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Ureaplasma Spp. Were Investigated The Development of Methods to Investigate the Mechanisms underlying Serum Resistance of Ureaplasma Species By Ali Farag Aboklaish BSc of Medical Laboratory Technology MSc of Biomedical Sciences (Medical Microbiology) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy October 2014 Department of Child Health Institute of Molecular and Experimental Medicine School of Medicine Cardiff University United Kingdom DECLARATION i DEDICATION This thesis is dedicated to my beloved mother, wife and children (Fatma, Enaas, Aya, and Mayar) ii Aboklaish A.F. Summary of Thesis Summary of Thesis The human Ureaplasma species are among the smallest and simplest self-replicating bacteria known to date. These microbes cause infection in humans, particularly in the upper genital tract during pregnancy, leading to several adverse outcomes including preterm birth, chorioamnionitis, and respiratory diseases of neonates. Little is known about the pathogenesis of Ureaplasma and mechanisms by which they avoid recognition and killing by the complement system. In this thesis, some mechanisms underlying serum resistance of Ureaplasma spp. were investigated. This goal was achieved by creating serum-resistant models of serum-sensitive laboratory Ureaplasma strains and developing and using some proteomic and molecular biology methods to study the role of potential factors, which mediate serum resistance and play a role in pathogenesis of Ureaplasma. My original contribution to the knowledge in this work was the development of transposon mutagenesis method that can now be used to study virulence genes of Ureaplasma. This method will also allow genetic manipulation of Ureaplasma for future studies. Monitoring and investigating induced serum-resistant strains using immunoblot analysis and proteomics revealed significant changes in two candidate proteins coincident with serum resistance. The first was the elongation factor Tu protein that found to be immunogenic and had altered pI isoforms. The observed change in this protein was consistent in all serum-resistant strains, which suggests a possible role in mechanism of serum resistance, possibly as a mediator for binding complement regulators, such as factor H and C4BP, at the cell surface of Ureaplasma. The second candidate protein was a novel 41 kDa protein that was uniquely expressed in all induced serum-resistant strains. Expression of this protein in all resistant strains strongly indicates its involvement in mechanism(s) of serum resistance of Ureaplasma. The possible gene that encodes for this 41 kDa protein has putatively been identified as UUR10_0137 in the genome of U. urealyticum serovar 10 (strain ATCC 33699) using the transposon mutagenesis method developed in this study. Although the gene product of UUR10_0137 gene is not known (hypothetical protein), this protein is now identified and proposed to have a role in serum resistance of Ureaplasma. The product of the UUR10_0137 gene could function as a complement regulator or inhibitor that prevents the activation of complement system, protecting Ureaplasma from the complement attack. The contribution of the multiple- iii Aboklaish A.F. Summary of Thesis banded antigen, MBA, was proven to be unimportant to serum resistance. Sole antigenic variations in this major surface antigen of Ureaplasma did not play any role in mediating serum resistance. Confirmation of a gene that mediates complement resistance would dramatically increase our understanding of Ureaplasma pathogenicity and provide a target for future human studies with preterm birth and Ureaplasma infection. iv Aboklaish A.F. Acknowledgments Acknowledgements ﴿In the name of Allah, the Most Gracious, the Most Merciful﴾ First and foremost, all praises be to Allah, for his guidance, help and blessing, and who has given me the strength I needed to finish this thesis. I would like to express my sincere appreciation and deep gratitude to my main supervisor, Dr. Brad Spiller, for his great supervision, guidance, and patience throughout my PhD study. I would also like to thank him for the generosity of his time and for being very supportive, especially during the hard and devastating time I have gone through, during the war on my country Libya. Without his understanding, encouragement and support, I would not have been able to carry on and accomplish this work. My sincere gratitude also extends to my second supervisor, Dr. Ian Brewis for his scientific advice and help, especially with the proteomic work. I would like to express my sincere thanks and deep appreciation to my wife for her patience, support, care and encouragement at all times, and for the sacrifices she have made on my behalf. Words cannot express how grateful I am to my mother, who always praying and making supplication to God on my behalf, and who provided the courage I needed to finish this work. I am forever thankful for her unconditional love and endless care and patience. I would also like to express my grateful and appreciation to my sister Aisha for her support and care. I also thank all my family members, who have been supportive and caring throughout. I am also very grateful to my family in-law (father, mother, brothers and sisters) for their support and encouragement. I would also like to acknowledge all my colleagues and staff members at in the Department of Child Health for their assistance. I am also grateful to all staff members at the postgraduate office for their support. I am very grateful to Dr. Mark Toleman, Mr Sanjay Khanna and Mrs Vickie Nixon for their help with some experiments in this thesis. I also thank Dr. Nicola Maxwell for providing the clinical samples from Deriford Hospital in Plymouth used in this work. I would like to thank all my friends back home and in the UK, who have been helpful and supportive. Finally, I wish to acknowledge my sponsor, the Libyan Higher Education and Sebha University (Faculty of Engineering and Technology, Brack) for giving me this opportunity to complete my PhD study program. May God Bless You All v Aboklaish A.F. Abbreviations Abbreviations 5’ 5 prime 3’ 3 prime 16S rRNA 16S ribosomal RNA 23S rRNA 23S ribosomal RNA 1-DE One Dimensional Electrophoresis 2-DE Two Dimensional Electrophoresis A Adenine Ab Antibody Ag Ag AP Alternative pathway ATCC American Type Culture Collection BAL Bronchioalveolar lavage BPD Bronchopulmonary dysplasia BCA Bicinchoninic acid bp Base pair BSA Bovine serum albumin C Cytosine C1 INH C1 inhibitor C4BP C4-binding protein CCU Colour changing unit CBS Central Biotechnology Services CFU Colony forming unit CLD Chronic Lung Disease CNS Central nervous system CP Classical pathway CR1 Complement receptor 1 CRP C-reactive protein CSF Cerebrobral spinal fluid Da Dalton DAF Decay accelerating factor DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate vi Aboklaish A.F. Abbreviations DR Direct repeats DTT Dithiothreitol E. coli Escherichia coli EDTA Ethylene diamine tertraacetic acid ETA Endotracheal aspirate FH Factor H FHL-1 Factor H- like protein 1 FHRs Factor H-related proteins G Guanine GPI Glycosylphosphatidylinositol HAE Hereditary angioedema HBSS Hanks buffered saline solution HI-NHS Heat-inactivated normal human serum HPA Health Protection Agency HPE Heath Protection England HRPO Horse radish peroxidase ICE Integrative conjugative element IEF Isoelectric focusing IgA Immunoglobulin A IgG Immunoglobulin G IgM Immunoglobulin M IL Interleukin IR Inverted repeats JCVI J. Craig Venter Institute Kb Kilo bases kDa Kilo Dalton Kg Kilo grams LDS lithium dodecyl sulphate LPS Lipopolysaccharide MAb Monoclonal antibody MAC Membrane attack complex MASP-2 MBL-associated serine peortease-2 MBA Multiple banded antigen MBL Mannose binding lectin MCP Membrane cofactor protein MIC Minimal inhibitory concentration vii Aboklaish A.F. Abbreviations M. genitalium Mycoplasma genitalium M. hominis Mycoplasma hominis M. pneumoniae Mycoplasma pneumoniae MS Mass spectrophotometry MALDI TOF/TOF Matrix- assisted laser desorption ionization time-of-flight mRNA Messenger RNA NGU Non-gonococcal urethritis NHS Normal human serum NICU Neonatal Intensive Care Unit PAMP Pathogen-associated molecular pattern PBS Phosphate buffered saline PBST Phosphate buffered saline with Tween-20 PCR Polymerase chain reaction PEG Polyethylene glycol PI Isoelectric point PMN Polymorphonuclear Ply Plymouth PMF Protein mass fingerprint PRRs Pattern recognition receptors RCA Regulators of complement activation RNA Ribonucleic acid rRNA Ribosomal RNA SAK Staphylokinase S. aureus Staphylococcus aureus SEM Standard error of the mean SNP Single nucleotide polymorphism Spp. Species SV Serovar SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis T Thymine TEAB Tri-ethyl ammonium Bicarbonate TFA Trifluoroacetic acid TNF-α Tumour necrosis factor alpha TLRs Toll-like receptors U Uracil U. parvum Ureaplasma parvum viii Aboklaish A.F. Abbreviations Upvmp376 Ureaplasma phase-variable membrane protein 376 U. urealyticum Ureaplasma urealyticum USM Ureaplasma Selective Medium VBS Veronal buffered saline W.B Western blot ix Aboklaish A.F. Table of Contents Table of Contents DECLARATION ............................................................................................ i DEDICATION ............................................................................................... ii Summary of Thesis ...................................................................................... iii Acknowledgements .......................................................................................
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