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Francesca P. Rothenbacher
B.S. Biology, Delaware State University -2006
Thesis Advisor: Nancy A. Woychik, Ph.D.
Graduate Program Molecular Genetics, Microbiology
School of Public Health, Room 258
Wednesday, November 2, 2011
Clostridium difficile is an important, emerging nosocomial pathogen. The transition from harmless colonization to disease is typically preceded by antimicrobial therapy that alters the balance of the intestinal flora, enabling C. difficile to proliferate in the colon. One of the most perplexing aspects of the C. difficile infectious cycle is its ability to survive antimicrobial therapy and transition from inert colonization to active infection. In general, toxin-antitoxin (TA) systems appear to enable short-term stress survival or plasmid maintenance depending on whether they are harbored on the bacterial chromosome or as stable plasmids, respectively. The goals of my thesis work involved both basic research as well as clinically-oriented projects. The first goal was to understand the biochemical characteristics of TA toxins in C. difficile. We identified only one TA system in C. difficile strain 630 (epidemic type X), designated MazE-cd and MazF-cd, a counterpart of the well-characterized Escherichia coli MazEF TA system. E. coli MazF cleaves mRNA at ACA sequences leading to global mRNA degradation, growth arrest and death. Primer extension analysis revealed that MazF-cd cleaved RNA at the five base consensus sequence UACAU. After detailed statistical analyses to identify mRNAs in which UACAU was either over- or under-represented, the major C. difficile virulence factor toxin B (TcdB) was among the top four transcripts predicted to be significantly resistant to MazF-cd cleavage along with the cell surface protein and predicted adhesin CwpV. The second goal of my thesis project was to initiate studies on the physiology of C. difficile in infected patients. This approach enabled us to investigate whether TA systems influence C. difficile pathogenesis and/or persistence after antibiotic treatment. Surprisingly, quantification of the levels of C. difficile vegetative cells and spores in stool revealed that although most patients have very high levels of both, there was no correlation between spore and/or vegetative cell load and disease severity. The third goal was to elucidate the mechanism of action of another TA toxin from bacteriophage P1 called Doc. We demonstrated that this TA toxin exhibits novel properties and has a unique intracellular target, 16S rRNA of the 30S ribosomal subunit.