Analysis of the Bactericidal Activity of Naive Rabbit Serum Against Staphylococcus Aureus
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Analysis of the Bactericidal Activity of Naive Rabbit Serum against Staphylococcus aureus by Beatriz González Muñoz Molecular Biotechnology Engineer, MSci (Universidad de Chile) A thesis submitted for the degree of Doctor of Philosophy November 2015 Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN Summary Staphylococcus aureus is a commensal bacterium that can also act as an opportunistic pathogen, causing a wide range of diseases in humans and in economically important livestock such as cows and rabbits. S. aureus quickly develops resistance against antibiotics and also evades the immune system of the host. As current treatments are difficult and expensive, new antibiotics, vaccines and the use of elements of the immune system are currently being studied as novel control approaches. A novel bactericidal mechanism able to kill S. aureus present in naive rabbit serum (NRS) was previously discovered in our laboratory. The bactericidal activity of the NRS had been characterized and the importance of the wall teichoic acids (WTA) on the bacteria for susceptibility was identified previously, but the mechanism involved in the bacterial killing remained unknown. In this study the role of the WTA as the bacterial receptor that interacts with the ‘killing factor’ present in the NRS was established. The importance of WTA modifications were also determined in pathogen resistance to the NRS killing mechanism. Use of bacterial cell wall material as an affinity matrix led to the identification of secreted phospholipase A2 (sPLA2) as a potential part of the bactericidal mechanism. The use of a range of inhibitors and specific antibodies confirmed sPLA2 as part of the killing mechanism. Purified sPLA2 from a number of species was tested and cobra sPLA2 (cvPLA2) was able to kill S. aureus in buffer but led to bacterial growth when added to NRS. This suggests that cvPLA2 interacts with rabbit serum components and this results in inactivation of both cvPLA2 and the bactericidal activity of the NRS. Annexin A1, a known regulator of PLA2, was identified through mass spectrometry as a serum protein also bound to the cell wall affinity matrix. This protein enhanced the bactericidal activity of cvPLA2 against S. aureus by direct protein-protein interaction. A model of NRS staphylococcal activity is presented. ii Acknowledgements First, I would like to thank Professor Simon Foster for all the support and guidance throughout this project, for allowing me be part of his research group and for all his help and understanding about everything during these four years. I would also like to thank Dr. Jorge García-Lara and Dr. Stephane Mesnage for their support and technical assistance, and for all the times I just needed a ‘science question’ answered; Dr. Fiona Shackley from the Royal Hallamshire Children’s Hospital for the samples of infant’s blood; Dr. Suzan Roiijakkers from University Medical Centre Utrecht (The Netherlands) for sharing her results confirming my hypothesis; Dr. Richard Beniston from the biOMICS Facility from the University of Sheffield for the LC-MS analysis; Dr. Cristina Teles and John Connolly for the gifts of different types of mammalian serum; and everybody in F18 who donated their blood for my studies. I need to thank all of the friends I made in the F18 and F25 labs, who made these years awesome (and not just for the science), especially Kasia Wacnik and Bartek Salamaga, Victoria Lund, Hannah Kelly, Felix Weihs, Bryony Cotterell, Kalpita Padwal and Joe Kirk. I need to also thank my friends from Chile (Astrid, Cristina, Daniela, Alejandra, Ximena, Inés y Maca), who where just a text away no matter the time difference. Finally and most importantly, I need to thank my family. They have supported me my whole life, and these four years in the UK have not been different. Thank you for all the pacience, the support, the love, and all the things that they did for me, from Skype calls to plane tickets so I could spent every Christmas with them. This is for my parents, Mónica and Alejandro, and my siblings, Javier and Lorena. iii Publications from this study Viana, D., Comos, M., McAdam, P.R., Ward, M.J., Selva, L., Guinane, C.M., González-Muñoz B.M., Tristan, A., Foster, S.J., Fitzgerald, J.R., Penadés, J.R. (2015) A single nucleotide mutation alters bacterial pathogen host tropism. Nature Genetics, 47, p.361-366. iv Abbreviations ºC Degree Celsius AX Absorbance at indicated wavelength x (nm) AACOCF3 Arachinodyl trifluoromethyl ketone ADP Adenosine diphosphate AGC Automatic gain control AMP Adenosine monophosphate Anx-A1 Annexin A1 APS Ammnoium persulphate ATP Adenosine triphosphate BCR B cells receptor BHI Brain heart infusion BLAST Basic local alignment search tool BODIPY Boron-dipyrromethene BPB 4-bromophenacyl bromide BPI Bactericidal/permeability increasing protein BSA Bovine serum albumin C5aR Complement component C5a receptor C55-P Undecaprenol phosphate CC Clonal complex CID Collision induced dissociation CFU Colony forming units CHIPS Chemotaxis inhibitory protein of S. aureus ClfA Clumping factor A Cm Chloramphenicol cPLA2 Cytosolic phospholipase A2 CRIT C2 receptor inhibitor trispanning cvPLA2 Cobra venom phospholipase A2 D-ala D-alanine DGlcDAG Diglucosyl-diacylglycerol dH2O Distilled water DNA Desoxyribonucleic acid DOPC 1,2-Dioleoyl-sn-glycero-3-phosphocholine DOPG 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) DTT Dithiothreitol v Eap Extracellular adherence protein Ecb Extracellular complement binding protein ECL Enhanced chemiluminescent Efb Extracellular fibrinogen binding protein EGTA Ethileneglycol tretaacetic acid Ery Erytromycin ETV Electron transfer dissociation FPR Formyl peptide receptor FRET Fluorescence resonance energy transfer g Grams Gal Galactose Glc2DAG Diglycosyl-diacylglycerol GlcNac N-acetylglucosamine Gro-P Glycerophosphate HF Hydrofluoric acid hr Hour HRP Horseradish peroxidase HS Human serum ICAM-1 Intracellular adhesion molecule-1 Ig Immunoglobulin IL Interleukin iPLA2 Calcium-independent phospholipase A2 Kb Kilobase pairs kDa Kilodaltons kV Kilovolts L Litre LB Luria-Bertani LC-MS Liquid Chromatography Mass Spectrometry LPS Lipopolysaccharide LTA Lipoteichoic acid µg Microgram µl Microlitre µm Micrometre µM Micromolar M Molar MAC Membrane attack complex ManNac N-acetylmannoseamine vi Map19 MBL-associated plasma protein of 19 kDa MASPs MBL-associated serine proteases MBL Mannose-binding lectin Mbp Megabase pairs MES 2-(N-morpholino) ethanesulphonic acid mg Miligram MGE Mobile genetic element MHC Major histocompatibility complex MIC Minimum inhibitory concentration min Minute ml Mililitre MLST Multilocus sequence typing mM Milimolar MRSA Methicillin-resistant S. aureus MS Mass spectrometry MSCRAMM Microbial surface components recognizing adhesive matrix molecules MurNac N-acetylmuramic acid MWCO Molecular weight cut-off m/z mass to charge ratio nm Nanometres NRS Naive rabbit serum ODX Optic density at indicated wavelength x (nm) PAF Platelet activating factor PAF-AH Platelet activating factor acetylhydrolase PAMPs Pathogen-associated molecular pattern PBPs Penicillin binding proteins PBS Phosphate buffered saline PG Peptidoglycan PGRP Peptidoglycan recognition protein PLA2 Phospholipase A2 ppm Parts per million PRRs Pathogen- or pattern-recognition receptor rhPLA2 Recombinant human phospholipase A2 RNA Ribonucleic acid rpm Revolutions per minute RT Room temperature vii SB Slotblot Sbi Staphylococcal immunoglobulin binding protein SCIN Staphylococcal complement inhibitor SDS Sodium dodecyl sulphate SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis sMAP Small MBL-associated protein SNP Single nucleotide polymorphism Spec Spectinomycin sPLA2 Secreted phospholipase A2 SSL Staphylococcal superantigen-like protein STs Sequence types TBS Tris buffered saline TCR T cell receptor TEMED Tetramethylethylenediamine Tet Tetracyclin TNF Tumour necrosis factor Tris Tris (hydroxymethyl) aminomethane TSA Tryptic soy agar TSB Tryptic soy broth V Volts v/v Volume for volume VISA Vancomycin-intermediate S. aureus VRSA Vancomycin-resistant S. aureus w/v Weight for volume WB Western blot WTA Wall teichoic acid viii Table of Contents Title page i Summary ii Acknowledgments iii Publications from this study iv Abbreviations v Table of Contents ix List of Figures xvi List of Tables xx CHAPTER 1: ...................................................................................................... 1 Introduction ....................................................................................................... 1 1.1 Staphylococci ..................................................................................................... 1 1.2 Staphylococcus aureus ...................................................................................... 1 1.2.1 Antibiotic resistance in S. aureus ................................................................ 2 1.2.2 S. aureus infections ..................................................................................... 5 1.2.3 S. aureus virulence factors .......................................................................... 6 1.3 The bacterial cell wall ......................................................................................... 8 1.3.1 Gram-negative bacteria ............................................................................... 8 1.3.2 Gram-positive bacteria .............................................................................. 10 1.3.3 Peptidoglycan