En Liv1023 (SH1000 mtlD::tet) and SH1000 in the presence of

En Liv1023 (SH1000 mtlD::tet) and SH1000 in the presence of a range of concentrations of NaCl, lauroyl sarcosine, SDS, dichlorophenyl and the human cathelicidin LL37 (Sigma). Liv1023 (SH1000 mtlD::tet) was observed to exhibit a lower MIC for H2O2 (1 mM) compared to SH1000 (4 mM) and Liv1024 (SH1000 mtlABFD::tet) (4 mM). The hydrophobicity and zeta potential of all of the strains was similar when tested using either hexadecane partitioning or measured using a zetasizer (Malvern, UK), respectively (data not shown). The levels of carotenoid in cell membranes were similarGC-MS was used to analyse cytoplasmic fractions from exponential growth phase cells. 131 unique metabolites were compared and chromatograms and mass spectra were evaluated as JWH-133 described previously [8,32] using the MSD ChemStation (Agilent, Palo Alto, CA, USA) and AMDIS (NIST, Gaithersburg, MD, USA) programs. The resulting data from triplicate samples (with 16574785 less than 10 variability) were analyzed using a t-test. Samples with greater than 2-fold variation (p,0.05) were analyzed using the MetPA enrichment pathway analysis web application (http://metpa.metabolomics.ca/) [45]. ND, not detectable. doi:10.1371/journal.pone.0067698.tS. aureus Mannitol Utilisation and SurvivalFigure 8. Virulence of mtlD in a murine infection. (A) Effect of WT SH1000 or Liv1023 (SH1000 mtlD::tet) on percentage change in weight of infected mice. There were no significant differences using Dunn’s test. (B) Effect of mutations of mtlD on cfu of S. aureus SH1000 in kidneys of infected mice. There were no significant differences using the Mann Whitney Test. doi:10.1371/journal.pone.0067698.gDiscussionThe intrinsic importance of S. aureus carriage and transmission in relation to disease and its hypothesized link with virulence [39] requires that determinants are identified and characterised that promote survival in its primary niche and during its transient residence on human skin. From the study of gene mutants S. aureus defence from AFAs is achieved via a variety of surface components (IsdA, WTA, SasF) and regulation of peptidoglycan biosynthesis (VraRS, VraE), where a reduction in hydrophobicity to minimize access of the AFA to the membrane explains the contribution of several of these components to survival [6,7,19]. In addition, the arginine deiminase pathway increases survival [6], where its various contributions to metabolic versatility and its potential to modify local pH could explain its role. Determining that an Mtl-1-P-dehydrogenase mutant, but not an mtlABFD transport operon mutant, has greatly reduced survival from AFAs implicates the accumulation of Mtl-1-P as being the causative factor. As the most abundant natural hexitol, Mtl is a carbon source for staphylococci and the inducible oxidation of Mtl-1-P generates fructose-6-P for entry into the EmbdenMeyerhoff and hexosemonophosphate glycolytic pathways [38,40]. All strains of S. aureus accumulate Mtl, despite not all being capable of using it for metabolism during aerobic growth. In S. aureus the cellular accumulation of Mtl was identified in resting cells when incubated in glucose or cultured in media without added 23977191 carbohydrate [38]. Mtl accumulation was proposed to enhance metabolic versatility in S. aureus, however its mechanistic role is incompletely understood [41]. Following MedChemExpress NT-157 stress, such as after exposure to AFAs, utilisation of the pathway for Mtl conversion to fructose-6-P would regenerate NADH, thereby alleviating the pressure upon.En Liv1023 (SH1000 mtlD::tet) and SH1000 in the presence of a range of concentrations of NaCl, lauroyl sarcosine, SDS, dichlorophenyl and the human cathelicidin LL37 (Sigma). Liv1023 (SH1000 mtlD::tet) was observed to exhibit a lower MIC for H2O2 (1 mM) compared to SH1000 (4 mM) and Liv1024 (SH1000 mtlABFD::tet) (4 mM). The hydrophobicity and zeta potential of all of the strains was similar when tested using either hexadecane partitioning or measured using a zetasizer (Malvern, UK), respectively (data not shown). The levels of carotenoid in cell membranes were similarGC-MS was used to analyse cytoplasmic fractions from exponential growth phase cells. 131 unique metabolites were compared and chromatograms and mass spectra were evaluated as described previously [8,32] using the MSD ChemStation (Agilent, Palo Alto, CA, USA) and AMDIS (NIST, Gaithersburg, MD, USA) programs. The resulting data from triplicate samples (with 16574785 less than 10 variability) were analyzed using a t-test. Samples with greater than 2-fold variation (p,0.05) were analyzed using the MetPA enrichment pathway analysis web application (http://metpa.metabolomics.ca/) [45]. ND, not detectable. doi:10.1371/journal.pone.0067698.tS. aureus Mannitol Utilisation and SurvivalFigure 8. Virulence of mtlD in a murine infection. (A) Effect of WT SH1000 or Liv1023 (SH1000 mtlD::tet) on percentage change in weight of infected mice. There were no significant differences using Dunn’s test. (B) Effect of mutations of mtlD on cfu of S. aureus SH1000 in kidneys of infected mice. There were no significant differences using the Mann Whitney Test. doi:10.1371/journal.pone.0067698.gDiscussionThe intrinsic importance of S. aureus carriage and transmission in relation to disease and its hypothesized link with virulence [39] requires that determinants are identified and characterised that promote survival in its primary niche and during its transient residence on human skin. From the study of gene mutants S. aureus defence from AFAs is achieved via a variety of surface components (IsdA, WTA, SasF) and regulation of peptidoglycan biosynthesis (VraRS, VraE), where a reduction in hydrophobicity to minimize access of the AFA to the membrane explains the contribution of several of these components to survival [6,7,19]. In addition, the arginine deiminase pathway increases survival [6], where its various contributions to metabolic versatility and its potential to modify local pH could explain its role. Determining that an Mtl-1-P-dehydrogenase mutant, but not an mtlABFD transport operon mutant, has greatly reduced survival from AFAs implicates the accumulation of Mtl-1-P as being the causative factor. As the most abundant natural hexitol, Mtl is a carbon source for staphylococci and the inducible oxidation of Mtl-1-P generates fructose-6-P for entry into the EmbdenMeyerhoff and hexosemonophosphate glycolytic pathways [38,40]. All strains of S. aureus accumulate Mtl, despite not all being capable of using it for metabolism during aerobic growth. In S. aureus the cellular accumulation of Mtl was identified in resting cells when incubated in glucose or cultured in media without added 23977191 carbohydrate [38]. Mtl accumulation was proposed to enhance metabolic versatility in S. aureus, however its mechanistic role is incompletely understood [41]. Following stress, such as after exposure to AFAs, utilisation of the pathway for Mtl conversion to fructose-6-P would regenerate NADH, thereby alleviating the pressure upon.