Staphylococcus Aureus Survival Mechanisms from Skin Antimicrobials

Staphylococcus aureus survival mechanisms from skin antimicrobials Thesis submitted for the degree of Doctor of Philosophy by Munirah Aldayel April 2015 Declaration I declare that this thesis entitled Staphylococcus aureus survival mechanisms from skin antimicrobial is my own work, and that neither the whole nor part has been submitted to another university. Munirah Aldayel April 2015 i Abstract 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 B. 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 B. To investigate the effects of ethanol and ethanol-solubilised cholesterol on pigment expression and B 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 B activity and regulation might be incomplete. ii Acknowledgement There have been many late nights and early mornings, and after all the work I can finally conclude that the thesis is finished. Through all of this there have been several helpful people I would like to thank for their support and encourage. My deepest sincere gratitude goes first of all to Almighty ALLAH. I would like to thank my supervisor, Dr. Mal Horsburgh, who accepted me as one of his research group and has steered me in the right direction and given me great support throughout the work process. I would also like to thank members of LabH who have come and gone over the years. Especially to Dave Rooks who assisted me at the beginning of my PhD and Marta Veses for helping me all the time in the lab especially with Q- RT-PCR and the most permanent of them all Paul Loughnane. I would also like to thank the many other members behind the project. The most important members of them, all my family, my parents and to my husband who has been very supportive and helpful and encouraged me every step of the way and also my daughter who has missed me all the time during my studying. I would finally like to thank the people who have support and making me who I am today: Thank You all. iii Table of Content Declaration ...........................................................................................................................i Abstract ............................................................................................................................... ii Acknowledgement .......................................................................................................... iii Table of Content ............................................................................................................... iv List of Tables .................................................................................................................. viii List of Figures ................................................................................................................... ix Abbreviations ................................................................................................................ xiii Units .................................................................................................................................... xv CHAPTER 1 .......................................................................................................................... 1 1. General Introduction ............................................................................ 1 1.1 The staphylococci: human and animal colonisers ............................. 1 1.2 Staphylococcus aureus. .................................................................... 3 1.2.1. Virulence factors (determinants) of S. aureus ............................... 6 1.2.2. Regulation the production of S. aureus virulence factors ............ 11 1.2.2.1. The accessory gene regulator (agr) locus ................................ 13 1.2.2.2. The staphylococcal accessory regulator (sar) locus ................ 16 1.2.2.3. S. aureus exoprotein expression (sae) locus ........................... 17 1.2.3. Diseases and infections caused by S. aureus ............................ 17 1.2.4. Antimicrobial chemotherapy of S. aureus infections ................ 20 1.3. The human skin ............................................................................... 21 1.3.1. Development and structure ........................................................... 22 1.3.2. Chemical features making skin a niche for bacterial colonisation ... 26 1.3.3. The lipid matrix of human skin ........................................................ 27 1- Composition and structural aspects of the matrix ................................. 27 2- Skin antimicrobial lipids (SAML), and their mode of action. ................. 30 3- Skin antimicrobial peptides (AMPs) ...................................................... 31 4- Bacterial defence mechanisms against skin AMPs and fatty acids. ..... 36 5- Regulation of S. aureus antimicrobial peptide defence ........................ 39 6- Bacterial competition ............................................................................ 41 7- Staphylococcal virulence/ the staphyloxanthin and membrane fluidity. 43 8- Osmotic stress and stress resistance in S. aureus .............................. 45 9- Regulation of stress resistance / SigB (accessory sigma factor) ......... 47 1.3.4 The rationale, objectives, and aims of this Thesis project ........ 49 CHAPTER 2: ...................................................................................................................... 50 2. Materials and Methods ....................................................................... 50 2.1. Growth Media and Antibiotics ........................................................ 50 2.2. Buffers stock solutions ................................................................... 51 iv 2.3. SDS-PAGE gel formulations and construction ............................. 52 Resolving gel components: ........................................................................................ 52 Stacking gel components: ..................................................................... 53 2.4. Staphylococcus aureus strains .......................................................................... 55 2.4.1 Bacterial Culture ............................................................................ 55 2.5 Methods ...................................................................................................................... 58 2.5.1 Determination of bacterial cell density ........................................ 58 2.5.1.1 Spectrophotometry (OD600) .......................................................... 58 2.5.1.2 Colony Forming Unit (CFU) .......................................................... 58 2.6 Minimum Inhibitory Concentration Test (MIC) ............................... 58 2.7 Staphyloxanthin extraction and assay ..........................................

Load more