Advisor(s)

Kim Lewis

Contributor(s)

Marin Vulić, Veronica S. Godoy-Carter, Gail S. Begley, Amy Spoering

Date of Award

2011

Date Accepted

12-2011

Degree Grantor

Northeastern University

Degree Level

Ph.D.

Degree Name

Doctor of Philosophy

Department or Academic Unit

College of Science. Department of Biology.

Keywords

biology, microbiology, AcrAB-TolC, heat shock, isolation, oxidative stress, persisters, superoxide

Subject Categories

Escherichia coli, Anti-infective agents, Oxidative stress

Disciplines

Biological Phenomena, Cell Phenomena, and Immunity

Abstract

Persisters are bacterial cells that survive antibiotic treatment without acquiring resistanceconferring mutations. Upon antibiotic removal, they form a population identical to the parental one, complicating treatment of infectious diseases. Persisters are multidrug-tolerant and form in response to stresses. Persisters can form in response to fluoroquinolone (FQ) treatment through the induction of the SOS response. FQ treatment produces DNA double strand breaks (DSBs), which induce the SOS response. TisB, part of a toxin-antitoxin (TA) module, is induced by the SOS response and causes persister formation by decreasing the proton motive force and ATP levels. Some persisters also form prior to antibiotic challenge. For example, the number of persisters increases with the rise of cell density during normal growth, probably due to the stress of nutrient limitation. We wonder if non-antibiotic environmental stresses cause persister formation. In this thesis, we investigated the influence of oxidative stress and heat shock on bacterial drug tolerance, respectively. Bacterial pathogens are routinely exposed to reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide (O2·) and hydroxyl radial (OH·) produced by the host immune system. Bactericidal antibiotics have been reported to generate ROS. These oxidants cause damage to cellular macromolecules and induce the oxidative stress response. By pretreating a growing Escherichia coli population with superoxide-producing paraquat, we found that tolerance to FQs increased significantly. This increase in tolerance is mainly due to the induction of the efflux pump AcrAB-TolC, a part of the soxRS regulon. The efflux pump AcrABTolC decreases the intracellular concentration of FQs, reducing the amount of DSBs. Consequently, more cells can survive by repairing the damage or reducing the ATP level by thePersisters are bacterial cells that survive antibiotic treatment without acquiring resistance-conferring mutations. Upon antibiotic removal, they form a population identical to the parental one, complicating treatment of infectious diseases. Persisters are multidrug-tolerant and form in response to stresses. Persisters can form in response to fluoroquinolone (FQ) treatment through the induction of the SOS response. FQ treatment produces DNA double strand breaks (DSBs), which induce the SOS response. TisB, part of a toxin-antitoxin (TA) module, is induced by the SOS response and causes persister formation by decreasing the proton motive force and ATP levels. Some persisters also form prior to antibiotic challenge. For example, the number of persisters increases with the rise of cell density during normal growth, probably due to the stress of nutrient limitation.

We wonder if non-antibiotic environmental stresses cause persister formation. In this thesis, we investigated the influence of oxidative stress and heat shock on bacterial drug tolerance, respectively.

Bacterial pathogens are routinely exposed to reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide (O2*) and hydroxyl radial (OH*) produced by the host immune system. Bactericidal antibiotics have been reported to generate ROS. These oxidants cause damage to cellular macromolecules and induce the oxidative stress response. By pretreating a growing Escherichia coli population with superoxide-producing paraquat, we found that tolerance to FQs increased significantly. This increase in tolerance is mainly due to the induction of the efflux pump AcrAB-TolC, a part of the soxRS regulon. The efflux pump AcrAB-TolC decreases the intracellular concentration of FQs, reducing the amount of DSBs. Consequently, more cells can survive by repairing the damage or reducing the ATP level by the expression of TisB through the induction of the SOS response. The effect of superoxide also contributes to the increased tolerance to a lesser extent. This finding demonstrates synergy between drug resistance and drug tolerance, thus improving bacterial survival against antibiotic treatment.

Bacteria endure temperature shifts upon host immune response like inflammation and fever, or during food processing. They respond to sharp increases in temperature by inducing over 100 heat shock proteins (HSPs). We found that heat shock response increased E. coli tolerance to ofloxacin. The screening of a single-gene knockout collection of HSPs resulted in the identification of potential candidate genes important for tolerance. These mutants, when pretreated with heat shock, demonstrated significantly less tolerance to ofloxacin compared to the wild type, indicating the role of these genes in drug tolerance.

Document Type

Dissertation

Rights Information

Copyright 2011

Rights Holder

Yanxia Wu


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