• Clostridioides difficile is a major public health threat.

    Clostridioides difficile causes severe diarrheal disease that is difficult to diagnose and treat. C. difficile infection (CDI) is the most common cause of antibiotic-associated nosocomial diarrhea [1]. In 2017, CDI resulted in over 200,000 hospitalizations, 12,000 deaths, and >$1 billion in estimated healthcare costs in the United States [2, 3]. 20-30% of CDI patients experience recurrent disease, and current prognostic testing has a poor positive predictive value for symptomatic C. difficile disease and severity [4, 5]. Up to 15% of healthy adults are asymptomatic carriers of C. difficile, contributing to spore transmission and infection of other individuals [6]. There is currently no reliable prognostic test for CDI that accurately can determine if a C. difficile isolate will cause symptomatic disease [7]. Current diagnostic testing relies on nucleic acid amplification tests (NAATs) to detect toxigenic C. difficile, or toxin assays to detect C. difficile toxins. However, NAATs can be positive for asymptomatic carriers, whereas positive toxin assays can miss clinically significant CDI. [7].

  • Flagella and toxin production in C. difficile confer fitness advantages during infection, but they are targets for host immune recognition.

    C. difficile disease symptoms, mediated by the cytotoxins TcdA and TcdB, can range from mild diarrhea to severe pseudomembranous colitis [8]. C. difficile produces flagella that confer swimming motility, promote adherence to intestinal epithelial cells in cell culture, and contribute to intestinal colonization in animal models [9, 10]. Although flagella and toxins both confer fitness advantages during CDI, they are also immunostimulatory and pose a fitness cost. C. difficile flagella stimulate host pattern recognition receptors, which may promote pathogen clearance [9-12]. Similarly, toxin production increases nutrient availability, but also stimulates the host immune response [11]. Consequently, while flagella and toxins confer fitness advantages during infection, their downregulation is likely vital for C. difficile to evade host immune recognition.

  • Phase variation of the flagellar switch controls the ON/OFF expression of genes encoding flagella and toxins in C. difficile.

    The production of toxins is linked to flagellar gene expression, and both are subject to phase variation. Phase variation is a strategy to introduce phenotypic heterogeneity that helps ensure survival by creating subpopulations that are differentially equipped to overcome selective pressures [13]. In C. difficile, several invertible switches have been identified that mediate phase variation by controlling the ON/OFF expression of downstream genes [13]. Phase variation that controls flagella and toxin production in C. difficile occurs when the “flagellar switch,” a 154 bp DNA sequence 5’ to the first gene in the flagellar gene operon, inverts through conservative site-specific DNA recombination, modulating the ON/OFF production of flagella and toxins [14]. However, the consequences of the phenotypic heterogeneity generated by phase variation during CDI remain poorly understood. Defining how phase variation-mediated expression of flagella and toxins impacts C. difficile physiology, virulence, and disease outcomes may reveal the basis for limitations in current diagnostic testing modalities and enable the development of improved therapeutics to inhibit intestinal colonization and toxin production.

  • References

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    2. Lessa, F.C., L.G. Winston, L.C. McDonald, and Emerging Infections Program C. difficile Surveillance Team. Burden of Clostridium difficile infection in the United States. N Engl J Med, 2015. 372(9): p. 825-834.

    3. Dubberke, E.R. and M.A. Olsen. Burden of Clostridium difficile on the healthcare system. Clin Infect Dis, 2012. 55 Suppl 2: p. S88-92.

    4. Cole, S. A. and T. J. Stahl. Persistent and recurrent Clostridium difficile colitis. Clin Colon Rectal Surg, 2015. 28(2): p. 65-69.

    5. Bagdasarian, N., Rao, K., and P. N. Malani. Diagnosis and treatment of Clostridium difficile in adults: a systematic review. J Amer Med Assoc, 2015. 313(4): p. 398-408.

    6. Curry, S.R., Muto, C.A., Schlackman, J.L., Pasculle, A.W., Shutt, K.A., Marsh, J.W., and L.H. Harrison. Use of multilocus variable number of tandem repeats analysis genotyping to determine the role of asymptomatic carriers in Clostridium difficile transmission. Clin Infect Dis 2013. 57(8): p. 1094-1102.

    7. Fang, F.C., Polage, C.R., and M.H. Wilcox. Point-counterpoint: what is the optimal approach for detection of Clostridium difficile infection? J Clin Microbio 2017. 55(3): p. 670-680.

    8. Kuehne, S.A., S.T. Cartman, J.T. Heap, M.L. Kelly, A. Cockayne, and N. P. Minton. The role of toxin A and toxin B in Clostridium difficile infection. Nature, 2010. 467(7316): p. 711-713.

    9. Twine, S.M., C.W. Reid, A. Aubry, D.R. McMullin, K. M. Fulton, J. Austin, and S. M. Logan. Motility and flagellar glycosylation in Clostridium difficile. J Bacteriol, 2009. 191(22): p. 7050-7062.

    10. Stevenson, E., N.P. Minton, and S.A. Kuehne. The role of flagella in Clostridium difficile pathogenicity. Trends Microbiol, 2015. 23(5): p. 275-282.

    11. Shen, A. Clostridium difficile toxins: mediators of inflammation. J Innate Immun 2012. 4(2): p. 149-158.

    12. Jarchum, I., M. Liu, L. Lipuma, and E.G. Pamer. Toll-like receptor 5 stimulation protects mice from acute Clostridium difficile colitis. Infect Immun, 2011. 79(4): p. 1498-1503.

    13. van der Woude, M.W. and A.J Bäumler. Phase and antigenic variation in bacteria. Clin Microbio Rev, 2004. 17(3): p. 581-611.

    14. Anjuwon-Foster, B.R. and R. Tamayo. A genetic switch controls the production of flagella and toxins in Clostridium difficile. PLoS Genet, 2017. 13(3): p. e1006701.