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VanRS and CroRS cross-talk revealed by coevolutionary modeling regulates antibiotic resistance in VanA-type vancomycin-resistant Enterococcus faecalis

Enterococcus faecalis is an opportunistic pathogen that can cause bacteremia and endocarditis. Previous studies have shown that concurrent treatment with cephalosporin and vancomycin antibiotics exhibit synergy in vancomycin-resistant E. faecalis to render the bacterium susceptible to antibiotic treatment whereas treatment with each antibiotic separately was not successful. Proteins responsible for mediating vancomycin and cephalosporin resistance are classified as two-component systems (TCS). TCS consist of a histidine kinase that phosphorylates a response regulator after environmental activation. These signaling networks have been shown to exhibit cross-talk interactions, and through direct coupling analysis, we identify encoded specificity between vancomycin resistance TCS, which are horizontally acquired, and cephalosporin resistance TCS, which are endogenous to E. faecalis. To verify cross-talk between these pathways is responsible for vancomycin and cephalosporin synergy, we use RNA-Seq to identify differentially expressed genes in VanA- and VanB-type vancomycin resistant enterococci after treatment with the cephalosporin antibiotic, ceftriaxone, and also with vancomycin. We find that cross-talk between VanSA and CroR in strain HIP11704 may be responsible for synergy, demonstrating that horizontally acquired TCS can have large impacts on pre-existing signaling networks. The presence of encoded specificity between exogenous TCS and endogenous TCS show that the systems co-evolve, and cross-talk between these systems may be exploited to engineer genetic elements that disrupt antibiotic resistance TCS pathways. Author Summary Bacteria may transmit genetic elements to other bacteria through the process known as horizontal gene transfer. In some enterococci, vancomycin resistance genes are acquired this way. Proteins encoded within the bacterial genome can interact with proteins acquired through horizontal gene transfer. The interaction that occurs between proteins VanSA and VanRA is known to mediate vancomycin antibiotic resistance in VanA-type vancomycin resistant enterococci (VRE), and the interaction between proteins CroS and CroR is an important pathway in cephalosporin antibiotic resistance. We show that the VanSA, which is obtained through horizontal gene transfer, inhibits CroR under treatment with antibiotics vancomycin and ceftriaxone. This interaction is responsible for the observed synergy between vancomycin and ceftriaxone in VanA-type VREs. These findings demonstrate how horizontally acquired genes may produce proteins that interrupt known protein interactions, including antibiotic resistance signaling pathways in bacteria. Furthermore, the specific mechanism found for VanA-type VREs provides a basis for engineering of horizontally acquired proteins that disrupt antibiotic resistance pathways.