Clostridium difficile Lipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination

dc.contributor.authorDiaz, Oscar R.en
dc.contributor.authorSayer, Cameron V.en
dc.contributor.authorPopham, David L.en
dc.contributor.authorShen, Aimeeen
dc.contributor.departmentBiological Sciencesen
dc.date.accessioned2019-08-22T12:48:17Zen
dc.date.available2019-08-22T12:48:17Zen
dc.date.issued2018-06-27en
dc.description.abstractClostridium 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-l-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-8-lactam and thus controls the ability of SleC to recognize its cortex substrate. By comparing the muropeptide profiles of AgerS spores to those of spores lacking either CwID or PdaA, both of which mediate cortex modification in Bacillus subtilis, we determined that C. difficile GerS, CwID, and PdaA are all required to generate muramic-3-lactam. Both GerS and CwID 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 CwID directly interact and that CwID modulates GerS incorporation into mature spores. Since AgerS, AcwID, and Delta pdaA 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 Delta gerS spores to the cortex composition of spores lacking two putative cortex-modifying enzymes, CwID and PdaA. These analyses revealed that GerS, CwID, and PdaA are all required to generate cortex-specific modifications. Since loss of these modifications in Delta gerS, Delta cwID, and Delta pdaA 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 CwID 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.en
dc.description.notesResearch described in the manuscript was funded by award R01GM108684 from the National Institutes of General Medical Sciences and by a Pew Scholar in the Biomedical Sciences grant from the Pew Charitable Trusts to A.S.; R21AI109111 from the National Institutes of Allergy and Infectious Disease (NIAID) to D.L.P.; and PREP training grant R25 GM066567 from NIGMS to O.R.D. The research benefitted from award R01AI22232 from the NIAID to A.S. The content is solely our responsibility and does not necessarily reflect the views of the Pew Charitable Trusts, NIAID, or the National Institutes of Health. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.en
dc.description.sponsorshipNational Institutes of General Medical Sciences [R01GM108684]; Pew Charitable Trusts; National Institutes of Allergy and Infectious Disease (NIAID) [R21AI109111]; PREP from NIGMS [R25 GM066567]; NIAID [R01AI22232]en
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1128/mSphere.00205-18en
dc.identifier.issn2379-5042en
dc.identifier.issue3en
dc.identifier.othere00205-18en
dc.identifier.pmid29950380en
dc.identifier.urihttp://hdl.handle.net/10919/93215en
dc.identifier.volume3en
dc.language.isoenen
dc.publisherAmerican Society for Microbiologyen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectClostridium difficileen
dc.subjectCwIDen
dc.subjectGerSen
dc.subjectPdaAen
dc.subjectcortex modificationen
dc.subjectgerminationen
dc.subjectlipoproteinen
dc.titleClostridium difficile Lipoprotein GerS Is Required for Cortex Modification and Thus Spore Germinationen
dc.title.serialmSphereen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
dc.type.dcmitypeStillImageen
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