Clostridium difficile, the most common cause of hospital-acquired infectious diarrhea, is responsible for 250,000 infections and 14,000 deaths each year in the United States alone. Despite the clinical impact of C. difficile relatively little is known about the factors of C. difficile required to colonize the host and evade host immune defenses. Several lines of evidence suggest components of the innate immune system play an important role in controlling C. difficile infections. Our long-term goal is to better understand how C. difficile resists the innate immune defenses during an infection. The bacterial cell envelope is essential for cell viability and is the target of many components of the innate immune system. Lysozyme is an important component of the innate immune system. We found that C. difficile is highly resistant to lysozyme. We have identified a C. difficile Extra-Cytoplasmic Function (ECF) ? factor ?V encoded by csfV, which is specifically induced by lysozyme and is critical to lysozyme resistance in C. difficile and is an important virulence factor in an animal model of C. difficile infection. ECF ? factors represent an important class of signal transduction systems which respond to cell envelope stresses. ?V is activated upon the sequential proteolytic destruction of the anti-? factor RsiV. Our data indicate that RsiV becomes sensitive to proteases upon binding directly to lysozyme. The X-ray crystal co- structure of the RsiV-lysozyme complex revealed not only the regions involved in binding lysozyme but also that RsiV likely functions as a lysozyme inhibitor. This finding raises several important questions: How is does RsiV binding to lysozyme binding control activation of ?V? How does RsiV avoid site-1 cleavage in the absence of lysozyme? What genes are important for mediating lysozyme resistance in C. difficile? Here we propose to 1) Use the RsiV-lysozyme structure to determine the features of RsiV and CD1560 required for interaction with lysozyme; 2) Determine the role of signal peptidase and signal peptides in processing of RsiV; and 3) Define the contribution of ?V-dependent genes in lysozyme resistance and virulence in C. difficile.

Public Health Relevance

The rapid emergence and spread of Clostridium difficile in hospitals and nursing homes presents a large and urgent threat to human health. Lysozyme is an important component of the innate immune system and C. difficile mutants with increased lysozyme sensitivity are less virulent. Our studies of how C. difficile resists the toxic effects of lysozyme are relevant to public health because they may lead to new ways to treat C. difficile infections.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Bacterial Pathogenesis Study Section (BACP)
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Ranallo, Ryan
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University of Iowa
Schools of Medicine
Iowa City
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Lewerke, Lincoln T; Kies, Paige J; Müh, Ute et al. (2018) Bacterial sensing: A putative amphipathic helix in RsiV is the switch for activating ?V in response to lysozyme. PLoS Genet 14:e1007527
Schlievert, Patrick M; Kilgore, Samuel H; Kaus, Gabriela M et al. (2018) Glycerol Monolaurate (GML) and a Nonaqueous Five-Percent GML Gel Kill Bacillus and Clostridium Spores. mSphere 3:
Castro, Ana N; Lewerke, Lincoln T; Hastie, Jessica L et al. (2018) Signal Peptidase Is Necessary and Sufficient for Site 1 Cleavage of RsiV in Bacillus subtilis in Response to Lysozyme. J Bacteriol 200:
Hastie, Jessica L; Williams, Kyle B; Bohr, Lindsey L et al. (2016) The Anti-sigma Factor RsiV Is a Bacterial Receptor for Lysozyme: Co-crystal Structure Determination and Demonstration That Binding of Lysozyme to RsiV Is Required for ?V Activation. PLoS Genet 12:e1006287
Ransom, Eric M; Weiss, David S; Ellermeier, Craig D (2016) Use of mCherryOpt Fluorescent Protein in Clostridium difficile. Methods Mol Biol 1476:53-67
Ho, Theresa D; Ellermeier, Craig D (2015) Ferric Uptake Regulator Fur Control of Putative Iron Acquisition Systems in Clostridium difficile. J Bacteriol 197:2930-40
Ransom, Eric M; Ellermeier, Craig D; Weiss, David S (2015) Use of mCherry Red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile. Appl Environ Microbiol 81:1652-60
Hastie, Jessica L; Williams, Kyle B; Sepúlveda, Carolina et al. (2014) Evidence of a bacterial receptor for lysozyme: binding of lysozyme to the anti-? factor RsiV controls activation of the ecf ? factor ?V. PLoS Genet 10:e1004643
Ho, Theresa D; Williams, Kyle B; Chen, Yan et al. (2014) Clostridium difficile extracytoplasmic function ? factor ?V regulates lysozyme resistance and is necessary for pathogenesis in the hamster model of infection. Infect Immun 82:2345-55
Ransom, Eric M; Williams, Kyle B; Weiss, David S et al. (2014) Identification and characterization of a gene cluster required for proper rod shape, cell division, and pathogenesis in Clostridium difficile. J Bacteriol 196:2290-300

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