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Regulation of amidase activity during spore development

Award Number: F32AI197474
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 04/30/2026
PERIOD OF PERFORMANCE END DATE: 04/29/2029

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Regulation of amidase activity during spore development - PROJECT SUMMARY/ABSTRACT Spore germination is essential for clostridial pathogens to initiate disease. During germination, spores degrade their protective cortex, a thick layer of modified peptidoglycan (PG) that maintains metabolic dormancy. The cortex lytic enzymes that specifically degrade the cortex recognize the cortex-specific PG modifications muramic- ∂-lactam (MAL). MAL is generated during spore formation by the successive actions of the CwlD amidase and a deacetylase. While CwlD has intrinsic activity in Bacillus subtilis, the Shen lab recently discovered that CwlD activity in Clostridioides difficile depends on the lipoprotein GerS. GerS activates CwlD by stabilizing its binding to its Zn²⁺ cofactor. Since they further showed that GerS binds a site >25 Å away from CwlD’s Zn²⁺-containing active site, GerS allosterically activates CwlD. Despite these insights into the role of CwlD during C. difficile spore formation, several aspects of its regulation remain unclear. We do not understand why C. difficile CwlD fails to bind Zn2+ in the absence of GerS unlike most CwlD orthologs in part because the conformational changes induced by GerS binding to CwlD are unknown. While I have shown that CwlD localizes around the forespore, the mechanisms driving this localization are also unclear. Since loss of CwlD leads to abnormally shaped forespores with loosely tethered proteinaceous coat layers, it also remains unclear how CwlD impacts cortex synthesis and/or coat assembly. This proposal will address these questions by determining the molecular mechanisms underlying CwlD activation, localization, and coordination during spore development in C. difficile. To define the allosteric circuit that leads to CwlD amidase activation, I will use genetic, biochemical, and structural approaches to identify key regions that regulate CwlD binding to Zn2+. To reveal the mechanisms that regulate CwlD localization around the forespore, I will use mutagenesis and fluorescence microscopy to identify regions within CwlD that are necessary for this localization pattern as well as sporulation factors that control CwlD localization. To determine how cortex modification impacts forespore development, I will identify and characterize CwlD partners using a high-throughput in silico screen combined with co-immunoprecipitation analyses. Because amidase structure and function are well conserved across bacteria and amidases play critical roles in essential cellular processes, these analyses will reveal key functional regions in these enzymes as well as uncover fundamental principles that govern enzymatic allostery more broadly. Finally, since the MAL modification is required for germination to proceed, inhibiting the function of CwlD should prevent disease transmission. Thus, this work will inform the development of anti-spore germination therapies.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $75,880 )
2026	2026	TRUSTEES OF TUFTS COLLEGE	136 HARRISON AVE	BOSTON	MA	02111	SUFFOLK	USA	Allergy and Infectious Diseases Research	000	1	3/30/2026	NEW	$74,632
2026	2026	TRUSTEES OF TUFTS COLLEGE	136 HARRISON AVE	BOSTON	MA	02111	SUFFOLK	USA	Allergy and Infectious Diseases Research	002	1	4/10/2026	NEW	$0
2026	2026	TRUSTEES OF TUFTS COLLEGE	136 HARRISON AVE	BOSTON	MA	02111	SUFFOLK	USA	Allergy and Infectious Diseases Research	001	1	4/10/2026	NEW	$1,248
														Subtotal = $75,880

Grand Total All Awards = $75,880

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Regulation of amidase activity during spore development

Award Number: F32AI197474

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 04/30/2026

PERIOD OF PERFORMANCE END DATE: 04/29/2029

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