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BackgroundDaptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus.MethodsWhole genome sequencing was performed on a unique collection of isogenic, clinical (21 strains) and laboratory (12 strains) derived strains that had been exposed to daptomycin and developed daptomycin-nonsusceptibility. Electron microscopy (EM) and lipid membrane studies were performed on selected isolates.ResultsOn average, six coding region mutations were observed across the genome in the clinical daptomycin exposed strains, whereas only two mutations on average were seen in the laboratory exposed pairs. All daptomycin-nonsusceptible strains had a mutation in a phospholipid biosynthesis gene. This included mutations in the previously described mprF gene, but also in other phospholipid biosynthesis genes, including cardiolipin synthase (cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (pgsA). EM and lipid membrane composition analyses on two clinical pairs showed that the daptomycin-nonsusceptible strains had a thicker cell wall and an increase in membrane lysyl-phosphatidylglycerol.ConclusionPoint mutations in genes coding for membrane phospholipids are associated with the development of reduced susceptibility to daptomycin in S. aureus. Mutations in cls2 and pgsA appear to be new genetic mechanisms affecting daptomycin susceptibility in S. aureus.

We investigated the hypothesis that methicillin-resistant

Protein localization is crucial for cellular morphogenesis and intracellular signal transduction cascades. Here we describe an interaction between two membrane proteins expressed in different cells of the Bacillus subtilis sporangium, the mother cell protein SpoIIIAH and the forespore protein SpoIIQ. We used affinity chromatography, coimmunoprecipitation, and the yeast two-hybrid system to demonstrate that the extracellular domains of these proteins interact, tethering SpoIIIAH to the sporulation septum, and directing its assembly with SpoIIQ into helical arcs and foci around the forespore. We also demonstrate that this interaction can direct proteins made in the same cell to active division sites, as when SpoIIQ is made in the mother cell, it localizes to nascent septa in a SpoIIIAH-dependent manner. Both SpoIIIAH and SpoIIQ are necessary for activation of the second forespore-specific transcription factor ( G ) after engulfment, and we propose that the SpoIIIAH-SpoIIQ complex contributes to a morphological checkpoint coupling G activation to engulfment. In keeping with this hypothesis, SpoIIIAH localization depends on the first step of engulfment, septal thinning. The SpoIIQ-SpoIIIAH complex reaches from the mother cell cytoplasm to the forespore cytoplasm and is ideally positioned to govern the activity of engulfment-dependent transcription factors.

Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Cadependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR. Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations in mprF, vraSR contributes to DAP resistance in the present group of clinical strains. Staphylococcus aureus is the most common Gram-positive pathogen among skin and soft tissue infections (2). Methicillin resistance in S. aureus is mediated by the acquisition of a penicillin-binding protein (PBP), PBP 2a, which has decreased affinity for ␤-lactam antibiotics but can continue to cross-link the cell wall once the native PBPs (i.e., PBPs 1 to 4) have been inactivated (23). A distinctive feature for most methicillin-resistant S. aureus (MRSA) strains is the heterogeneous expression of resistance, characterized by a small proportion (Յ0.1%) of the population expressing a high level of homogeneous resistance while most of the other isolates in the population express resistance to 10 g/ml (12, 15, 43). Daptomycin (DAP) is a cyclic anionic lipopeptide antibiotic that is produced by Streptomyces roseosporus (3) and is approved for treatment of skin and skin structure infections as well as treatment of bacteremia and right-side endocarditis caused by MRSA (1). The mechanism of action involves disruption of cytoplasmic membrane function, resulting in depolarization and cell death due to disruption of critical metabolic functions, such as protein, DNA, and RNA synthesis (2).The incidence of DAP resistance in clinical isolates is very low, and resistant strains display small increases in MIC (2). The exact m...

We analyzed the emergence of daptomycin nonsusceptibility in a patient with persistent vancomycin-intermediate Staphylococcus aureus (VISA) bacteremia. The daptomycin-nonsusceptible VISA's cell wall demonstrated a reduction in muramic acid O-acetylation, a phenotypic parameter not previously reported for VISA; some isolates also contained a single point mutation in the mprF gene.

In Bacillus subtilis, many membrane proteins localize to the sporulation septum, where they play key roles in spore morphogenesis and cell-specific gene expression, but the mechanism for septal targeting is not well understood. SpoIIQ, a forespore-expressed protein, is involved in engulfment and forespore-specific gene expression. We find that SpoIIQ dynamically localizes to the sporulation septum, tracks the engulfing mother cell membrane, assembles into helical arcs around the forespore and is finally degraded. Retention of SpoIIQ in the septum requires one or more mother cell-expressed proteins. We also observed that any forespore-expressed membrane protein initially localizes to the septum and later spreads throughout the forespore membrane, suggesting that membrane protein insertion occurs at the forespore septal region. This possibility provides an attractive mechanism for how activation of mother cell-specific gene expression is restricted to adjacent sister cells, since direct insertion of the signaling protein SpoIIR into the septum would spatially restrict its activity. In keeping with this hypothesis, we find that SpoIIR localizes to the septum and is transiently expressed.

Staphylococcus aureus is the second leading cause of bacteremia worldwide and the most important pathogen in endovascular infections (1, 2). S. aureus has a particular propensity for developing multidrug resistance (e.g., methicillin-resistant S. aureus [MRSA] and vancomycin-intermediate S. aureus [VISA]), and serious infections with such strains result in enhanced attributable mortality (3).Since its FDA approval in 2003, daptomycin (DAP) has been utilized in many clinical settings, especially for recalcitrant MRSA infections, such as endocarditis (4-6). However, there have been a number of recently reported cases of clinical S. aureus strains that have evolved in vitro DAP resistance in the context of failing DAP treatment regimens, especially in endovascular syndromes. (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation [7][8][9][10][11][12].) One of the major features of DAP-resistant (DAP r ) S. aureus strains is the acquisition of gain-in-function mutations in a relatively restricted cadre of genes, especially in the mprF locus (13-18). The MprF protein is responsible for the synthesis and translocation (flipping) of the unique positively charged phospholipid (PL) lysyl-phosphotidylglycerol (L-PG) from the inner-to-outer cell membrane (CM) leaflet (18)(19)(20). Increases in L-PG synthesis and flipping usually result in augmented positive surface charge; many investigators have speculated that this event leads to a charge-repulsive milieu for cationic molecules, such as host defense peptides (HDPs) and calcium-complexed DAP (13,(19)(20)(21)(22)(23). Over the past several years, a number of laboratories, including ours, have linked the presence of singlenucleotide polymorphisms (SNPs) within the mprF locus to the DAP r phenotype (13,14, 21,(24)(25)(26). These SNPs have occurred throughout the mprF open reading frame (ORF), although there are clearly hot spots within this locus for those SNPs linked to DAP resistance (13,14,19, 23). Investigations of mprF SNPs associated with DAP resistance in S. aureus have principally emerged from studies that used only a few or individual isogenic DAP-susceptible (DAP s ) and DAP r strain pairs. To address this limitation, in the current study, we assessed (i) the frequencies and distribution of putative mprF gain-in-function SNPs using 22 DAP s /DAP r isogenic clinical MRSA strain pairs, (ii) the relation-