Date of Award

Fall 12-2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

School

Biological, Environmental, and Earth Sciences

Committee Chair

Janet R. Donaldson

Committee Chair School

Biological, Environmental, and Earth Sciences

Committee Member 2

Fengwei Bai

Committee Member 2 School

Biological, Environmental, and Earth Sciences

Committee Member 3

Alex Flynt

Committee Member 3 School

Biological, Environmental, and Earth Sciences

Committee Member 4

Mohammed Elasri

Committee Member 4 School

Biological, Environmental, and Earth Sciences

Committee Member 5

Dmitri Mavrodi

Committee Member 5 School

Biological, Environmental, and Earth Sciences

Abstract

Listeria monocytogenes is a Gram-positive, facultative intracellular food-borne pathogen that causes the disease listeriosis. In order to establish an infection, L. monocytogenes must survive multiple stressors encountered within the gastrointestinal tract, including alterations in pH, bile, salt, and oxygen availability. This dissertation focused on understanding the stress response of L. monocytogenes to bile. Bile acts as a bactericidal agent by disrupting the membrane integrity and causing instability to macromolecules like DNA. Thus, a bacterium must be able to maintain its membrane architecture, composition and integrity.

Often times, bacteria will modulate their fatty acid composition in the membrane to cope with environmental changes. Our research through fatty acid methyl ester analyses showed that the fatty acid composition of the cell membrane of L. monocytogenes was altered after exposure to bile, suggesting that L. monocytogenes incorporates exogenous fatty acids from bile. Additionally, incorporation of exogenous fatty acids was subsequently found to increase bile survival in the bile sensitive strain HCC23 under aerobic conditions whereas improved the survival of the moderately bile resistant strain, 10403s under anaerobic conditions. Thus, suggesting oxygen availability plays a role in influencing survival. The incorporation of fatty acids was also found to increase the fluidity of the cell membrane following exposure to bile. Together, these data indicate that bile sensitive strains of L. monocytogenes may incorporate exogenous fatty acids from the host into their cell membrane as an attempt to survive membrane damage, such as that induced by bile but in turn lose intestinal fitness.

It is known that bile causes oxidative damage to bacterial cells. However, it is not known if oxidative stress occurs under physiologically relevant anaerobic conditions. Our results showed that bile exposure alters the redox potential of L. monocytogenes by increasing the membrane potential for the bile resistant strain F2365 and by reducing the NADH:NAD+ in F2365 and 10403S under anaerobic conditions. Though the decrease in NADH:NAD+ may suggest an oxidative environment, no signs of oxidative stress were observed as there was lack of lipid and protein oxidation under anaerobic conditions. This data correlates with our previous proteomics data. Further research is needed to understand the kind of damage induced by bile in Listeria monocytogenes under anaerobic conditions.

Bile is also known to cause DNA damage. We wanted to see if L. monocytogenes’ ability to repair bile induced DNA damage is what aids in bile resistance. Since recA is the inducer of SOS response, we analyzed the expression of various DNA repair and bile resistant genes in the L. monocytogenes strain, EGD-e and the mutant of EGDe lacking recA. We also analyzed the ability of EGD-e and the recA mutant to survive bile stress under aerobic and anaerobic conditions. Our research suggests that there may be a recA independent DNA repair mechanism involved in the bile induced DNA repair in L. monocytogenes strain EGD-e.

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