Perturbing fatty acid metabolism in Enterococcus faecalis disrupts responses to exogenous fatty acids and the antibiotic daptomycin

Enterococcus faecalis is an opportunistic pathogen. In the host, it is exposed to fatty acids which impact cellular physiology and induce tolerance to the antibiotic daptomycin. To determine the requirements for induction of daptomycin tolerance, I examined the impacts of blocking de novo fatty acid synthesis or protein synthesis and removing cell wall. I observed that removal of the cell wall induced daptomycin tolerance, indicating that peptidoglycan is necessary for daptomycin to function. As specific exogenous fatty acids induce protection against daptomycin in E. faecalis, I also opted to examine whether incorporation of these free fatty acids was necessary to induce drug tolerance. To do this, I deleted the genes predicted to encode fatty acid binding proteins (FakBs). I found that combined deletion of specific fakB genes caused altered physiology, including distorted morphology, increased levels of free fatty acids, and altered growth kinetics in the presence of exogenous fatty acids. This led me to investigate whether a thioesterase, TesS, which cleaves fatty acids from acyl-ACP, was causing a buildup of free fatty acids within the fakB deletion strains. Deletion of tesS in the fakB deletion strains reverted free fatty acid levels to wild-type levels. Additionally upon deletion of tesS, I was able to delete all four fakB homologs, generating a quintuple deletion strain. I followed up on the physiology of this strain and found that the growth kinetics and free fatty acid levels associated with deletion of the fakB genes were reverted to the wild-type phenotype. Further, I observed that the quintuple deletion strain could not grow in the presence of a de novo fatty acid biosynthesis inhibitor, even when supplemented with oleic acid. These data indicated that the quintuple deletion strain had a reduced ability to incorporate exogenous fatty acids. Additionally, deletion of fakB genes led to increased basal daptomycin tolerance, yet exogenous fatty acids were still able to induce daptomycin protection. These data indicated that the fakB genes play a role in normal cellular physiology, and that incorporation of fatty acids into membrane phospholipids is likely not the driver for fatty acid-mediated daptomycin tolerance