The bacterial pathogen, Clostridioides difficile, has been designated an urgent threat to the US healthcare system by the Centers for Disease Control. In order for C. difficile to initiate infection, its infectious spore form must germinate in the gut of susceptible individuals. Since spore germination depends on the spore cell wall undergoing specific modifications, determining how this process occurs in C. difficile could inform the development of strategies for preventing C. difficile disease transmission and recurrence.

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Spore germination is necessary for many bacterial pathogens, particularly anaerobes, to initiate infection. A critical step during germination is the degradation of the cortex, a thick layer of modified peptidoglycan that maintains metabolic dormancy. Digestion of this protective layer is mediated by cortex lytic enzymes that recognize the spore-specific peptidoglycan modification, muramic-?-lactam. As a result, mutants lacking muramic-?-lactam fail to degrade their cortex layer and exhibit severe defects in germination. Previous work in Bacillus subtilis has shown that muramic-?-lactam is generated through the sequential action of the CwlD amidase and PdaA deacetylase, which are conserved in all spore-forming bacteria. While B. subtilis CwlD has intrinsic amidase activity, we recently determined that the CwlD amidase produced by the bacterial pathogen, Clostridioides difficile, requires the GerS lipoprotein to deamidate N-acetylmuramic acid residues. Notably, GerS is only conserved in members of the Peptostreptococcaceae family, and our preliminary data indicate that only CwlD homologs from the Peptostreptococcaceae family can complement a C. difficile ?cwlD mutant, while B. subtilis and C. perfringens CwlD homologs cannot. These observations suggest that the Peptostreptococceae family uses an alternative mechanism to mediate cortex modification during spore formation. Since our data also indicate that GerS and CwlD interact stoichiometrically, and we have recently crystallized this complex, the goal of this proposal is to determine the mechanism by which GerS regulates CwlD amidase function in C. difficile. We will test the hypothesis that GerS binding to CwlD allosterically activates CwlD function and distinguish between different models for why C. difficile CwlD function depends on GerS. These models include that GerS (i) functions as a co-factor to license CwlD?s amidase activity, recruits CwlD to the forespore, and/or (ii) facilitates CwlD?s recognition of C. difficile?s unique cortex structure. To accomplish these goals, we will use mutational analyses, heterologous expression systems, biochemical analyses, and cell biological techniques to determine the requirements for not only C. difficile CwlD amidase activity but also B. subtilis CwlD amidase function. These comparative analyses will provide mechanistic insight into the regulation of cortex modification in spore-forming bacteria. The proposed work may also expand our understanding of how bacterial amidases can be allosterically regulated by binding partners.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Gaillard, Shawn R
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Tufts University
Schools of Medicine
United States
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