Investigating the Survival Response of Staphylococcus aureus to the Antimicrobial Lipid Sphingosine Yiyun Chen Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy November 2017 Acknowledgement I would like to express my deepest thanks to my supervisor Mal Horsburgh for his invaluable assistance, support and guidance, as well as his smile and patience, throughout my PhD studies. I would like to thank all members in Horsburgh’s group for their kindly support and assistance, especially Josephine Moran who trained me a lot with bioinformatics and data analysis. I would like to thank all members of LabH who created such a great environment for working and studying, with special thanks to lab manager Paul Loughnane who was always around to help. I would like to thank my family for their unconditional support, encouragement and love in my life. Abstract The human skin surface is covered with a lipid film that forms the interface between the viable epidermal layers and outer environment, and influences the colonisation of bacteria. Hydrolysis of the epidermal lipid ceramide via ceramidase yields sphingosine, an antimicrobial lipid that is able to kill staphylococci rapidly. Studies of patients with atopic dermatitis have indicated a correlation between levels of sphingosine and S. aureus colonisation of human skin, yet the contribution of sphingosine to the composition of the human skin microbiome and microbiota colonisation is unclear. This study investigates the effect of the host epidermal antimicrobial lipid D-sphingosine on S. aureus and S. epidermidis, with a focus on discovering components that contribute to survival of the former. The methods RNA-Seq and qPCR were used to dissect the transcriptional responses of S. aureus Newman and S. epidermidis Tü3298 after challenge with D-sphingosine. S. aureus displayed a large set of differentially expressed genes (1331), which contrasted with a smaller set for S. epidermidis (340). There was a clearly defined stimulon that was common between the species that included pathways for energy maintenance, amino acid and ion transport and metabolism, and protein repair. Previously reported transcriptomic responses to multiple cell wall and membrane-targeting antimicrobials shared commonalities with the response to sphingosine that supports its reported mode of action at the cell surface with membrane damage. The VraSR cell wall stress stimulon was upregulated in both species. Each species displayed pronounced upregulation of a putative phosphate-specific ABC transporter system encoded by the pstS-pstCAB operons. Experiments identified a contribution of this transport system to sphingosine survival and phosphate supplementation was shown to markedly increase sphingosine survival. Previous studies by the Horsburgh group identified experimentally evolved S. aureus strains with high levels of D-sphingosine resistance after serial passage with increasing concentrations of the lipid. SNPs associated with increased resistance were determined by genome resequencing and some of these were localised to the farER genes encoding a FarR-regulated FarE (mmpL) lipid efflux transporter. An isogenic farR SNP mutant of S. aureus was generated using molecular genetics approaches and showed a 10-fold higher MIC for D-sphingosine compared with the wild type Newman strain, indicating that the purported FarER fatty acid efflux system may have the capacity to mediate transport of sphingosine. The study findings of a link between sphingosine challenge and phosphate metabolism indicates future research potential to study host spingosine-1-phosphate in staphylococcal lifecycles, during health and disease. Contents Chapter 1: Introduction………………………………………………………………………..1 1.1 Staphylococci………………………………………………………………………………..1 1.1.1 Staphylococcus aureus………………………………………………………..……1 1.1.2 Staphylococcus epidermidis……………………………………………………...3 1.2 Human skin……………………………………………………………………………….….4 1.2.1 Structure of human epidermis………………………………………………....4 1.2.2 Human skin microbiome………………………………………………………….6 1.3 Staphylococcal skin survival………………………………………………………...9 1.3.1 Colonisation and persistence…………………………………………………....9 1.3.2 Osmotic stress and resistance…………………………………………………..17 1.3.3 Acid stress and resistance………………………………………………………..19 1.3.4 Antimicrobial fatty acids and resistance…………………………………...21 1.3.5 Antimicrobial peptides and resistance……………………………………...23 1.3.6 Competitive exclusion……………………………………………………………...25 1.3.7 Two-component systems……………………………………………………........27 1.4 Sphingosine and staphylococci……………………………………………………..31 1.4.1 Atopic dermatitis: sphingosine defence against S. aureus colonisation……………………………………………………………………………………..31 1.4.2 Sphingoid bases and antimicrobial activities…………………………….33 1.5 Thesis aims…………………………………………………………………………………....35 Chapter 2: Methods and Materials………………………………………………………36 2.1 Bacterial strains and culture………………………………………………………..36 2.2 Survival tests………………………………………………………………………………..37 2.2.1 Growth curves ………………………………………………………………………..37 2.2.2 Growth in phosphate deficient media………………………………………37 2.2.3 Minimum inhibitory concentration (MIC) assay……………………....39 2.2.4 Peroxide survival assay …………………………………………………………39 2.2.5 Sphingosine survival assay………………………………………………...…....40 2.3 Construction of allelic replacement variants……………………………....40 2.3.1 Extraction of plasmid DNA………………………………………………………40 2.3.2 PCR…………………………………………………………………………………...........40 2.3.3 Restriction digests and ligation………………………………………………..40 2.3.4 Transformation of chemically competent E. coli………………………..41 2.3.5 Preparation of electrocompetent S. aureus…………………………….....41 2.3.6 Electroporation of S. aureus……………………………………………………..41 2.3.7 Phage transduction……………………………………………………………........42 2.3.8 Nebraska transposon library mutants………………………………………42 2.4 Agarose gel electrophoresis……………………………………………………........43 2.5 RNA sequencing…………………………………………………………………………….44 2.5.1 Bacteria growth and challenge condition…………………………………..44 2.5.2 Cell lysis…………………………………………………………………………………..44 2.5.3 RNA extraction………………………………………………………………………...44 2.5.4 DNase treatment……………………………………………………………………...45 2.5.5 RNA quality control………………………………………………………………….45 2.5.6 RNA library preparation…………………………………………………………..45 2.5.7 RNA sequencing differential expression analysis………………………46 2.5.8 COG analysis……………………………………………………………………………46 2.6 qPCR………………………………………………………………………………………………47 2.6.1 First-strand cDNA synthesis………………………………………………........47 2.6.2 Primer design…………………………………………………………………………47 2.6.3 qPCR conditions……………………………………………………………………..49 2.7 Staphyloxanthin expression and extraction………………………………...49 Chapter 3: Comparative transcriptomics of the staphylococcal survival response to the epidermal lipid sphingosine…………………….50 3.1 Introduction…………………………………………………………………………………50 3.1.1 Transcriptomics technologies…………………………………………………50 3.1.2 Analysis tools…………………………………………………………………………51 3.2 Chapter aims………………………………………………………………………………..53 3.3 Results………………………………………………………………………………………….54 3.3.1 D-Sphingosine challenge titration…………………………………………...54 3.3.2 RNA quality control………………………………………………………………...57 3.3.3 Comparison of S. aureus Newman and S. epidermidis Tü3298 transcriptional responses to D-sphingosine challenge……………………...60 3.3.4 Comparison of DE gene COGs…………………………………………………..61 3.3.5 Comparison of DE metabolic pathways……………………………………66 3.3.6 Comparison of sphingosine transcriptome to other S. aureus transcriptome data sets…………………………………………………………………..84 3.3.7 Comparative transcriptomic responses shared between S. aureus and S. epidermidis ……………………………………………………………………..........94 3.3.8 Quantitative PCR validation…………………………………………………..100 3.4 Discussion…………………………………………………………………………………..102 3.4.1 Virulence factors………………………………………………………………..…102 3.4.2 Cell wall stress……………………………………………………………………...104 3.4.3 Membrane disruption…………………………………………………………...105 3.4.4 Capsule biosynthesis…………………………………………………………….107 3.4.5 Oxidative stress …………………………………………………………………....107 3.4.6 General stress response of both S. aureus and S. epidermidis……108 Chapter 4: Characterisation of DE genes in the Transcriptome of S. aureus Challenged with D-Sphingosine………………………………………..….110 4.1 Introduction……………………………………………………………………………….110 4.1.1 Staphyloxanthin biosynthesis…………………………………………….....110 4.1.2 Phosphate-specific transport (Pst) system………………………….…112 4.1.3 PhoRP alkaline phosphatase synthesis regulatory system………113 4.1.4 Heme-sensor system (HssRS) and heme regulated transporter (HrtAB) ………………………………………………………………………………………..113 4.2 Chapter aims …………………………………………………………………………...…115 4.3 Results………………………………………………………………………..……………….116 4.3.1 Phosphate specific transport (pst) system…………………………..….119 4.3.2 Construction of a pst operon mutant …………………………………...…122 4.3.3 Characterising transposon library mutants for survival phenotypes……………………………………………………………………………………125 4.3.4 DE comparison of various S. aureus strains by qPCR……………..…128 4.3.5 Sphingosine-induced changes in pigment gene expression and H2O2 survival……………………………………………………………………………...……….…131 4.4 Discussion…………………………………………………………………..…………….…135 4.4.1 Inorganic phosphate (Pi) and S. aureus survival to D- sphingosine………………………………………………………………...……………...…136 Chapter 5: The individual contributions of farE/R genes and SNPs that increase S. aureus survival to D-sphingosine………………………..…141 5.1 Introduction……………………………………………………………….…………….…141 5.1.1 Mechanism of action of sphingosines and AFAs………………………141 5.1.2 FarRE