All Publications

  • Oxidative ornithine metabolism supports non-inflammatory C.difficile colonization. Nature metabolism Pruss, K. M., Enam, F., Battaglioli, E., DeFeo, M., Diaz, O. R., Higginbottom, S. K., Fischer, C. R., Hryckowian, A. J., Van Treuren, W., Dodd, D., Kashyap, P., Sonnenburg, J. L. 1800


    The enteric pathogen Clostridioidesdifficile (Cd) is responsible for a toxin-mediated infection that causes more than 200,000 recorded hospitalizations and 13,000 deaths in the United States every year1. However, Cd can colonize the gut in the absence of disease symptoms. Prevalence of asymptomatic colonization by toxigenic Cd in healthy populations is high; asymptomatic carriers are at increased risk of infection compared to noncolonized individuals and may be a reservoir for transmission of Cd infection2,3. Elucidating the molecular mechanisms by which Cd persists in the absence of disease is necessary for understanding pathogenesis and developing refined therapeutic strategies. Here, we show with gut microbiome metatranscriptomic analysis that mice recalcitrant to Cd infection and inflammation exhibit increased community-wide expression of arginine and ornithine metabolic pathways. To query Cd metabolism specifically, we leverage RNA sequencing in gnotobiotic mice infected with two wild-type strains (630 and R20291) and isogenic toxin-deficient mutants of these strains to differentiate inflammation-dependent versus -independent transcriptional states. A single operon encoding oxidative ornithine degradation is consistently upregulated across non-toxigenic Cd strains. Combining untargeted and targeted metabolomics with bacterial and host genetics, we demonstrate that both diet- and host-derived sources of ornithine provide a competitive advantage to Cd, suggesting a mechanism for Cd persistence within a non-inflammatory, healthy gut.

    View details for DOI 10.1038/s42255-021-00506-4

    View details for PubMedID 34992297

  • A lipoprotein allosterically activates the CwlD amidase during Clostridioides difficile spore formation. PLoS genetics Alves Feliciano, C., Eckenroth, B. E., Diaz, O. R., Doublié, S., Shen, A. 2021; 17 (9): e1009791


    Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to Zn2+, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.

    View details for DOI 10.1371/journal.pgen.1009791

    View details for PubMedID 34570752

  • Chromatin Landscape Underpinning Human Dendritic Cell Heterogeneity. Cell reports Leylek, R. n., Alcántara-Hernández, M. n., Granja, J. M., Chavez, M. n., Perez, K. n., Diaz, O. R., Li, R. n., Satpathy, A. T., Chang, H. Y., Idoyaga, J. n. 2020; 32 (12): 108180


    Human dendritic cells (DCs) comprise subsets with distinct phenotypic and functional characteristics, but the transcriptional programs that dictate their identity remain elusive. Here, we analyze global chromatin accessibility profiles across resting and stimulated human DC subsets by means of the assay for transposase-accessible chromatin using sequencing (ATAC-seq). We uncover specific regions of chromatin accessibility for each subset and transcriptional regulators of DC function. By comparing plasmacytoid DC responses to IFN-I-producing and non-IFN-I-producing conditions, we identify genetic programs related to their function. Finally, by intersecting chromatin accessibility with genome-wide association studies, we recognize DC subset-specific enrichment of heritability in autoimmune diseases. Our results unravel the basis of human DC subset heterogeneity and provide a framework for their analysis in disease pathogenesis.

    View details for DOI 10.1016/j.celrep.2020.108180

    View details for PubMedID 32966789

  • Clostridium difficile Lipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination. mSphere Diaz, O. R., Sayer, C. V., Popham, D. L., Shen, A. 2018; 3 (3)


    Clostridium difficile, also known as Clostridioides difficile, is a Gram-positive, spore-forming bacterium that is a leading cause of antibiotic-associated diarrhea. C. difficile infections begin when its metabolically dormant spores germinate to form toxin-producing vegetative cells. Successful spore germination depends on the degradation of the cortex, a thick layer of modified peptidoglycan that maintains dormancy. Cortex degradation is mediated by the SleC cortex lytic enzyme, which is thought to recognize the cortex-specific modification muramic-δ-lactam. C. difficile cortex degradation also depends on the Peptostreptococcaceae-specific lipoprotein GerS for unknown reasons. In this study, we tested whether GerS regulates production of muramic-δ-lactam and thus controls the ability of SleC to recognize its cortex substrate. By comparing the muropeptide profiles of ΔgerS spores to those of spores lacking either CwlD or PdaA, both of which mediate cortex modification in Bacillus subtilis, we determined that C. difficile GerS, CwlD, and PdaA are all required to generate muramic-δ-lactam. Both GerS and CwlD were needed to cleave the peptide side chains from N-acetylmuramic acid, suggesting that these two factors act in concert. Consistent with this hypothesis, biochemical analyses revealed that GerS and CwlD directly interact and that CwlD modulates GerS incorporation into mature spores. Since ΔgerS, ΔcwlD, and ΔpdaA spores exhibited equivalent germination defects, our results indicate that C. difficile spore germination depends on cortex-specific modifications, reveal GerS as a novel regulator of these processes, and highlight additional differences in the regulation of spore germination in C. difficile relative to B. subtilis and other spore-forming organisms.IMPORTANCE The Gram-positive, spore-forming bacterium Clostridium difficile is a leading cause of antibiotic-associated diarrhea. Because C. difficile is an obligate anaerobe, its aerotolerant spores are essential for transmitting disease, and their germination into toxin-producing cells is necessary for causing disease. Spore germination requires the removal of the cortex, a thick layer of modified peptidoglycan that maintains spore dormancy. Cortex degradation is mediated by the SleC hydrolase, which is thought to recognize cortex-specific modifications. Cortex degradation also requires the GerS lipoprotein for unknown reasons. In our study, we tested whether GerS is required to generate cortex-specific modifications by comparing the cortex composition of ΔgerS spores to the cortex composition of spores lacking two putative cortex-modifying enzymes, CwlD and PdaA. These analyses revealed that GerS, CwlD, and PdaA are all required to generate cortex-specific modifications. Since loss of these modifications in ΔgerS, ΔcwlD, and ΔpdaA mutants resulted in spore germination and heat resistance defects, the SleC cortex lytic enzyme depends on cortex-specific modifications to efficiently degrade this protective layer. Our results further indicate that GerS and CwlD are mutually required for removing peptide chains from spore peptidoglycan and revealed a novel interaction between these proteins. Thus, our findings provide new mechanistic insight into C. difficile spore germination.

    View details for DOI 10.1128/mSphere.00205-18

    View details for PubMedID 29950380

    View details for PubMedCentralID PMC6021603