The Staphylococcus aureus Two-Component Regulatory System, GraRS, Senses and Confers Resistance to Selected Cationic Antimicrobial Peptides

Abstract

The two-component regulatory system, GraRS, appears to be involved in staphylococcal responses to cationic antimicrobial peptides (CAPs). However, the mechanism(s) by which GraRS is induced, regulated, and modulated remain undefined. In this study, we used two well-characterized MRSA strains (Mu50 and COL) and their respective mutants of graR and vraG (encoding the ABC transporter-dependent efflux pump immediately downstream of graRS), and show that (i) the expression of two key determinants of net positive surface charge (mprF and dlt) is dependent on the cotranscription of both graR and vraG, (ii) reduced expression of mprF and dlt in graR mutants was phenotypically associated with reduced surface-positive charge, (iii) this net reduction in surface-positive charge in graR and vraG mutants, in turn, correlated with enhanced killing by a range of CAPs of diverse structure and origin, including those from mammalian platelets (tPMPs) and neutrophils (hNP-1) and from bacteria (polymyxin B), and (iv) the synthesis and translocation of membrane lysyl-phosphatidylglycerol (an mprF-dependent function) was substantially lower in graR and vraG mutants than in parental strains. Importantly, the inducibility of mprF and dlt transcription via the graRS-vraFG pathway was selective, with induction by sublethal exposure to the CAPs, RP-1 (platelets), and polymyxin B, but not by other cationic molecules (hNP-1, vancomycin, gentamicin, or calcium-daptomycin). Although graR regulates expression of vraG, the expression of graR was codependent on an intact downstream vraG locus. Collectively, these data support an important role of the graRS and vraFG loci in the sensing of and response to specific CAPs involved in innate host defenses.

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

“…106 Interestingly, the GraSR locus encodes a third subunit (GraX) that is predicted to form a part of the sensing mechanism. 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 ).…”

Low pH Enhances the Action of Maximin H5 against Staphylococcus aureus and Helps Mediate Lysylated Phosphatidylglycerol-Induced Resistance

“…It has recently been shown that increasing levels of Lys-PG and other lysylated phospholipids promote the rigidification and stabilisation of bacterial membranes through the reduction of electrostatic repulsion between anionic lipid head-groups, thereby inhibiting the ability of AMPs to penetrate these membranes 35,36 . Moreover, it is generally accepted that a contribution to the protective mechanism provided by Lys-PG derives from a 'charge effect' whereby the positively charged head-group carried by the lipid is able to reduce the net negative charge of host bacterial membranes thus inhibiting the binding of cationic AMPs to these membranes 27,28,31,[33][34][35][36][37][38] . It therefore seems likely that the contribution made by 'charge effects' to this protective mechanism is further enhanced by low pH given that the Lys-PG head-group is predominantly cationic under acid conditions but becomes more zwitterionic in character as pH is increased 29,93 .…”

“…According to this mechanism of resistance, cationic AMPs activate a sensor histidine kinase (ApsS) of the ApsSR (GraSR) regulon, which leads to the up-regulated expression of the virulence factor mprF. The protein product of this gene, mprF, is a Lys-PG synthase that catalyses the transfer of lysine from lysyl-tRNA to the PG head-group and flipping of the resulting Lys-PG to the outer leaflet of the S. aureus CM 23,[31][32][33][34] . The presence of the lipid in the CM of S.…”

“…aureus attenuates a number of membrane associated properties that inhibit the ability of cationic AMPs to bind and exert their membranolytic action against these membranes 27,28,31,[33][34][35][36][37][38] . This effect in enhanced by low pH conditions, when the production of Lys-PG is upregulated and where levels of the Lys-PG as high as 80 % of total membrane lipid have been reported for the organism 29,[39][40][41][42] .…”

“…In general, the role of Lys-PG in mediating resistance to cationic AMPs in S. aureus has been well characterised 27,31,33,35,[43][44][45] but there is limited information on what if any role this mechanism of resistance has against anionic AMPs [46][47][48] . Accordingly, here, we have characterised the action of MH5 against S. aureus and investigated the role of Lys-PG its activity.…”