Site-Specific Mutation of the Sensor Kinase GraS in Staphylococcus aureus Alters the Adaptive Response to Distinct Cationic Antimicrobial Peptides

Abstract

The Staphylococcus aureus two-component regulatory system, GraRS, is involved in resistance to killing by distinct host defense cationic antimicrobial peptides (HD-CAPs). It is believed to regulate downstream target genes such as mprF and dltABCD to modify the S. aureus surface charge. However, the detailed mechanism(s) by which the histidine kinase, GraS, senses specific HD-CAPs is not well defined. Here, we studied a well-characterized clinical methicillin-resistant S. aureus (MRSA) strain (MW2), its isogenic graS deletion mutant (⌬graS strain), a nonameric extracellular loop mutant (⌬EL strain), and four residuespecific ⌬EL mutants (D37A, P39A, P39S, and D35G D37G D41G strains). The ⌬graS and ⌬EL strains were unable to induce mprF and dltA expression and, in turn, demonstrated significantly increased susceptibilities to daptomycin, polymyxin B, and two prototypical HD-CAPs (hNP-1 and RP-1). Further, P39A, P39S, and D35G-D37G-D41G ⌬EL mutations correlated with moderate increases in HD-CAP susceptibility. Reductions of mprF and dltA induction by PMB were also found in the ⌬EL mutants, suggesting these residues are pivotal to appropriate activation of the GraS sensor kinase. Importantly, a synthetic exogenous soluble EL mimic of GraS protected the parental MW2 strain against hNP-1-and RP-1-mediated killing, suggesting a direct interaction of the EL with HD-CAPs in GraS activation. In vivo, the ⌬graS and ⌬EL strains displayed dramatic reductions in achieved target tissue MRSA counts in an endocarditis model. Taken together, our results provide new insights into potential roles of GraS in S. aureus sensing of HD-CAPs to induce adaptive survival responses to these molecules.

Frequency and Distribution of Single-Nucleotide Polymorphisms within mprF in Methicillin-Resistant Staphylococcus aureus Clinical Isolates and Their Role in Cross-Resistance to Daptomycin and Host Defense Antimicrobial Peptides

“…The most common mprF SNP observed in the study strains resulted in either an L826F or T345A/K/I substitution, constituting 27% and 18%, respectively. These SNPs are well-known mprF hot spot mutations linked to DAP resistance (see below) (14,19,22,23,25,38,41,42). Although the C8, C23, and C35 strains had DAP MICs of 3 to 4 mg/liter, the mprF ORFs and promoter sequences of these strains were identical to those of the each respective DAP s parental strain (data not shown).…”

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

“…A recent study shed light on the molecular mechanism used by GraS to sense certain CAMPs with more sensitivity than others. 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.…”

The GraS Sensor in Staphylococcus aureus Mediates Resistance to Host Defense Peptides Differing in Mechanisms of Action

“…MW2 (USA400) is a prototypic and well-characterized clinical isolate with a known genome that has been demonstrated to be virulent in multiple animal models. graS mutant strains were generated by in-frame deletion of target genes by allelic replacement using the temperature-sensitive plasmid pMAD as previously detailed (7). Organisms were cultured in brain heart infusion broth (BHI) (Becton Dickinson) at 37°C with shaking overnight (ϳ16 h), subcultured under identical conditions for 3 h to logarithmic phase, harvested by centrifugation, washed, and resuspended to the appropriate CFU (culture confirmed) by spectrophotometry for each specific assay.…”

“…RP-1 was synthesized, purified, and authenticated as previously detailed (9). Each study peptide has demonstrated in vitro antimicrobial activity against S. aureus (3,7,8,10). sEL sensor domain.…”

“…Recently, we showed that the two-component regulatory system (TCRS) GraSR plays a key role in S. aureus survival in the face of HDPs (6)(7)(8). Also known to be a component of the antibiotic peptide sensor (APS) (9,10), GraSR upregulates adaptive resistance genes such as mprF and dltA, which encode proteins that modulate net surface charge and influence the composition of the S. aureus envelope (11,12).…”