Clostridium difficile is a major public health threat, causing disease ranging from mild diarrhea to pseudomembranous colitis. C. difficile disease symptoms are largely mediated by the secreted cytotoxins, TcdA and TcdB. Many aspects of the pathogenicity of this bacterium remains poorly understood, including how C. difficile regulates the production of the toxins and other virulence factors. Toxin gene expression is linked to expression of flagellar genes via the flagellar sigma factor sigma-D, which is encoded in the flgB operon. Thus, factors that regulate the expression of the flgB operon will impact toxin production in addition to flagellar motility. Our long-term goal is to determine how C. difficile coordinately regulates flagellum and toxin gene expression, as these mechanisms are likely fundamental to pathogenesis. We recently showed that flagellar gene expression is subject to phase variation through site-specific DNA recombination of an invertible sequence upstream of the flgB operon. Notably, inversion of the DNA sequence, which we term the ?flagellar switch?, also impacts the production of toxins essential for virulence. We identified RecV as the DNA recombinase that mediates inversion of the flagellar switch, and we have begun to define the mechanism by which the orientation of the flagellar switch sequence controls gene expression. Our central hypothesis is that phase variation of flagellum and toxin production is critical to C. difficile pathogenesis, and consequently interfering with the ability of C. difficile to phase vary will attenuate one or more aspects of virulence. While flagella and/or motility may be important for some aspects of infection, downregulation of flagella is likely vital for C. difficile to evade host immune recognition. Toxin production may similarly be beneficial or detrimental to C. difficile during different stages of infection. The objective of this proposal is to define the molecular mechanisms underlying phase variation and to evaluate the impact of phase variation on host colonization and virulence. This proposal is innovative because it represents an entirely new area of investigation toward understanding C. difficile disease: phase variable expression of flagella and toxins. Completion of the proposed studies will provide much needed mechanistic information on how C. difficile controls virulence factor production during infection, and may expose potential targets for inhibition of intestinal colonization and toxin production to facilitate efforts to combat this increasingly problematic pathogen.

Public Health Relevance

The proposed research is relevant to the mission of the NIH because it will define mechanisms by which Clostridium difficile, an important human diarrheal pathogen, regulates the production of factors necessary for causing disease. The objective of the proposed research is to define a newly-identified mechanism impacting the expression of genes encoding flagella and the toxins that drive disease development. Understanding how C. difficile regulates these virulence factors may reveal new targets for development of preventive and therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI107029-08
Application #
9991721
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Ranallo, Ryan
Project Start
2013-05-20
Project End
2023-08-31
Budget Start
2020-09-01
Budget End
2021-08-31
Support Year
8
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
McKee, Robert W; Aleksanyan, Naira; Garrett, Elizabeth M et al. (2018) Type IV Pili Promote Clostridium difficile Adherence and Persistence in a Mouse Model of Infection. Infect Immun 86:
McKee, Robert W; Harvest, Carissa K; Tamayo, Rita (2018) Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in Clostridium difficile. mSphere 3:
Sekulovic, Ognjen; Mathias Garrett, Elizabeth; Bourgeois, Jacob et al. (2018) Genome-wide detection of conservative site-specific recombination in bacteria. PLoS Genet 14:e1007332
Anjuwon-Foster, Brandon R; Tamayo, Rita (2018) Phase variation of Clostridium difficile virulence factors. Gut Microbes 9:76-83
Anjuwon-Foster, Brandon R; Maldonado-Vazquez, Natalia; Tamayo, Rita (2018) Characterization of Flagellum and Toxin Phase Variation in Clostridioides difficile Ribotype 012 Isolates. J Bacteriol 200:
Purcell, Erin B; McKee, Robert W; Courson, David S et al. (2017) A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase. Infect Immun 85:
Anjuwon-Foster, Brandon R; Tamayo, Rita (2017) A genetic switch controls the production of flagella and toxins in Clostridium difficile. PLoS Genet 13:e1006701
Purcell, Erin B; McKee, Robert W; Bordeleau, Eric et al. (2016) Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile. J Bacteriol 198:565-77
Purcell, Erin B; Tamayo, Rita (2016) Cyclic diguanylate signaling in Gram-positive bacteria. FEMS Microbiol Rev 40:753-73
Edwards, Adrianne N; Tamayo, Rita; McBride, Shonna M (2016) A novel regulator controls Clostridium difficile sporulation, motility and toxin production. Mol Microbiol 100:954-71

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