Infect Immun. 2021 Jul 6:IAI0034721. doi: 10.1128/IAI.00347-21. Online ahead of print.
GraS is a membrane sensor in S. aureus that induces mprF and dltABCD expression to alter the surface positive charge upon exposure to cationic human defense peptides (HDPs). The sensing domain of GraS likely resides in the 9-residue extracellular loop (EL). In this study, we assessed a hospital-acquired (HA)-MRSA strain (COL) for the specific role of two distinct EL mutations: F38G (bulk) and D/35/37/41K (charged inversion). Activation of mprF by polymyxin B (PMB) was reduced in the D35/37/41K mutant vs the D35/37/41G mutant, correlating with reduced surface positive charge; in contrast, these effects were less prominent in the F38G mutant, although still lower than the parent. These data indicated that both electrostatic charge and steric bulk of the EL of GraS influence induction of genes impacting HDP resistance. Using mprF expression as a readout, we confirmed GraS signaling was pH-dependent, increasing as pH was lowered (pH 7.5 ≫ 5.5). In contrast to PMB activation, reduction of mprF was comparable at pH 5.5 between the P38G and D35/37/41K point mutants, indicating a likely mechanistic divergence between GraS activation by acidic pH vs. cationic peptides. Survival assays in human blood and purified PMNs revealed lower survival of the D35/37/41K mutant vs. the F38G mutant, with both lower than the parent. Virulence studies in the rabbit endocarditis model mirrored whole blood and PMN killing assay data above. Collectively, these data confirmed the importance of specific residues within the EL of GraS in conferring essential bacterial responses for MRSA survival in infections.