title: Staphylococcus aureus survival mechanisms from skin antimicrobials
Creators: name: Al-Dayel, Munirah
Publisher: name: University of Liverpool Repository
Year: 2015

description: S.aureus is highly adaptable to environmental conditions and has the ability to colonise and infect a range of tissues within the host. The ability to colonise skin requires survival mechanisms to counter an array of abiotic factors that includes epidermal and sebaceous skin lipids. This study sought to investigate the effects of cholesterol, an epidermal lipid produced in substantial quantities, on S. aureus growth and survival. Previous studies have reported that cholesterol addition reduces the growth inhibition of antimicrobial fatty acids (AFAs) and this phenomenon was investigated further to identify the underlying mechanism. The addition of ethanol-solubilised cholesterol to broth cultures of S. aureus increased bacterial survival in the presence of growth inhibitory levels of linoleic acid and the lipid sphingosine. This effect was confirmed in strains SH1000 and Newman. The pigmentation of S. aureus when grown in the presence of ethanol-solubilised cholesterol was greatly reduced. Initially this study focused on these effects being mediated by cholesterol, however ethanol concentration was not controlled effectively when designing the experiments and ethanol could also be the major mediator of pigmentation changes. It was initially hypothesised that cholesterol would affect S. aureus cell membrane properties since it is known to be incorporated when added extracellularly. From this hypothesis, studies were designed to examine factors controlling survival and pigmentation changes in response to ethanol-solubilised cholesterol. Separate screens of transposon libraries were performed to identify mutants that: i) produced pigment in the presence of ethanol-solubilised cholesterol; ii) did not show enhanced growth with ethanol-solubilised cholesterol supplementation in the presence of growth-inhibitory levels of linoleic acid. The majority of the transposon mutants identified and localised using arbitrary-primed PCR sequencing revealed insertions into genes previously associated with modulating activity of the accessory sigma factor σB. Subsequent experiments with ethanol controls indicated a clear solvent effect on pigment expression, confounding the previous hypothesis and contrary to published reports about the activation of σB. To investigate the effects of ethanol and ethanol-solubilised cholesterol on pigment expression and σB activity a series of qRT-PCR assays were established. In the presence of ethanol-solubilised cholesterol the expression of crtM was decreased, whereas, extracellular protease, aur and sspA expression were increased in the presence of 0.3 mM ethanol-solubilised cholesterol. However the effect of ethanol, as a solvent control, was substantial resulting in decreased transcription of sigB and crtM while correspondingly aur and sspA transcription were increased. These data from the study of ethanol and ethanol-solubilised cholesterol identify novel effects on the cell membrane of S. aureus that require further study to dissect the individual roles of each component and indicate that current literature reports of σB activity and regulation might be incomplete.