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Doctoral Dissertations
Date of Award
12-2018
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Microbiology
Major Professor
Elizabeth M. Fozo
Committee Members
Tessa Calhoun, Todd Reynolds, Erik Zinser
Abstract
Enterococcus faecalis is a Gram-positive commensal bacterium that resides in the human gastrointestinal tract. Unfortunately, E. faecalis can also cause infections in humans and is notoriously difficult to treat due to drug resistance. One treatment that is used to treat enterococcal infections is the cell membrane targeting lipopeptide antibiotic, daptomycin. However, daptomycin resistant strains of E. faecalis have been isolated. Studies aimed at understanding these resistant strains show that mutations in genes associated with membrane homeostasis are involved. E faecalis can also incorporate exogenous fatty acids from environments in which it thrives, bile (GI tract) and serum (wounds), which cause increased physiological tolerance to daptomycin. The host fatty acids, oleic acid (C18:1 cis 9) and linoleic acid (C18:2 cis 9, 12), that are prevalent in serum and bile, are the major factors that contribute to this induction of daptomycin tolerance. Within this work, I determined that the cis bond at the 9th carbon of oleic acid is critical for increased tolerance. Moreover, I found that when the carboxyl group of oleic acid or linoleic acid was replaced with an amide group, tolerance was lost. To assess if increased tolerance induced by these host fatty acids was a consequence of a membrane stress response, I examined a strain of E. faecalis which lacks the response regulator of the LiaFSR three-component system and concluded that host fatty acid induced tolerance was not mediated by LiaFSR. Finally, I investigated whether or not supplementation with host fatty acids was altering the membrane phospholipid composition leading to increased tolerance. After mass spectrometry analysis, I discovered alterations in the composition of the major phospholipids in E. faecalis. To test these alterations, I deleted genes responsible for production of lysyl-phosphatidylglycerol (mprF2) and cardiolipin (cls1 and cls2). After supplementation with oleic acid and linoleic acid, I still observed increased tolerance to daptomycin. However, long term exposure to daptomycin resulted in no recovery even after supplementation with host fatty acids. These data suggest that oleic acid and linoleic acid can induce lipid alterations, but alteration in the composition of L-PG and CL are not responsible for acute daptomycin tolerance.
Recommended Citation
Harp, John Robert, "Incorporation of host fatty acids promotes tolerance to membrane stressors by modifying the lipid content of Enterococcus faecalis. " PhD diss., University of Tennessee, 2018.
https://trace.tennessee.edu/utk_graddiss/5228