The two-component system CroRS regulates isoprenoid flux to mediate antimicrobial tolerance in the bacterial pathogen Enterococcus faecalis

Antimicrobial tolerance is the ability of a microbial population to survive, but not proliferate, during antimicrobial exposure. Significantly, it has been shown to precede the development of bona fide antimicrobial resistance. We have previously identified the two-component system CroRS as a critical regulator of tolerance to antimicrobials like teixobactin in the bacterial pathogen Enterococcus faecalis. To understand the molecular mechanism of this tolerance, we carried out RNA-seq analyses in the E. faecalis wild-type and isogenic croRS mutant to determine the teixobactin-induced CroRS regulon. We identified a 132 gene CroRS regulon and show CroRS upregulates expression of all major components of the enterococcal cell envelope in response to teixobactin challenge. To gain further insight into the function of this regulon we isolated and characterized croRS mutants recovered for wild-type growth and tolerance. We show introduction of a single stop codon in a heptaprenyl diphosphate synthase ( hppS ), a key enzyme in the synthesis of the quinone electron carrier demethylmenaquinone (DMK), is sufficient to rescue loss of cell envelope integrity in the croRS deletion strain. Based on these findings, we propose a model where CroRS acts as a gate-keeper of isoprenoid biosynthesis, mediating flux of isoprenoids needed for cell wall synthesis (undecaprenyl pyrophosphate; UPP) and respiration (DMK) to maintain cell wall homeostasis upon antimicrobial challenge. Dysregulation of this flux in the absence of croRS leads to a loss of tolerance, which is rescued by loss of function mutations in HppS, allowing an increase in isoprenoid flow to UPP and subsequently cell wall synthesis.