Regulation of mprF in Daptomycin-Nonsusceptible Staphylococcus aureus Strains

Abstract

We used a well-characterized isogenic set of clinical bloodstream Staphylococcus aureus strains to study (i) regulation of mprF-mediated phosphatidylglycerol lysinylation in the contexts of in vitro daptomycin (DAP) nonsuceptibility and (ii) the role of mprF mutation in endovascular virulence. We observed a correlation between increased expression of a mutant mprF gene and reduced in vitro DAP susceptibility. There were no detectable fitness differences between strains in experimental infective endocarditis.

Several groups have reported an association between an increase in daptomycin (DAP) MICs and single-point mutations in the Staphylococcus aureus mprF gene acquired during in vitro or in vivo exposure to DAP (1, 4, 9). Since mprF contributes substantially to the staphylococcal net positive surface charge by lysinylation of membrane phosphatidylglycerol (PG), a gain-in-function mutation was postulated to impart DAP nonsusceptibility via a charge-repulsive mechanism (3, 7, 9, 12). However, detailed genotypic studies to define the transcriptional profiles and regulation of the mprF gene in the context of DAP nonsusceptibility have not been reported.

In the present study, we used a well-defined and previously characterized series of S. aureus isolates from a patient with persistent endocarditis (3, 10). The initial isolate (strain 616), the first strain to exhibit increases in DAP MICs (strain 629, termed the “transitional strain”), and two later isolates with DAP MICs within the nonsusceptible range (strains 701 and 703) were included in this investigation (3). The MICs of this strain set to DAP, as determined by standard Etest, were 0.5, 0.75, 2, and 2 μg/ml for strains 616, 629, 701, and 703, respectively (3).

Sequencing of the mprF open reading frames revealed a single-point mutation (C to A) at position 884 in the two DAP-nonsusceptible strains (701 and 703), resulting in an amino acid substitution at position 295 from serine-to-leucine (S295L) (data not shown). In contrast, analyses of the mprF promoter region showed no difference in sequence between DAP-susceptible and DAP-nonsusceptible strains (data not shown).

As shown in Fig. 1, Northern blot analysis revealed increased mprF transcripts in the two DAP-nonsusceptible strains (701 and 703) during stationary growth (12 h of incubation) compared to the DAP-susceptible (616) strain. This increased transcription could not be explained by altered mprF mRNA stability (half-lives) during the stationary phase (data not shown).

FIG. 1.

Northern blot analyses of mprF transcription. Total cellular RNA samples from strain 616 (lane 1), 629 (lane 2), 701 (lane 3), and 703 (lane 4) cells grown in Trypticase soy broth media were isolated at 2, 6, and 12 h postinoculation, as described previously (16, 17). Five micrograms of each RNA sample was separated in a 1% (wt/vol) agarose-formaldehyde gel, transferred to a nylon membrane, and hybridized to mprF-specific digoxigenin (DIG)-labeled probe synthesized using a PCR-based DIG probe synthesis kit (Roche) with primers mprF-F (5′-GTAGTAATCACATTGTATCGGGAGT-3′) and mprF-R (5′-GATGCATCGAAAACATGGAATAC-3′).

We next determined whether DAP-nonsusceptible strains are more or less fit than DAP-susceptible strains in vivo. One measurement of organism fitness is its ability to colonize, propagate, and disseminate within relevant animal models. Since many of the DAP-nonsusceptible clinical isolates have emerged during DAP therapy of vascular infections (2, 10, 11), we chose the infective endocarditis (IE) model (13, 14) to assess in vivo virulence. In agreement with previous observations (14, 15), as shown in Fig. 2, S. aureus cell densities achieved in vegetations were significantly higher than those in kidneys and spleens for each strain at 24 h postinfection (P < 0.05). Interestingly, both the DAP-susceptible and DAP-nonsusceptible strains were equivalent in their capabilities to induce experimental IE, proliferate within the vegetative lesion, and then disseminate hematogenously to distant target organs (Fig. 2).

FIG. 2.

In vivo virulence of DAP-susceptible and DAP-nonsusceptible strains. IE was produced using the DAP-susceptible (strain 616) or DAP-nonsusceptible (strain 703) strain, and microbiologic evaluation was performed at 24 h after infection as described previously (13, 14). IE was produced by intravenous injection of the selected S. aureus isolate (2 × 10⁴ CFU/animal) at 48 h postcatheterization.

Our prior data strongly suggested that DAP nonsusceptibility was associated with MprF gain in function, especially enhanced flippase activity (increasing the amount of lysyl-PG translocated to the outer cell membrane leaflets), rather than increased synthase function of this protein (3). This paradigm is compatible with the enhanced net positive surface charge and reduced DAP binding in the DAP-nonsusceptible strains previously documented and supported a charge repulsion mechanism for this strain set (3, 6, 8, 12). Importantly, Ernst et al. have localized the putative synthase domain of MprF to the hydrophilic C terminus plus the last six transmembrane helices of its C terminus, while the translocase domain appears to involve at least the first eight or perhaps all of the transmembrane helices of the N terminus (C. Ernst, P. Staubitz, G. Hornig, D. Kraus, and A. Peschel, presented at the International Symposium on Staphylococci and Staphylococcal Infections, Cairns, Australia, 2008). Based on this mapping and the location of the single nucleotide polymorphism (SNP) we observed in the present study, the amino acid substitution we identified likely resides within the translocase domain of mprF and accounts for an mprF gain in function, as reflected by increased lysyl-PG flipping in our DAP-nonsusceptible mutant strains.

Our Northern blot data, in combination with the sequencing data, indicate that DAP-nonsusceptible strains, 701 and 703, express more net mprF transcripts [in its mutated form, MprF(S295L)] over a standard in vitro growth cycle compared to the parental strain, 616. Recent studies have shown that neither exposure to DAP nor host defense cationic peptides from mammalian polymorphonuclear leukocytes or platelets (hNP-1 or RP-1, respectively) (8) induced expression of mprF in strains 616 and 703 (data not shown). Thus, it appears that DAP-nonsusceptible S. aureus strains with mprF SNPs and basal mprF gain-in-function phenotypes do not require additional mprF induction to affect nonsusceptibility to DAP.

Current studies are in progress to further examine the relationship of mprF SNPs and DAP resistance, as well as to define the genetic regulatory pathway or pathways that influence DAP resistance. Of note, Li et al. have recently shown that the aps (graRS) system in S. aureus positively regulates mprF expression via a cationic sensor function (5). The potential involvement of this latter locus in the mprF gain-in-function expression in our DAP-resistant mutants is under active investigation. Finally, the equivalent fitness of our strain set in vivo emphasizes the role of DAP treatment in divulging the clinical impacts of DAP nonsusceptibility.