Staphylococcal Responses to Skin Surface Lipid Squalene Xizhang Zhao Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy September 2017 Acknowledgement I would like to express my deepest appreciation to my supervisor Mal Horsburgh who was abundantly helpful and offered invaluable assistance, support and guidance. Without his knowledge and assistance this study would not have been complete. I would like to thank all members in Horsburgh’s lab for their unfailing support and assistance, but especially Josephine Moran who was never tired of training me in the world of bioinformatics. I would like to expression my gratitude to all those who have helped me in the lab, with special thanks to lab manager Paul Loughnane who was always willing to help me and maintained this cracking place to work! I would like to thank all my friends and family for the moral and emotional support in my life. Your encouragement is very important for me. Abstract The human skin surface is covered with a lipid film that represents the actual interface between the viable epidermal layers and outer environment and is crucial for our understanding of the colonisation of bacteria. The relationship between staphylococci and the abundant skin lipid squalene, which comprises about 12% of total skin surface lipids, remains poorly understood. This study therefore aimed to comparatively investigate the effects of the lipid squalene upon S. aureus and S. epidermidis. Here, it was determined that culture of S. aureus with squalene dramatically reduced expression of staphyloxanthin, its eponymous golden pigment . Culture of cells with squalene lowered the survival of S. aureus, but not S. epidermidis, to both H2O2 and nisin, while squalene mediated enhanced survival of S. epidermidis to LL- 37, but did not alter S. aureus survival. The transcriptional response of both species to squalene challenge was investigated using RNA-Seq and the proteome of S. aureus at late exponential phase of growth with squalene was studied using quantitative methods. While major similarities in the response of both species to squalene were observed with large changes to cellular pathway gene expression, reduced carotenoid (crt) operon expression was shown to be mediated at the level of transcription. Unexpectedly, challenge with squalene caused derepression of all the Fur-iron regulated uptake transporter genes in both S. aureus and S. epidermidis. This induction was accompanied by significantly decreased cellular iron. The means by which squalene causes iron starvation was not determined. Similar to squalene, ethanol was found to reduce pigment expression in S. aureus and major similarities in the crt operon and overall transcriptional profiles of expression were revealed after challenge. The experimental evolution of S. aureus with a sub-bactericidal concentration of ethanol and selection for increased growth yield identified three SNPs, with one SNP potentially implicating altered cell wall meso-diaminopimelic acid biosynthesis in enhanced survival. This study revealed that squalene has potential to play a role in colonisation of staphylococci by decreasing pigment expression, modulating resistance to skin antimicrobials and through starvation for iron. The mechanistic basis for these phenomena requires further study. Contents Chapter 1 Introduction...................................................................................................1 1.1 Staphylococcus...................................................................................................................1 1.2 Methicillin-resistant S. aureus (MRSA).....................................................................2 1.3 Global regulators of S. aureus.......................................................................................3 1.3.1 SigB...............................................................................................................................................................3 1.3.2 SaeRS............................................................................................................................................................4 1.3.3 Agr.................................................................................................................................................................6 1.4 The structure of human skin........................................................................................9 1.5 Skin surface lipids (SSLs)............................................................................................11 1.6 Squalene............................................................................................................................17 1.7 Staphylococcal skin survival......................................................................................15 1.7.1 Adhesion..................................................................................................................................................15 1.7.2 Osmotic stress resistance.................................................................................................................16 1.7.3 Acid resistance......................................................................................................................................18 1.7.4 Antimicrobial peptides and proteins resistance....................................................................20 1.7.5 Resistance to antimicrobial lipids.................................................................................................21 1.7.6 Competition on human skin............................................................................................................25 1.8 Staphylococcal disease.................................................................................................28 1.8.1 Atopic dermatitis..................................................................................................................................28 18..2 Abscess formation...............................................................................................................................29 1.9 Thesis aims......................................................................................................................32 Chapter 2 Methods and Materials.............................................................................33 2.1 Bacterial strains and growth conditions................................................................33 2.2 Growth curves.................................................................................................................35 2.3 H2O2, sapienic acid, nisin and LL-37 survival assays.........................................35 2.4 Staphyloxanthin expression and extraction.........................................................36 2.5 Agarose gel electrophoresis.......................................................................................36 2.6 RNA sequencing..............................................................................................................37 2.6.1 Notes..........................................................................................................................................................37 2.6.2 Growth and preparation of cells for RNA extraction............................................................37 2.6.3 Lysis of cells for RNA extraction....................................................................................................37 2.6.4 RNA extraction......................................................................................................................................37 2.6.5 DNase treatment of RNA...................................................................................................................38 2.6.6 RNA quality control.............................................................................................................................38 2.6.7 RNA library preparation...................................................................................................................38 2.6.8 RNA sequencing differential expression analysis..................................................................39 2.6.9 COG analysis...........................................................................................................................................39 2.7 Experimental evolution...............................................................................................40 2.7.1 Evolution passaging............................................................................................................................40 2.7.2 DNA extraction.....................................................................................................................................40 2.7.3 DNA Quality Control..........................................................................................................................40 2.7.4 Pooled DNA samples..........................................................................................................................41 2.7.5 DNA library preparation..................................................................................................................41 2.7.6 Genome assembly...............................................................................................................................41 2.7.7 SNP analysis..........................................................................................................................