Regulation of to xin Synthesis By Clostridium Difficile

<p><em>Clostridium difficile</em> is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of <em>C. difficile</em> infection (CDI) has been increasing exponentially in the last decade. Virulent strains of <em>C. difficile</em> produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent <em>C. difficile</em> strains that produce active toxins from non-virulent strains that do not produce toxins or produce inactive toxins. Based on the knowledge that <em>C. difficile</em> toxins A and B cleave a substrate that is stereochemically similar to the native substrate of the toxins, uridine diphosphoglucose, a quantitative, cost-efficient assay, the Cdifftox activity assay, was developed to measure <em>C. difficile</em> toxin activity. The concept behind the activity assay was modified to develop a novel, rapid, sensitive, and specific assay for <em>C. difficile</em> toxins in the form of a selective and differential agar plate culture medium, the Cdifftox Plate assay (CDPA). This assay combines in a single step the specific identification of <em>C. difficile</em> strains and the detection of active toxin(s). The CDPA was determined to be extremely accurate (99.8% effective) at detecting toxin-producing strains based on the analysis of 528 <em>C. difficile</em> isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. This new assay advances and improves the culture methodology in that only <em>C. difficile</em> strains will grow on this medium and virulent strains producing active toxins can be differentiated from non-virulent strains. This new method reduces the time and effort required to isolate and confirm toxin-producing <em>C. difficile</em> strains and provides a clinical isolate for antibiotic susceptibility testing and strain typing. The Cdifftox activity assay was used to screen for inhibitors of toxin activity. Physiological levels of the common human conjugated bile salt, taurocholate, was found to inhibit toxin A and B <em>in vitro</em> activities. When co-incubated <em>ex vivo</em> with purified toxin B, taurocholate protected Caco-2 colonic epithelial cells from the damaging effects of the toxin. Furthermore, using a caspase-3 detection assay, taurocholate reduced the extent of toxin B-induced Caco-2 cell apoptosis. These results suggest that bile salts can be effective in protecting the gut epithelium from <em>C. difficile</em> toxin damage, thus, the delivery of physiologic amounts of taurocholate to the colon, where it is normally in low concentration, could be useful in CDI treatment. These findings may help to explain why bile rich small intestine is spared damage in CDI, while the bile salt poor colon is vulnerable in CDI.</p> <p>Toxin synthesis in <em>C. difficile</em> occurs during the stationary phase, but little is known about the regulation of these toxins. It was hypothesized that <em>C. difficile</em> toxin synthesis is regulated by a quorum sensing mechanism. Two lines of evidence supported this hypothesis. First, a small (KDa), diffusible, heat-stable toxin-inducing activity accumulates in the medium of high-density <em>C. difficile</em> cells. This conditioned medium when incubated with low-density log-phase cells causes them to produce toxin early (2-4 hrs instead of 12-16 hrs) and at elevated levels when compared with cells grown in fresh medium. These data suggested that <em>C. difficile</em> cells extracellularly release an inducing molecule during growth that is able to activate toxin synthesis prematurely and demonstrates for the first time that toxin synthesis in <em>C. difficile</em> is regulated by quorum signaling. Second, this toxin-inducing activity was partially purified from high-density stationary-phase culture supernatant fluid by HPLC and confirmed to induce early toxin synthesis, even in <em>C. difficile</em> virulent strains that over-produce the toxins. Mass spectrometry analysis of the purified toxin-inducing fraction from HPLC revealed a cyclic compound with a mass of 655.8 Da. It is anticipated that identification of this toxin-inducing compound will advance our understanding of the mechanism involved in the quorum-dependent regulation of <em>C. difficile</em> toxin synthesis. This finding should lead to the development of even more sensitive tests to diagnose CDI and may lead to the discovery of promising novel therapeutic targets that could be harnessed for the treatment <em>C. difficile</em> infections.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2012

Author dc:creator

• Darkoh, Charles

Contributors dc:contributor

• Herbert L. DuPont
• Heidi B. Kaplan
• Steven J. Norris

• Clostridium difficile
• C. difficile
• C. difficile toxins
• Clostridium toxins
• Clostridium quorum sensing
• quorum sensing
• toxin activity
• difficile toxin activity
• Quorum sensing in C. difficile
• Taurcholate
• Bile salts
• Bacteriology
• Biochemistry
• Biology
• Immunology of Infectious Disease
• Immunopathology
• Medicine and Health Sciences
• Microbial Physiology
• Molecular Biology
• Other Immunology and Infectious Disease
• Pathogenic Microbiology

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/250

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1316