Date of Graduation

5-2016

Document Type

Dissertation (PhD)

Program Affiliation

Microbiology and Molecular Genetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Barbara E. Murray

Committee Member

Ambro van Hoof

Committee Member

Barrett R. Harvey

Committee Member

Danielle A. Garsin

Committee Member

Hung Ton-That

Committee Member

Jeffrey K. Actor

Abstract

Enterococcus faecalis and Enterococcus faecium are increasingly common as causative agents of human infections, many of which are very difficult to treat due to multi-drug resistance. The work presented in the first part of this dissertation elucidates a mechanism for the regulation of pilus expression. I showed that ATT is the initiation codon of ebpA, the first gene of an operon that codes for the endocarditis and biofilm-associated pili (Ebp), a recognized virulence factor in E. faecalis. The presence of this rare start codon downregulates EbpA translation and protein levels, diminishing biofilm and binding abilities of E. faecalis, as compared to an engineered ATG codon. My studies also extended to the ortholog of Ebp in E. faecium, known as the E. faecium pili (Emp), where the role of each Emp subunit in biofilm formation, adherence and experimental infection was demonstrated. This study highlighted the relevance of the tip subunit, EmpA, in pilus biogenesis and pilus-associated functions. Due to the rising clinical importance of E. faecium and the fact that enterococcal infections are commonly preceded by intestinal colonization, the second part of this dissertation focused on the dynamics of gastrointestinal tract (GIT) colonization of the three known E. faecium clades. This work found that clade B, composed of strains that are part of the normal human microbiota, outcompeted most of the clade A strains, which include strains linked to human infections or associated with animals, when present together in a mouse model of GIT colonization. Last, I demonstrated that the pbp5 gene, part of the E. faecium core genome that shows sequence variation between the clades and encodes a penicillin binding protein important for β-lactam resistance, is differentially expressed between strains, with higher PBP5 protein levels in clade A ampicillin-resistant strains as compared to clade B and subclade A2 ampicillin-susceptible strains. Furthermore, I found evidence that there are extensive differences within the region upstream of pbp5 among the clades that correlate with the differential abundance of PBP5 and ampicillin resistance. Together these studies provide further insight into determinants that contribute to colonization, virulence and resistance in enterococci.

Keywords

Enterococcus faecalis, Enterococcus faecium, pili, gastrointestinal tract colonization, ampicillin resistance, Penicillin-Binding Protein 5

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