Low-Level Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein 1 In Vitro Associated with qacA Gene Carriage Is Independent of Multidrug Efflux Pump Activity

Abstract

ABSTRACT Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus , the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1 r ) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1 r and (ii) tPMP-1 r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1 r qacA- bearing strains, the parental strain and its isogenic qacB - and qacC -containing strains were tPMP-1 susceptible (tPMP-1 s ). Efflux pump inhibition by cyanide m -chlorophenylhydrazone abrogated Et r , but not tPMP-1 r , in the qacA- bearing strain. In synergy assays, exposure of the qacA -bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA -bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA -bearing strain exhibited greater membrane fluidity than that of the parental, qacB -, or qacC -bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1 r phenotype. These data suggest that qacA -encoded tPMP-1 r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux.