Award Date

12-15-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Committee Member

Ernesto Abel-Santos

Second Committee Member

Gary Kleiger

Third Committee Member

Hui Zhang

Fourth Committee Member

Eduardo Robleto

Number of Pages

199

Abstract

Clostridioides (Clostridium) difficile infections (CDI) have become the leading cause of nosocomial antibiotic-associated diarrhea worldwide. Under normal circumstances, bacteria found in the gastrointestinal tract provide a barrier against C. difficile colonization. Upon antibiotic therapy, the protective barrier is lost as the microbial community becomes depleted thus providing the opportunity for C. difficile to colonize the human gut. Exposure to taurocholate, a bile acid produced within the mammalian gastrointestinal tract, causes C. difficile spores to begin their transition, a process known as germination, from metabolically dormant structures to toxin-producing cells. As germination is required for the onset of CDI, anti-germination compounds may serve as disease prophylactics.

CamSA, a synthetic analog of taurocholate, was previously shown to inhibit C. difficile strain 630 spores in vitro with an IC50 of 58.3 µM. CamSA was also shown to protect mice from strain 630-induced CDI by inhibiting spore germination in vivo without any overt toxicity. Since antibiotic treatment is a trigger for CDI, we examined the iv pharmacokinetic parameters of CamSA using an antibiotic-treated mouse model. Our data suggests that CamSA is localized primarily to the murine intestinal lumen and liver, while systemic absorption, distribution, and degradation of CamSA is minimal. Our data also suggests that CamSA is being recognized as a bile acid and undergoes enterohepatic circulation. This could be important in establishing a CamSA reservoir in our animal model. Since CamSA was found in the liver, we also evaluated a potential interaction between CamSA and CYP3A4, a P450 isozyme involved in drug metabolism. We observed a weak interaction between CamSA and CYP3A4.

While CamSA may be a feasible prophylactic treatment, CDI has been further complicated by the emergence of highly virulent variants of C. difficile. CamSA, interestingly, is inactive against C. difficile strain R20291, a hypervirulent strain of C. difficile. To increase the scope of anti-germination therapies for CDI prophylaxis, a library of CamSA analogs was synthesized and assayed against C. difficile strain R20291 spores. We were able to determine structure-activity relationships (SAR) from this chemical library. Our SAR analyses suggest shorter alkyl chains between the amide nitrogen and the aromatic side chain, as well as more constrained ring structures, allow for better germination inhibition of C. difficile strain R20291 spores. These SAR analyses would be used to further develop potent anti-germinants against C. difficile in a rationalized manner. We identified 24 anti-germinants with IC50 values between 0.35 µM and 37 µM. Several of these compounds are equally effective against strain 630 and account for the most potent anti-germinants reported so far against C. difficile.

The germination response difference between C. difficile strains R20291 and 630 suggests that there are strain-specific mutations that alter bile acid recognition. Indeed, v several clinically relevant C. difficile strains have been reported to exhibit a variety of germination responses. We attempted to elucidate the genetic basis behind the germination variations. By generating C. difficile knockouts and comparing a subset of phenotypically different C. difficile strains, we verified three essential germination proteins believed to function in germinant/anti-germinant recognition. We were, however, unable to establish a correlation between these germination proteins and the different germination phenotypes.

Lastly, conventional CDI treatments can often lead to the development of antibiotic-resistant C. difficile. Several clinically relevant C. difficile strains have already shown resistance to metronidazole. While vancomycin-resistant C. difficile have not yet been reported, the possibility of vancomycin resistance exists as shown in other species of bacteria. Thus, we determined the frequencies at which C. difficile spores could become resistant to our anti-germinants. We also determined the frequencies where our anti-germinants could trigger the germination and outgrowth of C. difficile spores.

Keywords

Bacterial spore germination; Bile salt analogs; Pharmacokinetics

Disciplines

Biochemistry | Microbiology | Molecular Biology

File Format

pdf

File Size

6.9 MB

Degree Grantor

University of Nevada, Las Vegas

Language

English

Rights

IN COPYRIGHT. For more information about this rights statement, please visit http://rightsstatements.org/vocab/InC/1.0/

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