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Bacterial Persistence and the HIPBA Toxin-Antitoxin System

by
Lauren A. DeStefano
BA, 2004
Drew University

Thesis Advisor: Nancy Woychik, PhD
Graduate Program of Molecular Genetics, Microbiology & Immunology

Robert Wood Johson Medical School
7th Floor Conference Room

Thursday, August 27, 2009
10:00 am


Abstract

Bacterial populations contain a small fraction of cells capable of surviving antibiotic exposure and persisting through mechanisms that may include a reduced growth rate or entering a state of dormancy. These persisters represent a clinically important subpopulation involved in multidrug tolerance, but the molecular switches that lead to the persister phenotype are poorly understood. The function of Escherichia coli HipA, the toxin component of the HipBA toxin-antitoxin (TA) system, has been directly linked to persistence based on the ability of a hipA mutant to enhance persister formation.

TA modules/systems are autoregulated operons comprising contiguous cognate TA genes that are found in free-living bacterial genomes, on extrachromosomal bacterial plasmids, or in bacteriophage genomes that lysogenize as low copy plasmids. The toxin components of chromosomal TA systems in E. coli appear to be responsible for the switch to a state of growth arrest, referred to as quasidormancy, which enables cell survival during stress.

We further characterized the HipBA TA system through a series of biochemical experiments to confirm its role as a bona fide TA system. We employed several in vivo and in vitro methods to search for the target of HipA. We also solved the crystal structure of the HipBA complex with our collaborators at Columbia University. We then examined a series of HipA mutants in an attempt to identify its intracellular targets in E. coli using a series of in vivo toxicity assays, persistence assays, and in vitro phosphorylation assays.

Finally, to further explore the role of TA systems in persister development, we performed a series of standardized assays in isogenic E. coli strains using inducible plasmids containing identical promoters. The results demonstrate that expression of any of six TA toxins (HipA, ChpBK, YafQ, YoeB, MazF or Doc) contributes to surviving antibiotic exposure by two mechanistically distinct antibiotics, ampicillin and ofloxacin, measured as CFU and by vital staining. Conversely, deletion of individual TA modules containing the respective toxin genes resulted in a striking reduction of persisters upon ampicillin treatment. Therefore, despite having distinctly different enzymatic activities and disparate intracellular targets, these TA toxins share the ability to induce persistence. Our results suggest that TA toxins in general may underlie the formation of persisters and/or the elusive ‘phenotypic switch’ linked to persister formation.


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