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Immunoproteomic Identification of Biomarkers for Diagnosis of Legionellosis

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Immunoproteomic Identification of Biomarkers for Diagnosis of Legionellosis Immunoproteomic identification of biomarkers for diagnosis of legionellosis Submitted in total fulfilment of the requirements for the degree of Doctor of philosophy by Kaylass Poorun Department of Chemistry and Biotechnology Faculty of Science, Engineering and Technology Swinburne University of Technology Australia 2014 Abstract Abstract Legionellosis, a disease with significant mortality and morbidity rates, is considered to be the second most frequent cause of severe community-acquired pneumonia. It is difficult to distinguish from other types of pneumonia due to similar clinical manifestations. Several studies have demonstrated the inadequacies of current diagnostic tests for confirming Legionella infections. This study was aimed at identifying biomarkers that can be used in an improved test. A comparative proteomic analysis, using DIGE, was carried out between L. pneumophila ATCC33152 and L. longbeachae NSW150 and D4968 isolates. While many homologous proteins were found to be commonly expressed, numerous others were identified to be differentially expressed under similar in vitro conditions suggesting that the two species have different lifestyles and infection strategies. The bacterial immunoglobulin domain containing protein, found to share sequence homology to Type V secretion proteins intimin and invasin, is not known to be present in Legionella. Human sera containing antibodies against Legionella from a set of blind samples were identified by ELISA. Downstream analyses revealed that diverse immunogens may be responsible for eliciting immune response in different Legionella species which in turn show little to no congeneric cross-reactivity. To the best of our knowledge, this is a unique finding not previously reported. Several serological diagnostic tests currently in use do not include many Legionella species in their testing panel, which may be a reason for many Legionella species being under-reported. Use of pooled specific antigens from different Legionella species for genus-level diagnosis, and a panel of individual species-specific antigens for species identification were proposed. In this pursuit, affinity purification of antibodies and antigens were attempted for epitope mapping. This was rather unsuccessful, probably because of low amounts of antibodies in sera. Peptides based on in silico prediction were assessed for antigenicity to determine suitability for application in diagnostic tests. However, it was inconclusive whether the peptides were not immunogenic or did not bind because of their small size. Moreover, only two genus- specific peptides were tested as the design was based on linear epitope prediction of conserved hydrophilic regions of the proteins. This approach may be too rigid and the method was also affected by the limited genome information available for most of the Legionella species. Overall, this study demonstrates that considerable variation exists in the proteomes of L. pneumophila and L. longbeachae, which may be responsible for the I | Page Abstract differential immunogenic reactions observed. Based on these findings, it appears that the serological tests currently being used may have limitations for genus-level diagnosis. Therefore, this study may serve as a pilot test for a better research design to further elucidate on the pathogenesis strategies of L. longbeachae and identify potential biomarkers. II | Page Acknowledgement Acknowledgements Since it all started and until I completed this thesis, the journey would not have been possible without the help of many people and I would like to acknowledge them for their contributions. Firstly, I would like to express my gratitude to my principal supervisor, Dr François Malherbe for helping me embark on this journey. I am thankful to you for believing in me and for the support and guidance you provided during my PhD. I would like to thank my co-supervisor, Professor Enzo Palombo for his prompt valuable feedback, advice and kind words. I am grateful to the Swinburne academic and technical staff, Professor Linda Blackall, Dr Daniel Eldridge, Mr John Fecondo, Ms Sheila Curtis, Ms Angela Mckellar, Mr Cameron Young, Mr Chris Key, Ms Soula Mougos, Mr Ngan Nguyen, Mr Chris Anthony, Mr Jemison Escalona, Ms Andrea Chisholm, Dr Huimei Wu and Ms Savithri Galappathie for their considerable assistance. I would also like to show my appreciation to the Swinburne Higher Research Degree personnel for their support. This thesis would not have been possible without the bacteria and serum samples that I obtained with the help of various people and I am indebted to them. I would like to thank Dr Natalia Kozak and Dr Barry Fields from CDC, USA, Professor Elizabeth Hartland from University of Melbourne, Australia, Dr Rodney Ratcliff, Dr Ivan Bastian, Mr Richard Lumb and Ms Lisa Shephard from IMVS, Australia, Mr David Dickeson from SWAHS-ICPMR, Australia and Dr John Stenos and Ms Chelsea Nguyen from ARRL, Australia. To the Bio21 Institute staff, Dr Nicholas Williamson, Dr Ching-Seng Ang and Mr Paul O!Donell and to Ms Irene Hatzinisiriou from Monash University, I thank you for your technical assistance. To Shanthi, you deserve a special thank you for always being a good friend with whom I could share my joy and more importantly my woes. A thank you goes to all my other university friends and colleagues, Bita, Rue, Azadeh, Jafar, Jun, Jiawey, Abdullah, Hayden, Saifone, Shahanee, Elisa, Vanu, Dave, Shaku, Suchetna, Babu, Dhivya, Snehal, Qudsia, Rashida, Chris, Ha, Vy, Hadi, Maho, Roslyn, James and Matthew. III | Page Acknowledgement To my friends and colleagues outside university, Aurelie, Kush, Anthony, Dan, Sanhit and Hans to mention a few who have been supportive along the way, I thank you all. Above all, I thank the Almighty for having bestowed the blessings on me. A special thank you goes to all my family members. To my two brothers, Vishal and Satyam, thank you for your encouragement and support. Last but not the least, and more importantly I express my deepest gratitude to my parents for their consistent encouragement, love and support, and to whom I dedicate this thesis. IV | Page Declaration Declaration I, Kaylass Poorun, declare that this PhD thesis entitled "Immunoproteomic identification of biomarkers for the diagnosis of legionellosis! is no more than 100,000 words in length, exclusive of tables, figures, appendices, references and footnotes. This thesis contains no materials that has been submitted previously, in whole or in part, for the award of any other academic degree or diploma, and has not been previously published by another person. Except where otherwise indicated, this thesis is my own work. Kaylass Poorun 2014 V | Page Table of contents Abbreviations A Ampere ACES N-(2-acetamido)-2-aminoethanesulfonic acid ATCC American Type Culture Collection AYE ACES-buffered yeast extract BCYE Buffered charcoal yeast extract BLAST Basic Local Alignment Search Tool CAP Community-acquired pneumonia Cat# Catalog number CD4 Cluster of differentiation 4 CHAPS 3-[(cholamidopropyl)dimethylammonio]-propanesulfonate Da Dalton DMF Dimethylformamide Dot/Icm Defect in organelle trafficking/Intracellular multiplication DTT Dithiothreitol EIA Enzyme immunoassays ELISA Enzyme linked immunosorbent assay HRP Horseradish peroxidase ICT Immunochromatographic test IEF Isoelectric focussing IFA Indirect fluorescence antibody test IgG Immunoglobulin G IPG Immobilised pH gradient kDa Kilodalton KX 1000 times LB Luria Bertani M Molar Mag Magnification MHC Major histocompatibility complex MS Mass spectrometry OD Optical density PCR Polymerase Chain Reaction pI Isoelectric point VI | Page Table of contents ppm parts per million PVDF Polyvinylidene SDS Sodium dodecyl sulphate SG Serogroup TBS Tris buffered saline TBS-T Tris-buffered saline-Tween20 Tris Tris (hydroxymethyl)aminomethane V Volt v/v volume by volume vs. Versus w/v weight by volume WD Width VII | Page Table of contents TABLE OF CONTENTS Abstract………………………………………………………………………………....I Acknowledgements…………………………………………………………………...III Declaration……………………………………………………………………………..V Abbreviations……………………………………………………………………….…VI Table of contents……………………………………………………………………VIII List of Figures……………………………………………………………………..….XV List of Tables………………………………………………………………..……...XVII 1 INTRODUCTION ........................................................................................................... 1 1.1 Overview ..................................................................................................................................... 1 1.2 Aims and objectives .................................................................................................................... 2 1.3 Thesis overview........................................................................................................................... 2 2 LITERATURE REVIEW ................................................................................................ 4 2.1 Legionnaires’ disease: An overview ............................................................................................. 4 2.2 Classification of Legionella .......................................................................................................... 5 2.3 Cell-Structure and Metabolism.................................................................................................... 6 2.4 Legionella hosts and life cycle ..................................................................................................... 9 2.5 Legionella Ecology ......................................................................................................................11
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