Modulating Activity of Vancomycin and Daptomycin on the Expression of Autolysis Cell-Wall Turnover and Membrane Charge Genes in hVISA and VISA Strains

et al. (2012) Modulating Activity of Vancomycin and Daptomycin on the Expression of Autolysis Cell-Wall Turnover and Membrane Charge Genes in hVISA and VISA Strains. PLoS ONE 7(1): e29573. doi:10.1371/journal.pone.0029573 Modulating Activity of Vancomycin and Daptomycin on the Expression of Autolysis Cell-Wall Turnover and Membrane Charge Genes in hVISA and VISA Strains Viviana Cafiso 0 Taschia Bertuccio 0 Daniela Spina 0 Simona Purrello 0 Floriana Campanile 0 Cinzia Di 0 Pietro 0 Michele Purrello 0 Stefania Stefani 0 Sarah K. Highlander, Baylor College of Medicine, United States of America 0 1 Unit of Microbiology, Department of Bio-Medical Sciences University of Catania , Catania , Italy , 2 Unit of Genome and Molecular Complex Systems BioMedicine G Sichel, Department Gian Filippo Ingrassia , Catania , Italy Glycopeptides are still the gold standard to treat MRSA (Methicillin Resistant Staphylococcus aureus) infections, but their widespread use has led to vancomycin-reduced susceptibility [heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA) and Vancomycin-Intermediate-Staphylococcus aureus (VISA)], in which different genetic loci (regulatory, autolytic, cell-wall turnover and cell-envelope positive charge genes) are involved. In addition, reduced susceptibility to vancomycin can influence the development of resistance to daptomycin. Although the phenotypic and molecular changes of hVISA/VISA have been the focus of different papers, the molecular mechanisms responsible for these different phenotypes and for the vancomycin and daptomycin cross-resistance are not clearly understood. The aim of our study was to investigate, by real time RT-PCR, the relative quantitative expression of genes involved in autolysis (atl-lytM), cell-wall turnover (sceD), membrane charges (mprF-dltA) and regulatory mechanisms (agr-locus-graRS-walKR), in hVISA and VISA cultured with or without vancomycin and daptomycin, in order to better understand the molecular basis of vancomycinreduced susceptibility and the modulating activity of vancomycin and daptomycin on the expression of genes implicated in their reduced susceptibility mechanisms. Our results show that hVISA and VISA present common features that distinguish them from Vancomycin-Susceptible Staphylococcus aureus (VSSA), responsible for the intermediate glycopeptide resistance i.e. an increased cell-wall turnover, an increased positive cell-wall charge responsible for a repulsion mechanism towards vancomycin and daptomycin, and reduced agr-functionality. Indeed, VISA emerges from hVISA when VISA acquires a reduced autolysis caused by a down-regulation of autolysin genes, atl/lytM, and a reduction of the net negative cellenvelope charge via dltA over-expression. Vancomycin and daptomycin, acting in a similar manner in hVISA and VISA, can influence their cross-resistance mechanisms promoting VISA behavior in hVISA and enhancing the cell-wall pathways responsible for the intermediate vancomycin resistance in VISA. Daptomycin can also induce a charge repulsion mechanism both in hVISA and VISA increasing the activity of the mprF. - Funding: This study was funded by grants from the Italian Minister of Health (MIUR PRIN 20087SM5HM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Emerging resistance to glycopeptides in Methicillin-Resistant Staphylococcus aureus (MRSA) poses a great threat to antimicrobial chemotherapy worldwide. Together with the recent discovery, in 2002, of the first clinical isolate of fully Vancomycin-Resistant S.aureus (VRSA) with VAN MIC .128 mg/L [1], numerous other isolates of homogeneous Vancomycin-Intermediate Staphylococcus aureus (VISA) or heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA) have been isolated worldwide. In these strains, this reduced susceptibility has been attributed to various cell-wall abnormalities, evolving in a multi-step fashion. These abnormalities include accumulation of D-Ala-D-Ala targets due to decreased cross-linking of peptidoglycan [2], the increased proportion of non-amidated muropeptides [3], and decreased alanylation of teichoic acids [4]. VISA does not emerge from vancomycin-susceptible MRSA but from hVISA, as was previously demonstrated: hVISA spontaneously produces VISA cells within its cell population at a frequency of 1026 or above [5] that is the same frequency of hVISA onset from a susceptible background [6]. Recent publications have added to the knowledge of the complex changes taking place in Staphylococci evolving towards the reduced glycopeptide susceptibility phenomenon. A reduced content of Lysyl-phosphatidylglycerol (LPG), synthesized by fmtC (also named mprF) encoded protein, has recently been demonstrated affecting some cationic antimicrobial agents including vancomycin (VAN), but also the Ca2+- daptomycin (DAP) [7]. In addition, a possible role of the autolytic enzymes has also been suggested, among which the major autolysin atl [8], the peptidoglycan hydrolase lytM [9], and the transglycolase sceD [10] variously involved in the physiology of the cell-wall as mediators of cell division, autolysis and peptidoglycan turnover. Moreover, different regulatory loci have been found to be involved in vancomycin intermediate resistance such as agr-locus (accessory gene regulator locus) encoding d-hemolysin and also considered as its functionality indicator; GraRS and WalKR, both encoding Two-Component Regulatory Systems TCRSs, involved in the regulation of mprF/dltA and atl/lytM/sceD, respectively [1120]. GraRS has been involved in the VISA phenotype, because these lead to increased autolytic rates and a more negative net surface charge, which increase the sensitivity to cationic antimicrobial peptides [15]. Recently, Dubrac et al. emphasized the importance of the walKR system in cell-wall metabolism in S.aureus [19]. Moreover, Jansen et al. reported that walKR was highly up-regulated due to an insertion mutation in the walKR promoter in a VISA clinical isolate, suggesting that the increment of vancomycin resistance was mediated by activation of this system [17]. On the contrary, in a recent paper, Hiramatsu et al. reported a deletion mutation in walKR [deletion of 3 nucleotides (CAA) from the position 1111 to 1113] and a truncating mutation in a proteoliytic regulatory gene, clpC, responsible for the raised vancomycin resistance in a laboratory derivate strain, but they did not find any significant changes in the expression of walKR in any of the resistant mutants studied. Thus, the cause of raised resistance due to the walKR mutation still remains unknown [21]. Moreover, Delaune et al. recently reported on the effect of walKR on cell morphology, showing that walKR depletion could raise the cell-wall thickness of S.aureus [20], but this regulatory pathway toward cell-wa (...truncated)