EnvZ, enabling analog tuning of gene expression via its response regulator OmpR. By regulating phosphatase expression with endogenous miRNAs, we demonstrate cell-type specific signaling responses and a new strategy for accurate cell type classification. Finally, we implement a tunable negative feedback controller via a small molecule-stabilized phosphatase, reducing output expression variance and mitigating the context-dependent effects of off-target regulation and resource competition. Our work lays the foundation for establishing tunable, precise, and robust control over cell behavior with synthetic signaling networks.

  View details for DOI 10.1038/s41467-022-29338-w

  View details for Web of Science ID 000777008300007

  View details for PubMedID 35361767

  View details for PubMedCentralID PMC8971529

• Evolution towards Virulence in a Burkholderia Two-Component System MBIO Schaefers, M. M., Wang, B. X., Boisvert, N. M., Martini, S. J., Bonney, S. L., Marshall, C. W., Laub, M. T., Cooper, V. S., Priebe, G. P. 2021; 12 (4): e0182321

  Abstract

  Bacteria in the Burkholderia cepacia complex (BCC) are significant pathogens for people with cystic fibrosis (CF) and are often extensively antibiotic resistant. Here, we assess the impacts of clinically observed mutations in fixL, which encodes the sensor histidine kinase FixL. FixL along with FixJ compose a two-component system that regulates multiple phenotypes. Mutations in fixL across two species, B. dolosa and B. multivorans, have shown evidence of positive selection during chronic lung infection in CF. Herein, we find that BCC carrying the conserved, ancestral fixL sequence have lower survival in macrophages and in murine pneumonia models than mutants carrying evolved fixL sequences associated with clinical decline in CF patients. In vitro phosphotransfer experiments found that one evolved FixL protein, W439S, has a reduced ability to autophosphorylate and phosphorylate FixJ, while LacZ reporter experiments demonstrate that B. dolosa carrying evolved fixL alleles has reduced fix pathway activity. Interestingly, B. dolosa carrying evolved fixL alleles was less fit in a soil assay than those strains carrying the ancestral allele, demonstrating that increased survival of these variants in macrophages and the murine lung comes at a potential expense in their environmental reservoir. Thus, modulation of the two-component system encoded by fixLJ by point mutations is one mechanism that allows BCC to adapt to the host infection environment. IMPORTANCE Infections caused by members of the Burkholderia cepacia complex (BCC) are a serious concern for patients with cystic fibrosis (CF) as these bacteria are often resistant to many antibiotics. During long-term infection of CF patients with BCC, mutations in genes encoding the FixLJ system often become prevalent, suggesting that these changes may benefit the bacteria during infection. The system encoded by fixLJ is involved in sensing oxygen and regulating many genes in response and is required for full virulence of the bacteria in a murine pneumonia model. Evolved fixL mutations seen later in infection improve bacterial persistence within macrophages and enhance infection within mice. However, these adaptations are short sighted because they reduce bacterial fitness within their natural habitat, soil.

  View details for DOI 10.1128/mBio.01823-21

  View details for Web of Science ID 000694775300002

  View details for PubMedID 34372701

  View details for PubMedCentralID PMC8406202

• Two-Component Signaling Systems Regulate Diverse Virulence-Associated Traits in Pseudomonas aeruginosa APPLIED AND ENVIRONMENTAL MICROBIOLOGY Wang, B. X., Cady, K. C., Oyarce, G. C., Ribbeck, K., Laub, M. T. 2021; 87 (11)

  Abstract

  Pseudomonas aeruginosa is an opportunistic pathogen that can cause problematic infections at different sites throughout the human body. P. aeruginosa encodes a large suite of over 60 two-component signaling systems that enable cells to rapidly sense and respond to external signals. Previous work has shown that some of these sensory systems contribute to P. aeruginosa pathogenesis, but the virulence-associated processes and phenotypic traits that each of these systems controls are still largely unclear. To aid investigations of these sensory systems, we have generated deletion strains for each of 64 genes encoding histidine kinases and one histidine phosphotransferase in P. aeruginosa PA14. We carried out initial phenotypic characterizations of this collection by assaying these mutants for over a dozen virulence-associated traits, and we found that each of these phenotypes is regulated by multiple sensory systems. Our work highlights the usefulness of this collection for further studies of P. aeruginosa two-component signaling systems and provides insight into how these systems may contribute to P. aeruginosa infection.IMPORTANCEPseudomonas aeruginosa can grow and survive under a wide range of conditions, including as a human pathogen. As such, P. aeruginosa must be able to sense and respond to diverse signals and cues in its environment. This sensory capability is endowed in part by the hundreds of two-component signaling proteins encoded in the P. aeruginosa genome, but the precise roles of each remain poorly defined. To facilitate systematic study of the signaling repertoire of P. aeruginosa PA14, we generated a library of deletion strains, each lacking one of the 64 histidine kinases. By subjecting these strains to a battery of phenotypic assays, we confirmed the functions of many and unveiled roles for dozens of previously uncharacterized histidine kinases in controlling various traits, many of which are associated with P. aeruginosa virulence. Thus, this work provides new insight into the functions of two-component signaling proteins and provides a resource for future investigations.

  View details for DOI 10.1128/AEM.03089-20

  View details for Web of Science ID 000655912400029

  View details for PubMedID 33771779

  View details for PubMedCentralID PMC8208149

• Mucin Glycans Signal through the Sensor Kinase RetS to Inhibit Virulence-Associated Traits in Pseudomonas aeruginosa CURRENT BIOLOGY Wang, B. X., Wheeler, K. M., Cady, K. C., Lehoux, S., Cummings, R. D., Laub, M. T., Ribbeck, K. 2021; 31 (1): 90-+

  Abstract

  Mucus is a densely populated ecological niche that coats all non-keratinized epithelia, and plays a critical role in protecting the human body from infections. Although traditionally viewed as a physical barrier, emerging evidence suggests that mucus can directly suppress virulence-associated traits in opportunistic pathogens including Pseudomonas aeruginosa. However, the molecular mechanisms by which mucus affords this protection are unclear. Here, we show that mucins, and particularly their associated glycans, signal through the Dismed2 domain of the sensor kinase RetS in P. aeruginosa. We find that this RetS-dependent signaling leads to the direct inhibition of the GacS-GacA two-component system, the activity of which is associated with a chronic infection state. This signaling includes downregulation of the type VI secretion system (T6SS), and prevents T6SS-dependent bacterial killing by P. aeruginosa. Overall, these results shed light on how mucus impacts P. aeruginosa behavior, and may inspire novel approaches for controlling P. aeruginosa infections.

  View details for DOI 10.1016/j.cub.2020.09.088

  View details for Web of Science ID 000614361000025

  View details for PubMedID 33125866

  View details for PubMedCentralID PMC8759707

• Home, sweet home: how mucus accommodates our microbiota FEBS JOURNAL Wang, B. X., Wu, C. M., Ribbeck, K. 2021; 288 (6): 1789-1799

  Abstract

  As a natural environment for human-microbiota interactions, healthy mucus houses a remarkably stable and diverse microbial community. Maintaining this microbiota is essential to human health, both to support the commensal bacteria that perform a wide array of beneficial functions and to prevent the outgrowth of pathogens. However, how the host selects and maintains a specialized microbiota remains largely unknown. In this viewpoint, we propose several strategies by which mucus may regulate the composition and function of the human microbiota and discuss how compromised mucus barriers in disease can give rise to microbial dysbiosis.

  View details for DOI 10.1111/febs.15504

  View details for Web of Science ID 000559459200001

  View details for PubMedID 32755014

  View details for PubMedCentralID PMC8739745

• The Pyruvate and alpha-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase JOURNAL OF BIOLOGICAL CHEMISTRY Glasser, N. R., Wang, B. X., Hoy, J. A., Newman, D. K. 2017; 292 (13): 5593-5607

  Abstract

  Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD+ (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD+ in LpdG and other enzymes, achieving the same end by a different mechanism.

  View details for DOI 10.1074/jbc.M116.772848

  View details for Web of Science ID 000397875500037

  View details for PubMedID 28174304

  View details for PubMedCentralID PMC5392700