MOLECULAR MECHANISMS OF ANTIMICROBIAL RESISTANCE IN MULTI-DRUG RESISTANT ENTEROCOCCI

Antibiotic resistance is a major global public health threat. Enterococci are recalcitrant, nosocomial pathogens that can be intrinsically resistant to valuable antibiotics, like beta-lactams, or evolve resistance to all existing antimicrobials. The LiaFSR system regulates resistance to cell membrane (CM) stressors like daptomycin (DAP), a front-line drug for multi-drug resistant infections. DAP resistance (DAP-R) in E. faecalis is mediated by CM phospholipid alterations. Emergence of DAP-R often leads to b-lactam resensitization, a phenomenon called the seesaw effect. The molecular mechanism of DAP-R and the seesaw effect are unknown. Here we show that LiaX is a surface exposed protein whose C-terminal domain inhibits LiaFSR in DAP-S strains. DAP-R strains highly secrete, both LiaX and the N-terminal sentinel domain alone, into the environment. The N-terminus binds DAP or innate immune antimicrobial peptides (AMPs) triggering activation of LiaFSR, CM remodeling, and enhanced virulence in vivo due to innate immune resistance. LiaX bridges CM adaptation and cell wall homeostasis through interactions with penicillin-biding proteins (PBPs). LiaX also bridges antimicrobial resistance and pathogenesis. The Fsr quorum sensing system regulates both virulence and processing of the N-terminal sentinel domain of LiaX. In DAP-R strains, the seesaw effect is a consequence of CM remodeling which causes mislocalization of select PBPs, like Pbp5 which is essential for beta-lactam resistance, increasing their beta-lactam affinity. Select class A PBPs that likely retain functionality and compensate are hypersusceptible to cephalosporins, thus contributing to the seesaw effect. This study shows that innovative therapeutic targeting of stress adaptation can restore utility of existing antimicrobials and enhance innate immune function. Dissecting mechanisms allows us to be able to deploy targeted DAP and beta-lactam combination therapy in the future to exploit the seesaw effect. We further translated our molecular findings to develop a diagnostic to utilize LiaX as a biomarker of DAP nonsusceptibility in E. faecalis and E. faecium, potentially circumventing limitations of existing platforms. This dissertation identifies unprecedented mechanisms of antimicrobial resistance, providing us key insight into bacterial evolution and shows that these insights can be applied to directly advance patient care