GraXSR Proteins Interact with the VraFG ABC Transporter To Form a Five-Component System Required for Cationic Antimicrobial Peptide Sensing and Resistance in Staphylococcus aureus

Abstract

The GraSR two-component system (TCS) controls cationic antimicrobial peptide (CAMP) resistance in Staphylococcus aureus through the synthesis of enzymes that increase bacterial cell surface positive charges, by D-alanylation of teichoic acids and lysylination of phosphatidylglycerol, leading to electrostatic repulsion of CAMPs. The GraS histidine kinase belongs to the "intramembrane-sensing kinases" subfamily, with a structure featuring a short amino-terminal sensing domain, and two transmembrane helices separated only by a short loop, thought to be buried in the cytoplasmic membrane. The GraSR TCS is in fact a multicomponent system, requiring at least one accessory protein, GraX, in order to function, which, as we show here, acts by signaling through the GraS kinase. The graXRS genes are located immediately upstream from genes encoding an ABC transporter, vraFG, whose expression is controlled by GraSR. We demonstrated that the VraFG transporter does not act as a detoxification module, as it cannot confer resistance when produced on its own, but instead plays an essential role by sensing the presence of CAMPs and signaling through GraS to activate GraR-dependent transcription. A bacterial two-hybrid approach, designed to identify interactions between the GraXSR and VraFG proteins, was carried out in order to understand how they act in detecting and signaling the presence of CAMPs. We identified many interactions between these protein pairs, notably between the GraS kinase and both GraX and the VraG permease, indicating the existence of an original five-component system involved in CAMP sensing and signal transduction to promote S. aureus resistance.

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances

“…102-104 Together with previous observations showing that expression of mprF and dltABCD is dependent on a functional VraFG, new studies have shown that GraX is the central component of a “5-component system” for sensing of CAMPs (Fig. 7, 107 ). The GraSR sensing system requires both GraX and the VraGF transporter to function.…”

“…CAMPs are sensed by VraFG and the signal is transduced to GraS through a mechanism that is likely to involve interaction between VraG and GraS. Activation of the GraSR system leads to increased transcription of the dltABCD operon and the mprF gene, leading to cell wall remodeling and CAMP resistance (figure adapted from [107]). …”

Sensor–response regulator interactions in a cross-regulated signal transduction network

“…Analogous results were obtained with the analogous NarP fusions (not shown). Thus, fusion design is important for monitoring sensor-response regulator BACTH interaction output, as noted also for other systems, such as VraS-VraR (McCallum et al, 2011) and GraSGraR (Falord et al, 2012).…”

Regulation of bacterial virulence gene expression by cell envelope stress responses

“…In fact, it is even more complex because the VraFG transporter might be involved in signal sensing rather than detoxification, leading to this being described as a 5-component system. 176,179 As mentioned above the staphylococcal ApsXSR/GraXSR system is a ESR that regulates virulence genes because it controls the dlt operon and mprF, decreasing cell surface negative charge and promoting resistance to CAMPs (Fig. 4).…”

“…133,[174][175][176] For example, the BceRS system of B. subtilis responds to bacitracin and promotes resistance to it. 177,178 However, a BceRS-like system in S. aureus and S. epidermidis known as GraSR (glycopeptide resistance associated) or ApsSR (antimicrobial peptide-sensing), respectively, has several unusual features.…”