The extracellular loop of the membrane permease VraG interacts with GraS to sense cationic antimicrobial peptides in Staphylococcus aureus

Host defense proteins (HDPs), aka defensins, are a key part of the innate immune system that functions by inserting into the bacterial membranes to form pores to kill invading and colonizing microorganisms. To ensure survival, microorganism such as S. aureus has developed survival strategies to sense and respond to HDPs. One key strategy in S. aureus is a two-component system (TCS) called GraRS coupled to an efflux pump that consists of a membrane permease VraG and an ATPase VraF, analogous to the BceRS-BceAB system of Bacillus subtilis but with distinct differences. While the 9 negatively charged amino acid extracellular loop of the membrane sensor GraS has been shown to be involved in sensing, the major question is how such a small loop can sense diverse HDPs. Mutation analysis in this study divulged that the vraG mutant phenocopied the graS mutant with respect to reduced activation of downstream effector mprF, reduction in surface positive charge and enhanced 2 hr. killing with LL-37 as compared with the parental MRSA strain JE2. In silico analysis revealed VraG contains a single 200-residue extracellular loop (EL) situated between the 7(th) and 8(th) transmembrane segments (out of 10). Remarkably, deletion of EL in VraG enhanced mprF expression, augmented surface positive charge and improved survival in LL-37 vs. parent JE2. As the EL of VraG is rich in lysine residues (16%), in contrast to a preponderance of negatively charged aspartic acid residues (3 out of 9) in the EL of GraS, we divulged the role of charge interaction by showing that K380 in the EL of VraG is an important residue that likely interacts with GraS to interfere with GraS-mediated signaling. Bacterial two-hybrid analysis also supported the interaction of EL of VraG with the EL of GraS. Collectively, we demonstrated an interesting facet of efflux pumps whereby the membrane permease disrupts HDP signaling by inhibiting GraS sensing that involves charged residues in the EL of VraG. Author summary TCS have been used by bacteria to sense and respond to environmental stimuli. In S. aureus, the bacterial defense against host HDPs is mounted from GraRS where GraS is the membrane sensor comprising two transmembrane segments framing a small extracellular loop, and a cytoplasmic tail. We have shown previously that the critical sensing moiety for HDPs in S. aureus is the 9-residue extracellular loop of the sensor GraS that contains 3 negatively charged aspartic acid residues critical to HDP sensing. However, the 9-residue loop is predicted to be too flexible to confer proper conformation for the specificity response to divergent HDPs in S. aureus. The genes encoding GraRS lies adjacent to an efflux system encoded by vraFG where VraG is the membrane permease and VraF is the ATPase. We showed that VraG contributes to sensing of HDPs mediated by GraS. More specifically, the 200-residue lysine rich extracellular loop of VraG interferes with GraS sensing of HDP via charge interaction mediated in part by K380. This finding represents another interesting facet of efflux pump whereby the membrane permease interferes with HDP sensing by inhibiting GraS signaling.

Automated summary

Staphylococcus aureus has developed survival strategies to respond to host defense proteins by utilizing a two-component system and an efflux pump, where the lysine-rich extracellular loop of VraG interferes with GraS sensing and signaling. This finding highlights an interesting facet of how efflux pumps disrupt host defense protein signaling in bacterial pathogens.