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

Abstract

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 7th and 8th 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.

Two-Component Systems of S. aureus: Signaling and Sensing Mechanisms

“…The GraR target gene vraG encodes a membrane permease, while vraF encodes an ATPase to provide energy for efflux. Interestingly, a vraG mutant phenocopied a graS mutant with respect to CAMP sensing [ 58 ]. Native VraG interacted with GraS to restrict its kinase activity [ 58 ].…”

“…Interestingly, a vraG mutant phenocopied a graS mutant with respect to CAMP sensing [ 58 ]. Native VraG interacted with GraS to restrict its kinase activity [ 58 ]. VraG contains a single 200-residue EL located between the seventh and eighth transmembrane segments.…”

“…Specific lysine residue(s) on the VraG-EL presumably reduces the sensing of cognate CAMPs by GraS-EL. Thus, VraG, more than an efflux pump, facilitates GraS-mediated sensing of HDPs [ 58 ].…”

“…Recent work has expanded the sensory capabilities of GraS to include the sensing of acidic pH [ 54 , 55 , 59 ], and GraS activation at low pH is mandatory for survival within macrophages [ 58 ]. Interestingly, pH-dependent modulation of GraS activity seems to be independent of the Gra-EL implicated in CAMP sensing [ 58 ]. Acidic pH reduces the charge on polar lipid head groups, which presumably impacts GraS activity.…”

New insights into the resistance mechanism for the BceAB-type transporter SaNsrFP

“…This was described i.e. for the GraRS-VraFG system 37 in S. aureus and also for several TCS-ABC transporters in B. subtilis (BceRS-AB, YxdJK-LM and YvcPQ-RS) 35 , 38 . The direct interaction between the BceAB transporter and the BceS histidine kinase was demonstrated in B. subtilis 36 .…”