Spore germination is essential for the major nosocomial pathogen Clostridium difficile to initiate and transmit infection, yet little is known about the molecular mechanisms regulating this complex developmental process. Since this gap in knowledge has prevented the development of therapies that can prevent dissemination of C. difficile, our long-term goal is to elucidate the molecular basis by which C. difficile spores germinate into vegetative cells. A critical step during germination is the enzymatic removal of the spore cortex, a protective layer of peptidoglycan that maintains spores in a dormant state. In the Clostridia, cortex degradation depends on the SleC cortex hydrolase being proteolytically activated by Csp family proteases. While only a single Csp protease is sufficient to induce cortex hydrolysis in Clostridium perfringens, we have shown that in C. difficile both CspC and the CspBA fusion protease are required to activate SleC upon germinant addition. Intriguingly, CspC and the CspA domain of CspBA are both pseudoproteases that we and others have shown regulate cortex hydrolysis;indeed, CspC was recently identified as a novel germinant receptor. These findings raise a number of important questions: how do pseudoproteases regulate the activity of the CspB protease? How does regulated proteolysis activate SleC? Our objective in this proposal is to determine the molecular mechanisms by which CspC, CspBA, and SleC coordinately control cortex hydrolysis. Using genetic, biochemical and structural methods, we will identify regions within the CspC and CspA pseudoproteases required for CspB activation. Targeted mutagenesis and crystallographic studies of SleC will be used to elucidate the molecular basis by which regulated proteolysis activates SleC. Lastly, interactions between CspC, CspBA, and SleC will be identified using complementary bacterial two-hybrid, immunoprecipitation, and affinity purification approaches. Collectively, the proposed studies will increase our understanding of how pseudoenzymes can control enzyme activity and how C. difficile spores sense and respond to bile salt germinants. These studies will lay the foundation for developing therapeutics that can reduce C. difficile disease transmission and recurrence.

Public Health Relevance

Clostridium difficile is a significant nosocomial pathogen and emergent public health threat that costs the US health care system ~$3 billion to treat each year. Since germination of C. difficile spores is essential for disease transmission, investigating the molecular mechanisms by which its spores transform into toxin-secreting vegetative cells will inform the development of therapeutics that can prevent C. difficile disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM108684-01A1
Application #
8760059
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Gaillard, Shawn R
Project Start
2014-07-10
Project End
2019-06-30
Budget Start
2014-07-10
Budget End
2015-06-30
Support Year
1
Fiscal Year
2014
Total Cost
$289,750
Indirect Cost
$99,750
Name
University of Vermont & St Agric College
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Diaz, Oscar R; Sayer, Cameron V; Popham, David L et al. (2018) Clostridium difficile Lipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination. mSphere 3:
Kevorkian, Yuzo; Shen, Aimee (2017) Revisiting the Role of Csp Family Proteins in Regulating Clostridium difficile Spore Germination. J Bacteriol 199:
Korza, George; Abini-Agbomson, Stephen; Setlow, Barbara et al. (2017) Levels of L-malate and other low molecular weight metabolites in spores of Bacillus species and Clostridium difficile. PLoS One 12:e0182656
Donnelly, M Lauren; Li, William; Li, Yong-Qing et al. (2017) A Clostridium difficile-Specific, Gel-Forming Protein Required for Optimal Spore Germination. MBio 8:
Doona, Christopher J; Feeherry, Florence E; Setlow, Barbara et al. (2016) Effects of High-Pressure Treatment on Spores of Clostridium Species. Appl Environ Microbiol 82:5287-97
Donnelly, M Lauren; Fimlaid, Kelly A; Shen, Aimee (2016) Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase. J Bacteriol 198:1694-1707
Kevorkian, Yuzo; Shirley, David J; Shen, Aimee (2016) Regulation of Clostridium difficile spore germination by the CspA pseudoprotease domain. Biochimie 122:243-54
Shen, Aimee; Fimlaid, Kelly A; Pishdadian, Keyan (2016) Inducing and Quantifying Clostridium difficile Spore Formation. Methods Mol Biol 1476:129-42
Fimlaid, Kelly A; Jensen, Owen; Donnelly, M Lauren et al. (2015) Identification of a Novel Lipoprotein Regulator of Clostridium difficile Spore Germination. PLoS Pathog 11:e1005239
Wang, Shiwei; Shen, Aimee; Setlow, Peter et al. (2015) Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy. J Bacteriol 197:2361-73

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