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

Abstract

MprF is responsible for the lysinylation of phosphatidylglycerol (PG) to synthesize the positively charged phospholipid (PL) species, lysyl-PG (L-PG). It has been proposed that the single-nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) are associated with a gain-in-function phenotype in terms of daptomycin resistance in Staphylococcus aureus. (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation.) Using 22 daptomycin-susceptible (DAP(s))/daptomycin-resistant (DAP(r)) clinical methicillin-resistant S. aureus (MRSA) strain pairs, we assessed (i) the frequencies and distribution of putative mprF gain-in-function SNPs, (ii) the relationships of the SNPs to both daptomycin resistance and cross-resistance to the prototypical endovascular host defense peptide (HDP) thrombin-induced platelet microbicidal protein (tPMP), and (iii) the impact of mprF SNPs on positive surface charge phenotype and modifications of membrane PL profiles. Most of the mprF SNPs identified in our DAP(r) strains were clustered within the two MprF loci, (i) the central bifunctional domain and (ii) the C-terminal synthase domain. Moreover, we were able to correlate the presence and location of mprF SNPs in DAP(r) strains with HDP cross-resistance, positive surface charge, and L-PG profiles. Although DAP(r) strains with mprF SNPs in the bifunctional domain showed higher resistance to tPMPs than DAP(r) strains with SNPs in the synthase domain, this relationship was not observed in positive surface charge assays. These results demonstrated that both charge-mediated and -unrelated mechanisms are involved in DAP resistance and HDP cross-resistance in S. aureus.

FIG 1

Location of the point mutations within the MprF protein. The multiple peptide resistance factor (MprF) of S. aureus is a bifunctional enzyme that synthesizes positively charged L-PG and facilitates L-PG translocation from the inner to the outer leaflet of the CM. MprF consists of a C-terminal L-PG synthase domain, an N-terminal L-PG translocase (flippase) domain, and a central bifunctional domain bridging the synthase and translocase domains. The predicted MprF topology was modified from previous publications (13, 19).

FIG 2

Binding of positively charged cytochrome c to whole S. aureus cells. The graph shows percentage of cytochrome c bound after 15 min of incubation with S. aureus cells at room temperature. The less cytochrome c bound, the more positively charged the bacterial surface. The data represent the means (± SD) from three independent experiments. Each DAP^s parental S. aureus strain was normalized to 100% (e.g., bar graph of DAP^r strain C2 is compared to its DAP^s parental strain, set at 100%). *, P < 0.01 versus respective DAP^s parental strains.