Take Home Exam Example 1: Determining the role of the novel compound WD6 in Clostridium difficile toxin A and B regulation.

Transcription

1 Take Home Exam Example 1: Determining the role of the novel compound WD6 in Clostridium difficile toxin A and B regulation. Microbial Pathogenesis MMIC 7050

2 Clostridium difficile is a Gram positive anaerobic bacterium that is responsible for most cases of antibiotic-associated diarrhea, and is the leading cause of nosocomial disease in developed countries (Heinlen and Ballard 2010). Symptoms associated with C. difficile infection (CDI) range from asymptomatic carriage and diarrhea to pseudomembranous colitis and toxic megacolon (Heinlen and Ballard 2010). The bacterium exists in both an antibiotic sensitive vegetative form and an antibiotic resistant spore form (Heinlen and Ballard 2010). Spores can withstand harsh environments, such as ultraviolet light, high temperatures and low ph, and are the main cause of recurrent infections (Heinlen and Ballard 2010). The incidence and severity of CDI is increasing in the United States and Canada (Matamouros et al 2007), with an estimated cases per year in the United States (Heinlen and Ballard 2010), and an estimated cost of $3.2 billion dollars per year (Carter et al 2010). The virulence of C. difficile can be attributed to the production of two large toxins, Toxin A, 250 kda, and Toxin B, 308 kda (Carter et al 2010). These toxins are found on the pathogenicity locus, which is a 19.6 kb portion of the bacterial genome that contains the two toxin genes, tcda and tcdb, as well as regulatory factors, tcdr, tcdc and tcde, a putative holin (Figure 1, Matamouros et al 2007). TcdA and TcdB have enterotoxic and cytotoxic abilities, respectively. Both are glucosyltransferases that target the Rho GTPases thereby preventing their activation, resulting in actin cytoskeleton disruption and cell death (Heinlen and Ballard 2010, Carter et al 2010). Recently an epidemic strain of C. difficile, NAP1/027, was been shown to have increased virulence due to a 10-fold increase in TcdA and a 23-fold increase in TcdB production and is associated with worse clinical outcomes (Heinlen and Ballard 2010, Carter et al 2010). Therefore, toxin production is an attractive target for therapeutic intervention. Toxin A was thought to be the only requirement for disease onset; however the discovery of virulent TcdA- TcdB+ strains challenges this theory. Interestingly, most genes on the pathogenicity locus are expressed as the bacterial cells enter the stationary phase, whereas tcdc is only expressed during the exponential growth phase (Hundsberger 1997). Regulation of toxin production is mainly through TcdR and TcdC. TcdR is an RNA polymerase sigma factor required for toxin gene expression (Dupuy et al 2008). On the other hand, TcdC has been thought to play a negative role in toxin regulation. To date is has not been found to be similar to any other regulatory molecules, however the 26 kda acidic protein has been found to be associated with the membrane through its N-terminal transmembrane domain (Govind et al 2006). Since TcdC is only expressed during exponential growth phase, opposite of toxin expression, and new virulent strains of C. difficile that produce higher levels of toxins have deletions in the tcdc gene further point to negative toxin regulation (Matamouros et al

3 2007). Recently, insight into how TcdC regulates toxin production has been provided. Purified TcdC was able to inhibit tcda transcription in a dose-dependent manner, but not shown to interact directly with the tcda promoter (Matamouros et al 2007). Instead it was shown, through surface plasmon resonance, to interact with the core RNA polymerase and inhibit complex formation with TcdR (Matamouros et al 2007). Current treatment of C. difficile infections are through vancomycin and metronidazole, but risk selecting for vancomycin resistant enterococci in treated individuals and other antibiotic resistant microbes (Ochsner et al 2009). Recent therapeutics have also been developed that target and block the action of toxins in C. difficile, synosorb 90 and tolevamer, but have been pulled form clinical trials due to poor efficacy in vivo (Carter et al 2010, Weiss 2009). Therefore finding alternate novel therapeutics that target an aspect of toxin production remains an attractive approach for treating CDI. A novel chemical, WD6, has recently been developed to prevent toxin formation by C. difficile. The objective of this study is to use in vitro and in vivo methods to evaluate the ability of WD6 to target and inhibit toxin production in C. difficile. I hypothesize that this compound directly interacts with TcdC and this interaction increases its ability to interfere with RNA polymerase complex formation, preventing toxin A and B production by C. difficile. In order to determine if WD6 interacts with TcdC, an inactivating mutation in the tcdc gene is required. Previous studies have been limited in their ability to develop C. difficile mutants due to a lack of a proper model system. However, it is now possible to generate isogenic mutants of the virulent C. difficile strain 630 (O Connor et al 2006). Here, I will use the ClosTron gene knockout system to inactivate the tcdc gene by the stable and permanent insertion of an intron near the C-terminus of the sequence, resulting in a non-functional protein (Heap et al 2010). To confirm insertion of the intron, DNA sequences will be analyzed for the insertion site. Once intron insertion is confirmed, the ability of the intron to produce nonfunctional protein must be analyzed. To do this, wild-type and tcdc mutants will be transfected and cultured in IMR-90 human fibroblasts where Western blots on cell lysates probed with an anti-tcdc antibody will confirm knockout of the tcdc gene compared to the wild-type protein levels. Confirmation of a successful tcdc knockout allows for the use of this strain to observe if there is an interaction between WD6 and TcdC and whether is can lower toxin levels. Since toxins are produced at the start of the stationary phase, cultures of C. difficile will be used only

4 once in a high cell density. Wild-type and knockout C. difficile will be cultured in tryptone yeast extract (TY) broth overnight until high cell density is reached, where cultures will be pelleted and washed to remove any pre-existing toxins. tcdc knockout and wild-type C. difficile strains will then be either treated with the compound WD6 in a dose and time course manner or left untreated, and toxin A and B levels in the supernatant detected by Western blot probed with anti-tcda and anti-tcdb antibodies. Untreated wild-type sample is a control representing the natural toxin A and B induction and expression levels. If WD6 treatment on wild-type cells decreases toxin expression compared to WD6 treatment on the knockout, then there is evidence that WD6 lowers toxin production through TcdC. Functional toxin will produce a round cellular morphology as the actin cytoskeleton is disrupted, and cells will eventually die (Ochsner et al 2009). To compliment the above experiment and test functionality of the toxins produced, supernatant of treated and untreated wild-type and knockout strains will be incubated with fibroblasts. After 24 hours, the percent of rounded cells will be determined through light microscopy (Kuehne et al 2010). IMR-90 human fibroblasts are particularly useful in this type of study since they normally have a filamentous morphology and the rounding of cells is clear to see. Comparing cell morphology after incubation with WD6 treated wild-type sample with cells incubated with untreated wildtype sample would provide insight on WD6 ability to decrease toxin production. A difference between cells incubated with WD6 treated wild-type supernatant and WD6 treated knockout supernatant would also provide evidence that the compound interacts with TcdC. Once the ability of WD6 to interact with TcdC and decrease toxin production is confirmed in vitro, its efficacy will be tested in vivo. The most widely used in vivo model system for C. difficile is the Syrian golden hamster because it most closely resembles the human disease (Carter et al 2010). 20 hamsters are given a dose of clindamycin, a protein synthesis inhibitor, to wipe out the natural flora. Half are then challenged with spores from wild-type and the other half with knockout C. difficile strains. Fecal samples will be monitored and cultured daily to confirm colonization with introduced strains of C. difficile. Once colonization is confirmed, 5 hamsters colonized by each strain will be treated with WD6 and monitored daily and time of death recorded. Controls used for this experiment will be an additional 5 hamsters that are uninfected, as well as the 5 hamsters that are infected with each strain but left untreated. Plotting the percent survival of animals for each test group for each day will provide insight into the effect of WD6 on TcdC and toxin production in vivo. Wild-type infected and untreated controls will die within 3 days, while knockout infected and untreated animals will die quicker if tcdc in fact plays a role in negative regulation of toxin genes. As well, knockout infected and

5 treated animals will not demonstrate a higher survival rate if WD6 acts on TcdC alone. However, wild-type infected and treated animals should show an increase in survival and more closely resemble survival of the uninfected controls. Providing the hypothesis is correct, the results of this study will reveal several things about C. difficile infection. First, it will provide further evidence that tcdc does in fact negatively regulate both toxin production. Earlier work on this issue has only looked at toxin A production because it was thought to be required to cause CDI, however the emergence of a virulent TcdA- TcdB+ strain demonstrates the need to study the production of both toxins. This study will also provide evidence that enhancing TcdC activity by Wd6 is sufficient to reduce toxin levels in vitro and in vivo. In other words, drug targeting the activity of TcdC is effective in lowering toxin levels; therefore the structure of WD6 can be modified to produce even more potent and higher efficiency drugs. CDI and associated symptoms are on the rise in developed countries and the cost of treatments that are not efficient and do not prevent recurrence is becoming a burden on the healthcare system. By targeting toxin regulation it may be possible to treat patients with C. difficile to reduce severity of disease and maintain patients as asymptomatic carriers. Also, if there is a way to control toxin production there may also be a way to control spore formation thereby increasing the efficacy of antibiotics to clear infection. References: Carter GP, Rood JI, and Lyras D The role of toxin A and B in Clostridium difficileassociated disease: Past and present perspectives. Gut Microbes. 1: Dupuy B, Govind R, Antunes A, and Matamouros S Clostridium difficile toxin synthesis is negatively regulated by TcdC. J Med. Microb. 57: Govind R, Vediyappen G, Rolfe RD, and Fralick JA Evidence that Clostridium difficile TcdC is a membrane-associated protein. J Bacteriol. 188: Heap JT, Keuhne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, and Minton NP The ClosTron: Mutagenesis in Clostridium refined and streamlined. J Microbiol. Methods. 80: Heinlen L and Ballard J Clostridium difficile infection. Am. J. Med. Sciences. 340:

6 Hundsberger T, Braun V, Weidmann M, Leukel P, Sauerborn M and von Eichel-Streiber C Transcription analysis of the genes tcda-e of the pathogenicity locus of Clostridium difficile. Eur. J Biochem. 244: Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, and Minton NP The role of toxin A and B in Clostridium difficile infection. Nature letter. 467: Matamouros S, England P, and Dupuy B Clostridium difficile toxin expression is inhibited by the novel regulator TcdC. Mol. Microb. 64: Ochsner UA, Bell SJ, O Leary AL, Hoang T, Stone KC, Young CL, Critchley IA, and Janjic N Inhibitory effect of REP3123 on toxin and spore formation in Clostridium difficile, and in vivo efficacy in a hamster gastrointenstinal infection model. J Antimicrob. Chemo. 63: O Connor JR, Lyras D, Farrow KA, Adams V, Powell DR, Hinds J, Cheung JK, and Rood JI Construction and analysis of chromosomal Clostridium difficile mutants. Mol. Microbiol. 61: Weiss K Toxin-binding treatment for Clostridium difficile: a review including reports of studies with tolevamer. Int. J Antimicrob. Agents. 33:4-7. Figures: Figure 1. Pathogenicity locus of C. difficile. Taken from Matamouros et al 2007.