Immunoproteomic Identification of Biomarkers for Diagnosis of Legionellosis

Home , Legionella adelaidensis, Legionella beliardensis, Legionella brunensis, Legionella cardiaca, Legionella gresilensis, Legionella impletisoli

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

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

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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.

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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 ODonell 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.

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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.

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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

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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

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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 2.5.1 Legionella habitat and risks of infection ...... 11 2.5.2 Biofilm formation and its clinical significance ...... 12

2.6 Mode of infection...... 13

2.7 Clinical manifestation in host ...... 13

2.8 Community-acquired pneumonia and economic burden ...... 14 VIII | Page Table of contents

2.9 CAP: typical and atypical pneumonia pathogens ...... 14

2.10 Identification of the etiological agent of CAP ...... 15

2.11 Treatment of CAP and Legionnaires’ disease...... 17

2.12 Implications of improper use of antibiotics ...... 18

2.13 Epidemiology of Legionellosis ...... 19

2.14 Factors Contributing to the Prevalence of Legionellosis ...... 22

2.15 Legionella Co-infection and Underlying Co-morbidities ...... 24

2.16 Legionella longbeachae and Legionnaires’ Disease ...... 24

2.17 Differences between L. pneumophila and L. longbeachae ...... 25

2.18 Infection and Immune Response ...... 27

2.19 Proteins as Antigens and Immunogens ...... 28

2.20 Bacterial Outer-Membrane and Outer-Membrane proteins...... 29

2.21 Antigenicity of Outer-Membrane Proteins ...... 30

2.22 Antigenic Proteins of Legionella ...... 31 2.22.1 Major outer membrane protein (24kDa) ...... 31 2.22.2 Macrophage Infectivity Potentiator (MIP) ...... 31 2.22.3 Peptidoglycan associated lipoprotein (PAL, 19 kDa) ...... 32 2.22.4 Heat shock protein (60 kDa) ...... 33

2.23 Cross-Reactivity of Immunogenic Proteins ...... 33

2.24 Protective Immunity of Antigenic Proteins ...... 34

2.25 Antigenicity of Secreted Proteins and Proteins from other Cellular Localisations ...... 35

2.26 Diagnostics...... 35 2.26.1 Microbiological diagnosis of community-acquired pneumonia ...... 35 2.26.2 Diagnosis of legionellosis ...... 38 2.26.2.1 Culture method ...... 39 2.26.2.2 Serology ...... 39 2.26.2.3 Polymerase Chain Reaction (PCR) ...... 41

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3 MATERIALS & METHODS ...... 43

3.1 Materials ...... 43 3.1.1 Sterilisation of hard goods ...... 43 3.1.2 Culture media ...... 43 3.1.2.1 BCYE (Buffered Charcoal Yeast Extract) agar culture plates ...... 43 3.1.2.2 ACES-buffered yeast extract (AYE) broth ...... 43 3.1.2.3 Luria-Bertani (LB) agar culture plates ...... 44 3.1.3 Common Solutions ...... 44 3.1.3.1 1.5M Tris-HCl pH 8.8 ...... 44 3.1.3.2 0.5M Tris-HCl pH 6.8 ...... 44 3.1.3.3 10% SDS ...... 45 3.1.4 Protein isolation buffers ...... 45 3.1.4.1 Cell wash buffer ...... 45 3.1.4.2 0.05% bromophenol blue ...... 45 3.1.4.3 10mM HEPES pH 7 ...... 45 3.1.5 2D sample buffers...... 46 3.1.5.1 Whole cell lysate proteome/Lysis buffer ...... 46 3.1.5.2 Secretome...... 46 3.1.5.3 2D base equilibration buffer ...... 46 3.1.5.4 Outer-membrane sub-proteome ...... 47 3.1.5.5 SDS sample loading buffer/sample reducing buffer (SRB) ...... 47 3.1.6 Electrophoresis buffer ...... 48 3.1.7 Gel fixing and staining solutions ...... 48 3.1.7.1 Gel fixing solution...... 48 3.1.7.2 Colloidal coomassie blue stock staining solution ...... 48 3.1.8 Polyacrylamide gel casting ...... 49 3.1.8.1 Hand casting gels ...... 49 3.1.8.2 Mini-Protean gels for 2-Dimensional gel electrophoresis ...... 49 3.1.8.3 Mini-Protean gels for 1-Dimensional SDS-PAGE ...... 49 3.1.8.4 Criterion gels for 2-dimensional gel electrophoresis ...... 49 3.1.8.5 Protean gels (18 cm by length and 1 mm in thickness) ...... 50 3.1.9 Mass Spectrometry solutions ...... 51 3.1.9.1 50 mM Tetraethylammonium bromide (TEAB) ...... 51 3.1.9.2 Gel plug destaining solution for Mass Spectrometry ...... 51 3.1.9.3 Gel plug reduction solution ...... 51 3.1.9.4 Gel plug alkylation solution ...... 52 3.1.9.5 Trypsin solution ...... 52

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3.1.9.6 Mass Spectrometry Eluents ...... 52 3.1.10 Immunoassays (ELISA, Dot-blot and Western blot) solutions ...... 52 3.1.10.1 Transfer buffer (Towbin buffer) ...... 52 3.1.10.2 Towbin buffer with Sodium dodecyl sulfate (SDS) ...... 52 3.1.10.3 Tris-buffered saline buffer (TBS): 20 mM Tris-HCl, 500 mM sodium chloride, pH 7.5...... 52 3.1.10.4 Tris-bufferd saline buffer-Tween20 (TBS-T) ...... 53 3.1.10.5 Blocking solution (5% skim milk-TBS) ...... 53 3.1.10.6 Antibody buffer ...... 53 3.1.11 Trypsin shaving ...... 53 3.1.12 Affinity purification solutions ...... 53 3.1.12.1 Coupling/Binding/Wash buffer: Phosphate buffered saline (PBS) solution ...... 53 3.1.12.2 Quenching buffer ...... 53 3.1.12.3 Elution buffer ...... 53 3.1.12.4 Neutralisation buffer ...... 53

3.2 Methods...... 54 3.2.1 Determination of post-exponential growth phase of Legionella species ...... 54 3.2.2 Protein extraction ...... 55 3.2.2.1 Total cell soluble proteins isolation ...... 55 3.2.2.2 Secretome isolation...... 55 3.2.2.3 Outer membrane proteins isolation ...... 55 3.2.2.4 Surfaceome/trypsin shaving ...... 56 3.2.3 Bradford Assay for total protein quantification ...... 56 3.2.3.1 Total soluble proteins ...... 56 3.2.3.2 Secretome sample clean-up and protein quantification ...... 57 3.2.3.3 Outer membrane sub-proteome proteins quantification...... 57 3.2.4 2D Gel Electrophoresis – Criterion gels ...... 57 3.2.4.1 11cm IPG strip rehydration ...... 57 3.2.4.2 Iso-electric focussing (IEF) – 1st dimension separation ...... 58 3.2.4.3 IPG strip equilibration ...... 58 3.2.4.4 2nd Dimension SDS-PAGE ...... 59 3.2.5 2D Gel Electrophoresis - Protean gels ...... 59 3.2.5.1 17cm IPG (Immobilised pH Gradient) strip rehydration ...... 59 3.2.5.2 DIGE sample preparation...... 59 3.2.5.3 Iso-electric Focussing (IEF) ...... 61 3.2.5.4 IPG strip equilibration ...... 61 3.2.5.5 2nd Dimension SDS-PAGE ...... 62 3.2.5.6 Gel staining ...... 62 3.2.5.7 Gel imaging ...... 62 XI | Page Table of contents

3.2.6 DIGE gel analysis ...... 63 3.2.6.1 Difference in-gel Analysis (DIA) ...... 63 3.2.6.2 Biological Variation Analysis (BVA) ...... 64 3.2.7 Protein identification ...... 64 3.2.7.1 In-gel tryptic digestion ...... 64 3.2.7.2 Mass spectrometry...... 65 3.2.7.3 MASCOT® Search for protein identity (MSILE) ...... 66 3.2.8 Basic Local Alignment Search Tool (BLAST) ...... 66 3.2.9 Prediction of subcellular localisation of proteins ...... 66 3.2.10 Immunoassays ...... 67 3.2.10.1 Dot blot ...... 67 3.2.10.2 Western blotting ...... 67 3.2.10.3 Indirect ELISA ...... 68 3.2.11 Affinity purification ...... 69 3.2.11.1 Affinity purification of IgG ...... 69 3.2.11.2 Affinity purification of antigen ...... 70 3.2.12 In silico prediction of epitopes ...... 70 3.2.13 Peptide design ...... 70

4 COMPARATIVE PROTEOMICS ANALYSIS OF L. PNEUMOPHILA VS. L. LONGBEACHAE ...... 71

4.1 Introduction ...... 71

4.2 Experimental design ...... 73 4.2.1 Aims and objectives ...... 73 4.2.2 Experimental procedures ...... 73

4.3 Results ...... 74 4.3.1 Determination of post-exponential growth phase of Legionella ...... 74 4.3.2 Protein quantitation (Bradford Assay) ...... 75 4.3.3 Differential in-gel analysis (DIA) ...... 76 4.3.4 Results of Biological Variation Analysis (BVA) analysis ...... 78 4.3.4.1 L. pneumophila vs. L. longbeachae differential expression of proteins ...... 78 4.3.4.2 L. longbeachae NSW150 vs. L. longbeachae D4968 differential expression ...... 81 4.3.5 Mass spectrometry ...... 82 4.3.6 Bioinformatics prediction of cellular localisation and specificity of identified proteins ...... 86 4.3.7 Potential virulence and pathogenesis related proteins ...... 88 4.3.8 Classifying identified proteins based on function ...... 96 XII | Page Table of contents

4.3.8.1 Grouping of proteins based on most probable cellular function ...... 96 4.3.8.2 Investigation of the glycolysis/gluconeogenesis pathway ...... 97 4.3.8.3 Investigation of the citric acid cycle ...... 98 4.3.8.4 Investigation of the Entner-Doudoroff and Pentose Phosphate pathway ...... 99

4.4 Discussion ...... 100 4.4.1 Optimisation of growth conditions ...... 100 4.4.2 Optimisation of 2D gel electrophoresis ...... 100 4.4.3 Comparative analysis of the two Legionella longbeachae isolates ...... 101 4.4.4 Comparative analysis between L. pneumophila and L. longbeachae ...... 101 4.4.5 Virulence and pathogenesis related proteins of Legionella ...... 104

4.5 Concluding Remarks ...... 104

5 IDENTIFICATION OF IMMUNOGENIC PROTEINS ...... 105

5.1 Introduction ...... 105

5.2 Experimental Design ...... 106 5.2.1 Aims and objectives ...... 106 5.2.2 Experimental procedures ...... 106

5.3 Results ...... 107 5.3.1 ELISA of human sera against Legionella proteins ...... 107 5.3.2 ELISA for serum samples B1G and C5D against Legionella proteins ...... 109 5.3.3 Dot-blot analysis with selected serum samples ...... 111 5.3.4 Western blot analyses ...... 112 5.3.4.1 Total soluble proteins with serum sample B1G ...... 112 5.3.4.2 Differentially reacting serum samples ...... 113 5.3.4.3 Secretome of Legionella species against serum sample B1G ...... 114 5.3.4.4 Total soluble proteins from L. longbeachae ...... 115 5.3.4.5 Pooled standard protein samples (DIGE) ...... 116 5.3.4.6 Differential detection of immunoreactive proteins of the outer-membrane sub-proteome of L. longbeachae with serum samples B1G and C5D ...... 117 5.3.5 Western blot data mining ...... 119 5.3.6 Immunogenic proteins of interest ...... 120

5.4 Discussion ...... 123 5.4.1 Optimisation of isolation of outer-membrane sub-proteome ...... 123 5.4.2 Differential immunoreaction of serum samples ...... 124

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5.4.3 Potential limitations of current diagnostic tests ...... 126 5.4.4 Surface-exposed proteins as potential biomarker ...... 127 5.4.5 Immunogenic proteins of L. longbeachae ...... 128

5.5 Conclusion ...... 129

6 IDENTIFICATION OF ANTIGENIC EPITOPES ...... 130

6.1 Introduction ...... 130

6.2 Experimental design ...... 131 6.2.1 Aims and objectives ...... 131 6.2.2 Experimental procedures ...... 131

6.3 Results ...... 132 6.3.1 Trypsin shaving ...... 132 6.3.2 Affinity purification of antibodies and antigens ...... 133 6.3.2.1 Affinity purification of IgG from serum sample...... 133 6.3.2.2 Affinity purification of antigens ...... 136 6.3.3 In silico prediction of antigenic epitopes ...... 136 6.3.4 Determination of antigenicity of peptides ...... 141

6.4 Discussion ...... 141 6.4.1 Challenges in affinity purification of proteins ...... 141 6.4.2 Limitations of in silico prediction of epitopes and peptide design ...... 142 6.4.3 Lack of genomic data and its implications ...... 142

6.5 Conclusion ...... 143

7 GENERAL DISCUSSION AND CONCLUSIONS ...... 144

7.1 Overview ...... 144

7.2 Recommendations for Future Work ...... 148

REFERENCES ...... 149

APPENDICES ...... 175

CONFERENCES ...... 190

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LIST OF FIGURES

Figure 1.1: Schematic representation of experimental design and workflow ...... 3 Figure 2.1: Scanning electron microscopy image of L. longbeachae ...... 6 Figure 2.2: Legionella life cycle ...... 10 Figure 2.3: Biofilm and its clinical significance ...... 12 Figure 2.4: Diagnosis based on clinical symptoms ...... 15 Figure 2.5: Legionellosis notification rate ...... 20 Figure 2.6: cases of legionellosis in Australia in 2010 ...... 21 Figure 2.7: percentage of cases of legionellosis in Australia in 2010 ...... 21 Figure 2.8: Gram-negative outer membrane structure...... 30 Figure 4.1: Downstream applications of genomic data ...... 72 Figure 4.2: Determination of post-exponential phase of growth of Legionella spp...... 74 Figure 4.3: BSA standard curve for protein quantitation of total soluble proteome samples pre and postendonuclease treatment...... 75 Figure 4.4: Histogram of differentially expressed proteins of L. pneumophila vs. L. longbeachae ...... 77 Figure 4.5: Histogram of differentially expressed proteins of L. longbeachae D4968 vs. NSW150 ...... 77 Figure 4.6: DIGE gel3 annotation for L. pneumophila differentially expressed proteins ...... 78 Figure 4.7: DIGE gel 3 annotation for L. longbeachae differentially expressed proteins ...... 79 Figure 4.8: Graphical representation and spot image of (A) spot 284 from Figure 4.6 and (B) spot 137 from Figure 4.7 ...... 80 Figure 4.9: DIGE gel 6 annotations for L. longbeachae NSW150 and D4968 differentially expressed proteins ...... 81 Figure 4.10: Graphical representation and spot image of spot 5 from Figure 10 ...... 82 Figure 4.11: Mass spectrometry data for spot 138 of L. longbeachae...... 83 Figure 4.12: Venn diagram of intra and inter-specific differentially expressed proteins...... 85 Figure 4.13: Pie charts of predicted cellular localisation of proteins ((A & B) and of predicted specificity of proteins identified (C & D) for L. pneumophila (A & C) and L. longbeachae (B & D) ...... 86 Figure 4.14: Most probable cellular localisation of identified virulence and pathogenesis related proteins ...... 87 Figure 4.15: Grouping of identified proteins according to most probable cellular function ...... 96 Figure 4.16: Proposed glycolysis/gluconeogenesis pathway ...... 97 Figure 4.17: Proposed citric acid cycle pathway ...... 98 Figure 4.18: Proposed Entner-Doudoroff and Pentose Phosphate pathway ...... 99 Figure 5.1: Indirect ELISA for selection of Legionella antibodies containing serum samples ...... 108 Figure 5.2: Indirect ELISA to determine cross-reactivity of serum samples across Legionella species .. 110 Figure 5.3: Dot-blot of protein from Legionella species against human sera ...... 111 Figure 5.4: Western blot analysis of Legionella species with Sample B1G ...... 112 Figure 5.5: Comparative Western blot analysis with serum samples B1G and C5D ...... 113 Figure 5.6: Western blot of secretomes of L. pneumophila and L. longbeachae with Sample B1G ...... 114 XV | Page

List of Tables

Figure 5.7: Western blot of total soluble protein from L. longbeachae against serum sample B1G ...... 115 Figure 5.8: Western blot of pooled total soluble proteins from L. pneumophila and L. longbeachae against serum sample C5D...... 116 Figure 5.9: Western blot of outer-membrane sub-proteome of L. longbeachae against serum sample C5D ...... 117 Figure 5.10: A: 2D gel image of outer membrane subproteome of L. longbeachae. B: Western blot of outer-membrane sub-proteome of L. longbeachae against serum sample B1G ...... 118 Figure 5.11: Venn diagram of differentially identified proteins per proteome sub-type against different serum samples ...... 119 Figure 6.1: Affinity purification of IgG from serum sample C5D and U1S ...... 134 Figure 6.2: Activity of affinity purified IgG ...... 136 Figure 6.3: Peptidyl-prolyl cis-trans isomerase (MIP) protein sequence alignment for identification of conserved regions ...... 140

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List of Tables

LIST OF TABLES

Table 2.1: Current list of Legionella species and their pathogenesis identity ...... 7 Table 2.2: Comparison of methods for laboratory diagnosis of Legionnaires' disease ...... 38 Table 3.1: 12% Resolving gel solution (Total volume 3.5mL) ...... 50 Table 3.2: 4% Stacking gel solution (Total volume 1.5mL) ...... 50 Table 3.3: 12% Resolving gel solution for Protean gels (total volume 40mL) ...... 51 Table 3.4: Total cellular soluble protein samples labelling for DIGE ...... 60 Table 3.5: Parameters used for DIGE gel imaging using Typhoon Trio ...... 63 Table 3.6: Non-proteinaceous spots exclusion criteria parameters ...... 64 Table 3.7: Parameters for protein identification using mass spectrometry data ...... 66 Table 4.1: Protein concentration of samples pre and post endonuclease treatment...... 76 Table 4.2: Results of mass spectrometry analysis of protein spots ...... 84 Table 4.3: Potential virulence and pathogenesis related proteins for L. pneumophila ...... 89 Table 4.4: Potential virulence and pathogenesis related proteins for L. longbeachae ...... 93 Table 5.1: Immunogenic proteins belonging to the secretome fraction only ...... 115 Table 5.2: Immunogenic proteins of interest from total soluble proteins and outer-membrane sub- proteome of L. longbeachae ...... 121 Table 6.1: Proteins identified from trypsin shaving ...... 133 Table 6.2: Protein identification by peptide mass fingerprinting ...... 135 Table 6.3: Proteins selected for further analysis as potential biomarker ...... 137 Table 6.4: Peptides designed on the basis of in silico epitope predictions ...... 138

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Chapter 1 Introduction

INTRODUCTION

1.1 OVERVIEW Legionellosis, also known as Legionnaires disease, a pneumonic illness caused by the accidental pathogen Legionella, is a notifiable disease under epidemiological surveillance programs in many countries. Although infections with Legionella are considered to be the second most frequent cause of community-acquired pneumonia, the general consensus among specialists is that the magnitude of reported cases is likely to be much higher due to under-reporting. This is probably the result of shortcomings of the current diagnostic tests. Infections with Legionella have been reported to require specific antibiotic treatment, but with the lack of appropriate test to identify Legionella as the causative agent of infection, empirical therapy is commonly used. The use of improper antibiotics may have serious implications such as the emergence of drug- resistant pathogens and an increased risk to the patients health. Improper diagnoses may eventually have significant repercussions on the epidemiology and may result in economic burden on individuals and communities. Given that infections with Legionella are indistinguishable from pneumonia caused by other pathogens due to similar symptoms, and that the use of specific antibiotic is necessary to curtail the risk of an outbreak, the availability of an unequivocal diagnostic test is critical.

Legionella pneumophila has been reported as the predominant species causing disease in human, and there is currently a highly sensitive and specific urinary antigen test for reliable diagnosis of infections with L. pneumophila. However, this test is only specific to L. pneumophila Serogroup 1. As a result, diagnosis of other Legionella species is performed through the culture method, serology or polymerase chain reaction (PCR). All these tests have their shortcomings such as the long turn-around time of the test results, and low sensitivity and specificity. Legionella longbeachae has only recently been increasingly reported as another major pathogenic species, especially in the

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Australasian region. The incidence of infections with L. longbeachae was found to be indeed underestimated, as observed by Murdoch et al. (2013) with their improved PCR testing method. However, this test also may have some limitations as sputum samples were used for the detection of the infecting organism, but the majority of the Legionnaires disease patients are known not to produce sputum.

1.2 AIMS AND OBJECTIVES

The main aim of this study is to identify genus-specific and species-specific protein biomarkers that can be used in the development of an improved diagnostic test for Legionnaires disease. This aim was set to be achieved through the following objectives: • Inter-specific (L. pneumophila vs. L. longbeachae) and intra-specific (L. longbeachae D4968 vs. L. longbeachae NSW150) comparative proteome analyses to determine differentially expressed proteins involved in the cellular physiology, virulence and pathogenesis. • Determining the immunoreactivity of blind serum samples to Legionella antigens by devising a suitable immunoassay for subsequent identification of immunogenic proteins. • Laboratory-based experiment and in silico epitope mapping to identify genus- specific and species-specific antigenic determinants.

The two Legionella species; L. pneumophila and L. longbeachae were chosen for the comparative analysis firstly because of higher prevalence of legionellosis throughout the world due to these two species, secondly because of the whole genome sequence availability for only these two species and isolates at the beginning of the study and thirdly because of the difference between the two species with regards to their natural habitat, morphology and dissimilarity in the infection rate in different parts of the world.

1.3 THESIS OVERVIEW

Chapter 1 introduces the research topic and the main objectives of the study. In Chapter 2, an extensive literature review regarding the objectives of this thesis is provided. It presents a description of the limitations in the tests used for diagnosis of infections with Legionella and their consequences. This chapter also includes a brief comparative study of Legionella species available from literature.

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Chapter 3 entails a detailed description of the materials and methods used. Several protocols from literature that were modified and optimised to perform experiments specific to subsequent chapter have also been explained in the corresponding sections.

Chapter 4 involves comparative analyses of the proteomes of L. pneumophila ATCC33152, L. longbeachae D4968 and L. longbeachae NSW150. This chapter offers an insight into the differentially expressed proteins between the Legionella species and isolates under similar growth conditions. Chapter 5 explores the immune response following potential Legionella infections for the identification of immunogenic proteins. This chapter provides a basis for the selection of immunogenic proteins of interest to target as potential genus-specific and species-specific biomarkers. Chapter 6 investigates the immunogenic proteins of interest to determine antigenic epitopes.

Chapter 7 concludes the thesis with a brief discussion and conclusions regarding the findings of the study and also some recommendations for future work.

Figure 1.1 shows a schematic representation of the experimental workflow of this study.

Figure 1.1: Schematic representation of experimental design and workflow 3 | Page Chapter 2 Literature Review

LITERATURE REVIEW

2.1 LEGIONNAIRES’ DISEASE: AN OVERVIEW

Legionnaires disease, also known as legionellosis, is a pneumonic illness first discovered in Philadelphia in 1976 when it caused an outbreak of pneumonia at an American Legion Convention. One-hundred-and-eighty-two people were infected and 29 died following further complications (Mcdade et al., 1977). Pontiac fever is another form of self-limited illness caused by Legionella.

Legionellosis is reported under the National Notifiable Disease Surveillance Systems in many countries, such as the USA (Garg, 2012) and Australia (Milton et al., 2012). Nosocomial legionellosis is also commonly reported, and infections can be sporadic or occur as an outbreak. Although Legionnaires disease is considered to be a rare disease, Lueck (2010) postulated that, according to estimates from previous studies, the incidence of infection with Legionella is considerably higher than reported, as many cases go undetected. Legionellosis is a disease with significant mortality and morbidity rates throughout the world, and is considered to be the second most frequent cause of severe community-acquired pneumonia (Diederen, 2008, Llorens et al., 2009). Whilst Legionella pneumophila accounts for approximately 90% of infections worldwide, L. longbeachae is responsible for an equal or even higher number of infections in Australasia (Amodeo et al., 2010, Phares et al., 2007).

Legionella is an accidental pathogen (Shuman et al., 1998) of humans. Amoebae and other protozoans have been described as the primary hosts for Legionella (Rowbotham, 1980, Molmeret et al., 2005) and it has been reported to multiply within human phagocytic alveolar macrophages (Horwitz and Silverstein, 1980) avoiding lysosomal digestion (Swanson and Isberg, 1995). Legionella is known to be involved in host organelle trafficking by secreting proteins known as effectors (Franco et al., 2012).

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Factors such as advanced age, alcohol abuse, smoking, chronic diseases and immunosuppression may contribute to higher vulnerability to infections (Falco et al., 1991, Che et al., 2008).

Legionnaires disease is most commonly spread through Legionella contaminated aerosols (Muder et al., 1986); Legionella is a ubiquitous organism in water environments. Fresh water is the major reservoir for most Legionella spp. (Fliermans et al., 1981) except for Legionella longbeachae which has been consistently isolated from soils, composts and potting mixes (Steele, 1989).

Legionellosis is difficult to distinguish from other types of pneumonia due to similar clinical manifestations (Garg, 2012). Higa et al., (2008) critically evaluated the inadequacies of currently available diagnostic tests, including the commonly used urine antigen test for confirming infection with Legionella. Of all the atypical pathogens that are considered to cause pneumonia in adults, Legionella is the only one that requires specific therapy (Robenshtok et al., 2008, Bartlett, 2011).

As infections with Legionella are indistinguishable from pneumonia caused by other pathogens due to similar symptoms, and that the use of specific antibiotic is necessary, the availability of an improved diagnostic test is needed.

2.2 CLASSIFICATION OF LEGIONELLA

Domain: Bacteria Phylum: Proteobacteria Class: Gammaproteobacteria Order: Legionellales Family: Legionellaceae Genus: Legionella

Legionella belongs to the class gammaproteobacteria which includes many pathogenic bacteria and several of which, such as Klebsiella pneumoniae and Haemophilus pneumoniae are known to cause pneumonia. Coxiella burnettii is closely related to Legionella and is also known to cause pneumonia. However, Coxiella infection is a zoonotic disease transmitted by animal vectors such as cattle. Legionella dumoffii had been proposed to be classified as Fluoribacter dumoffi, but this is still debatable. This Legionella species is also known to cause disease in human (Qin et al., 2012). 5 | Page Chapter 2 Literature Review

2.3 CELL-STRUCTURE AND METABOLISM

Legionella are Gram-negative bacteria. They are pleiomorphic organisms and have been identified as either rod or coccoid-shaped (Katz and Nash, 1978) and are 0.5 1 µm wide and 2 50 µm long (Brooks et al., 2004). Most species are motile, and have one to three polar or lateral flagellae (Rodgers et al., 1980). Interestingly, L. longbeachae has been described as non-flagellated but may have a capsule (Cazalet et al., 2010). Figure 2.1 shows an image acquired by scanning electron microscopy for L. longbeachae.

Figure 2.1: Scanning electron microscopy image of L. longbeachae

Legionellae are fastidious aerobic bacteria (Horwitz and Silverstein, 1980). They use amino acids as sources of energy, and thus the culture medium for Legionella is usually supplemented with L-cysteine (George et al., 1980). Legionellae are able to reproduce at 20°C to 40°C (Leoni et al., 2005).Whilst the list continues to grow, there are currently at least 60 species and 81 serogroups of Legionellae that have been described in literature. However, not all of the Legionella species have been reported to infect humans.

With the improvement in diagnostic tests, many new species are being identified to be implicated in infecting humans. However, the mode of infection and the incidence rate remains to be elucidated as little is known about the species. Moreover, many of the Legionella species are difficult to grow in vitro which is a hurdle in further investigations especially when culture confirmed diagnosis is required.

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Table 2.1 shows a list of Legionella species and the number of serogroups that have been reported in literature. Twenty-five species have been identified to cause infection in human.

Table 2.1: Current list of Legionella species and their pathogenesis identity

Serial Species No. No. of SG References No. of associated SG with disease 1 L. adelaidensis 1 0 (Benson et al., 1991) 2 L. anisa 1 1 (Bornstein et al., 1989b, Gorman et al., 1985) 3 L. beliardensis 1 0 (Lo Presti et al., 2001) 4 L. birminghamensis 1 1 (Wilkinson et al., 1987) 5 L. bozemanii 2 2 (Brenner et al., 1980) 6 L. brunensis 1 0 (Wilkinson et al., 1988) 7 L. busanensis 1 1 (Park et al., 2003) 8 L. cardiaca 1 1 (Pearce et al., 2012) 9 L. cherrii 1 0 (Brenner et al., 1985) 10 L. cincinnatiensis 1 1 (Thacker et al., 1988) 11 L. donaldsonii 1 1 (Fry and Harrison, 1998) 12 L. drancourtii 1 0 (La Scola et al., 2004) 13 L. dresdenensis 1 0 (Lück et al., 2010) 14 L. drozanskii 1 0 (Adeleke et al., 2001) 15 L. dumoffii 1 1 (Brenner et al., 1980) 16 L. erythra 2 1 (SG2) (Brenner et al., 1985) 17 L. fairfieldensis 1 0 (Thacker et al., 1991) 18 L. fallonii 1 0 (Adeleke et al., 2001) 19 L. feeleii 2 2 (Herwaldt et al., 1984) 20 L. geestiana 1 0 (Dennis et al., 1993) 21 L. genomospecies 1 0 (Benson et al., 1996) 22 L. gormanii 1 1 (Morris et al., 1980) 23 L. gratiana 1 0 (Bornstein et al., 1989a) 24 L. gresilensis 1 0 (Lo Presti et al., 2001) 25 L. hackeliae 2 2 (Brenner et al., 1985) 26 L. inipletisoli 1 0 (Kuroki et al., 2007) 27 L. israelensis 1 0 (Bercovier et al., 1986) 28 L. jamestowniensis 1 0 (Brenner et al., 1985) 29 L. jeonii 1 1 (Park et al., 2004) 30 L. jordanis 1 1 (Cherry et al., 1982)a 31 L. lansingensis 1 1 (Thacker et al., 1992) 32 L. londiniensis 1 0 (Dennis et al., 1993) 33 L. longbeachae 2 2 (Mckinney et al., 1981) 34 L. lytica 1 0 (Hookey et al., 1996) 35 L. maceachernii 1 1 (Brenner et al., 1985) 7 | Page

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Table 2.1: Current list of Legionella species and their pathogenicity identity (continued)

Serial Species No. No. of SG References No. of associated SG with disease 36 L. massiliensis 1 0 (Campocasso et al., 2012) 37 L. micdadei 1 1 (Hébert et al., 1980) 38 L. moravica 1 0 (Wilkinson et al., 1988) 39 L. nagasakiensis 1 1 (Yang et al., 2012) 40 L. natarum 1 0 (Dennis et al., 1993) 41 L. oakridgensis 1 1 (Orrison et al., 1983)a 42 L. parisiensis 1 1 (Brenner et al., 1985) 43 L. pneumohila 15 15 (Mcdade et al., 1977) 44 L. quateirensis 1 0 (Dennis et al., 1993) 45 L. quinlivanii 2 0 (Benson et al., 1989) 46 L. rowbothamii 1 0 (Adeleke et al., 2001) 47 L. rubrilucens 1 0 (Brenner et al., 1985, Matsui et al., 2010)b 48 L. sainthelensi 2 2 (Campbell et al., 1984) 49 L. santicrusis 1 0 (Brenner et al., 1985) 50 L. shakespearei 1 0 (Verma et al., 1992) 51 L. spiritensis 1 0 (Brenner et al., 1985) 52 L. steelei 1 ? (Edelstein et al., 2012) 53 L. steigerwaltii 1 0 (Brenner et al., 1985) 54 L. taurinensis 1 0 (Lo Presti et al., 1999) 55 L. tucsonensis 1 1 (Thacker et al., 1989) 56 L. tunisiensis 1 0 (Campocasso et al., 2012) 57 L. wadsworthii 1 1 (Edelstein et al., 1982) 58 L. waltersii 1 0 (Benson et al., 1996) 59 L. worsleiensis 1 0 (Dennis et al., 1993) 60 L. yabuuchiae 1 0 (Kuroki et al., 2007)

No.: number, SG: serogroup. a reference is for first isolation from water and not patient, as infection has been reported b a case of co-infection was found with L. pneumophila ? the species has been isolated from a clinical sample but its ability to cause infection is uncertain

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2.4 LEGIONELLA HOSTS AND LIFE CYCLE

Legionellae are non-sporulating bacteria (Duncan et al., 2011), and, in the primary host (protozoans and metazoans), the life cycle of the bacterium consists of two major phases. The first, during which the bacteria are non-motile and are less harmful, is called the replicative phase. In the second phase, called the infectious phase, the bacteria are shorter and thicker, have developed flagella and are highly infective (Rowbotham, 1986). In the opportunistic host (human), the life cycle of Legionella is also biphasic which involves a non-motile replicative phase and an infectious phase. In the latter stage, the organism develops flagella at exponential phase and induces apoptosis of the host cell, mediated through the mitochondrial signaling pathway (Neumeister et al., 2002), and is ready to infect new cells. Outside the host, Legionella may also exist in biofilms (Sheehan et al., 2005). Figure 2.2 shows a schematic representation of Legionella life cycle in the environment and in the host.

Legionella is an intracellular pathogen (Isberg et al., 2009). The pathogenesis strategy employed by L. pneumophila is quite remarkable: it survives within the phagocytic host cell by its ability to manipulate host cell processes. This involves organelle trafficking, avoiding fusion with lysosome and counteracting host immune defence pathways (Ge and Shao, 2011, Hubber and Roy, 2010). The ability of L. pneumophila to proliferate in evolutionarily different phagocytic host cells ranging from protozoans to human macrophages, suggests that the organism is capable of targeting conserved host metabolic pathways for survival and replication (Hubber and Roy, 2010).

L. pneumophila replicates in amoebae and in macrophages within a membrane-bound compartment termed the Legionella containing vacuole (LCV). A bacterial Type IV secretion system called the Dot/Icm (Defect in Organelle Trafficking/Intracellular multiplication) system, which fuses with the membrane of the phagosome and delivers bacterial proteins (called effectors) in the cytoplasmic compartment of the host cell, is required by L. pneumophila to create the LCV. It has been hypothesized that more than 150 effectors are translocated to the cytosol of the host cell by the Dot/Icm system, where they are involved in a variety of functions, of which the main one appears to be in the biogenesis of the LCV. The other effectors seem to function towards the inhibition of protein synthesis in host cell and eventually cause cell death (Vance, 2010). To replicate within the vacuole and escape lysosomal digestion, L. pneumophila modifies 9 | Page Chapter 2 Literature Review the vacuole by hijacking organelles to ultimately resemble an endoplasmic reticulum. After completion of replication, L. pneumophila disrupts the vacuole membrane and exit by rupturing the host cell. The bacteria are released in the extracellular environment, where they are ready to infect neighbouring cells and start a new replication cycle (Hubber and Roy, 2010). Similar mechanisms of replication and survival strategies have been found in L. longbeachae (Gerhardt et al., 2000).

Figure 2.2: Legionella life cycle

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2.5 LEGIONELLA ECOLOGY 2.5.1 Legionella habitat and risks of infection

Legionella is a ubiquitous organism in freshwater environments, inhabiting both natural (Fliermans et al., 1979, Fliermans et al., 1981) and artificial aquatic locations (Carvalho et al., 2007). Like many other bacterial species, Legionella can be equally found free living in the planktonic or sessile phase in water systems (Lin et al., 2011). Air- conditioning cooling towers, whirlpool spas, sink taps and shower heads are all major reservoirs for most of the Legionella spp. (Diederen, 2008) , whereas potting mixes, soil and compost are the major sources for L. longbeachae (Steele et al., 1990a). Drinking water has also been found to be an ecological niche of Legionella (Yoder et al., 2008). A study carried out by Sakamoto et al. (2009) demonstrated that car air-conditioners are potentially implicated in transmission of Legionnaires disease. Surveillance data suggest that travelers, for example on a cruise ship, can also develop the disease, and therefore, travel-associated Legionnaires disease is yet another prevalent reported source of legionellosis (De Jong et al., 2013).

Hospital-acquired legionellosis is also well documented and the presence of Legionella in the water systems can persist for a very long time (Kirby and Harris, 1987, Garcia- Nunez et al., 2008). Dental settings (Veronesi et al., 2007), steam towel warmers (Higa et al., 2012) and wash basins (Brulet et al., 2008) are a few examples of water systems in hospitals that have been found to be responsible for nosocomial Legionella pneumonia. The cases reported by Dhillon et al. (2009) and Grove et al. (2002) demonstrate how the soil dwelling L. longbeachae can also be implicated in causing nosocomial infections. As reported in the former case, the patient had a positive serum titre for L. longbeachae and disclosed to have been smoking discarded cigarette butts from a flower bed around the hospital premises. The area was found to be contaminated with L. longbeachae during an investigation to identify the causative organism.

Legionellae can persist at temperatures ranging from 0°C to 68°C and in pH range 5.0 8.5 (Diederen, 2008). The ability of Legionellae to remain viable at high temperatures and its tolerance to low nutritional availability account for its widespread and prolonged presence in many water systems, especially man-made water environments. These adaptation factors support multiplication of the organism in such environment which poses a serious risk to public health (Leoni et al., 2005, Yoder et al., 2008).

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2.5.2 Biofilm formation and its clinical significance Figure 2.3 shows a schematic representation of biofilm formation and being sloughed off which leads to re-colonisation of organisms at different locations.

Figure 2.3: Biofilm and its clinical significance In water environment, Legionellae are engulfed by grazing protozoa such as amoeba (primary host). Alternatively, the bacteria can exist in biofilms. The bacteria can colonise different locations by the displacement of sloughed off biofilms due to shear stress and also movement of protozoans. Adapted from Legionella and the prevention of Legionellosis (WHO) (Bartram J. Y., 2007)

Microbial activity (physical and chemical changes caused by microorganims) and colonization (biofilm formation) are surface-associated phenomena which occur in both natural and artificial environments on a variety of different surfaces. Microorganisms, including Legionella, form biofilms as a mechanism to withstand unfavorable conditions, such as inadequate/insufficient nutrients or temperature extremes (Rowbotham, 1980, Morton et al., 1998). The development of biofilms can be divided into three distinct phases; substratum-attachment of cells, growth of the cells to form a sessile biofilm colony and, finally, dissemination of detached cells from the colony into the surrounding environment. The last stage is crucial for the process of biofilm formation, as sloughing off of the cells contributes to the dispersal of organisms such as Legionella and also disease transmission (Kaplan, 2010).

It has been postulated concentrations of Legionellae undetectable by routine laboratory tests reach potable water distribution systems from mainstreams of fresh water (Garcia- Nunez et al., 2008). Microbial growth is nearly exclusively found as biofilms in the inner wall of pipes in these environments. The presence of amoebae or other ciliate protozoans adds to a flourishing ecological niche for Legionella (Lau and Ashbolt, 2009). Saby et al. (2005) demonstrated how different disinfection treatment protocols

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Chapter 2 Literature Review failed to completely eliminate Legionella found in a hot water distribution system, especially Legionellae found in biofilm present in the setting. This could possibly be due to the survival mechanism developed by the microbiota, making them resistant to biocides (Lau and Ashbolt, 2009). Moreover, amoebae have been demonstrated to help in the resuscitation of viable but non-culturable L. pneumophila after treatment with biocides. This could also possibly explain the re-emergence of Legionella in water systems after decontamination (Teresa Garcia et al., 2007). Legionellosis is a preventable disease and hot water systems are regularly tested for the presence of Legionellae for this purpose. Despite the advancements of detection methods and increased monitoring, contamination of hot water environments with Legionellae and their existence as biofilm remains a persistent challenge due to the incomplete elimination of the bacteria following disinfection treatments. This represents a serious threat to public health (Farhat et al., 2012).

2.6 MODE OF INFECTION

Legionellosis is a non-communicable disease. The route of transmission has been reported to be through the inhalation of Legionella contaminated aerosols for all of the Legionella species except for L. longbeachae. Although the mode of transmission of the disease by L. longbeachae is still unclear, it is believed to be associated with exposure to compost, potting mixes and soils, and may occur either by ingestion or inhalation (O'Connor et al., 2007).

2.7 CLINICAL MANIFESTATION IN HOST

The incubation period of pneumonia due to Legionella ranges from 2 to 18 days (Darby and Buising, 2008). The clinical symptoms of legionellosis are non-specific and therefore cannot be readily differentiated from other types of community-acquired pneumonia. This can be exhibited by multi-organ dysfunction and may include, but is not limited to, central nervous system abnormalities (headache, mental confusion, encephalopathy and lethargy), cardiac irregularities (relative bradycardia), gastrointestinal disorders (diarrhea, abdominal pain), hepatic manifestations (increased levels of serum transaminases) and renal anomalies (microscopic haematuria, elevated creatinine level) (Cunha, 2006). Systemic symptoms such as high fever and dry cough tend to predominate during the early stage of infection (Darby and Buising, 2008).

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2.8 COMMUNITY-ACQUIRED PNEUMONIA AND ECONOMIC BURDEN

Community-acquired pneumonia (CAP) remains a serious problem worldwide with a substantial economic burden of more than $17 billion per annum (estimated) in the USA (File and Marrie, 2010), 2.5 billion estimated cost involved with inpatients only in Europe (Gibson et al., 2013) and $20 million in Australia for only General Practitioner related costs (Li et al., 2012). A study carried out by Collier et al. (2012), estimated a cost of $33,366 per hospitalised case of Legionnaires disease in USA with an estimated yearly national insurance claim amounting to $434 million. It is estimated that in Europe, the economic burden associated with Legionnaires disease arising from the healthcare and absenteeism amounts to over 1 billion (Cossali et al., 2013). Engel et al. (2013), demonstrated that although the use of the Legionella urinary antigen test was associated with a considerable cost, the etiological agent was identified with a significant reduction in the amount of time required for a timely targeted treatment. As most of the cost (80%) involved in community acquired pneumonia is associated with inpatients (Martinez et al., 2009), early diagnosis and treatment may reduce the economic impact.

2.9 CAP: TYPICAL AND ATYPICAL PNEUMONIA PATHOGENS

The etiological agent of community-acquired pneumonia (CAP) remains unidentified in more than two-third of reported cases. Nevertheless, data suggest that CAP is mainly caused by bacterial species (Patterson and Loebinger, 2012). The bacteria responsible for community-acquired pneumonia have been divided into two groups: the typical and the atypical arms. The term atypical pneumonia is applied to a particular set of bacteria (respiratory pathogens), known to cause lower respiratory tract infections (Blasi, 2004), and comprises Chlamydia psittaci, Chlamydia pneumoniae, Coxiella burnetii, Francisella tularensis, Legionella spp. and Mycoplasma pneumoniae (Prasad, 2012).

Although typical pneumonia pathogens account for most (~50%) of the reported CAP cases, with Streptococcus pneumoniae being the leading cause (Lim et al., 2009, Patterson and Loebinger, 2012) and atypical pathogens representing about 15% of cases, the latter is more common in mild or ambulatory CAP in adults (Cunha, 2006). Atypical pneumonias can be further clinically classified as zoonotic and non-zoonotic.

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Psittacosis (Chlamydia psittaci), Q fever (Coxiella burnetti) and tularaemia (Francisella tularensis) are zoonotic pneumonias whereas pneumonia caused by Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella are non-zoonotic (Cunha, 2006).

2.10 IDENTIFICATION OF THE ETIOLOGICAL AGENT OF CAP

A simplistic approach for the prediction of the etiological agent of CAP is presented in Figure 2.4.

clinical pneumonia (confirmed by chest radiography)

Extrapulmonary features present No extrapulmonary features (atypical bacterial pneumoniae) (typical bacterial pneumoniae)

Zoonotic No zoonotic contact history S. pneumoniae contact history C. psittaci H. influenzae Mycoplasma C. burnetti M. catarrhalis C. pneumoniae F. tularensis K. pneumoniae Legionella

Group A streptococci Aspiration No Relative Relative Relative No Relative pneumonia bradycardia bradycardia bradycardia bradycardia Mycoplasma C. psittaci Legionella F. tularensis C. pneumoniae C. burnetti

Confirmatory laboratory tests

Figure 2.4: Diagnosis based on clinical symptoms Adapted from Cunha, 2006

Cunhas schematic representation of the clinical diagnosis of potential etiological agent of CAP appears simple and can be applied as the first line of diagnosis until a confirmatory test is carried out in the laboratory for initiation or change of antibiotic therapy. Assumptions about the etiological agent can be made referring to the chart and may also help to rule out other suspected organisms. This can especially be useful during outbreaks.

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The diagnosis of CAP involves a wide range of clinical, radiological, microbiological and biochemical techniques. Most of the CAPs are currently diagnosed in the primary care institutions where the limited use of the majority of available diagnostic tests makes clinical history and physical examination important in diagnosis. In the event of being challenged by treatment failure, the clinicians have to resort to more intensive and accurate investigations (Prasad, 2012). As such, the patients history regarding any potential exposure to Legionella in conjunction with physical examination and clinical findings may help to narrow down the diagnostic possibilities for physicians (Cunha, 2006).

Treatment of CAP at the site of care is generally managed using developed local or national guidelines that contain recommendations for evaluation, prognosis and treatment procedures. Different hospitals and healthcare institutions use different methods of diagnosing CAP cases before administering and monitoring medication. Several scoring systems; Pneumonia Severity Index (PSI) (Fine et al., 1997), Confusion-Urea-Respiratory Rate-Blood pressure-65 (CURB-65) (Lim et al., 2003) , Severe Community Acquired Pneumonia (SCAP) (Espana et al., 2006) and SMART- COP (Charles et al., 2008) are among a few that have been developed to guide physicians to identify patients at high risk of CAP. This may depend on the severity of the cases, the tests available and the clinical point scale systems used within the institutions based on a syndromic approach.

The severity scoring tool usually acts as the first point of management of CAP in most health institutions. However, a study carried out by Serisier et al. (2013) suggested that the severity scoring tool in management of CAP was infrequently used in Australia probably because of confusion in which protocol and guidelines to follow, and sometimes due to the inadequate training of the staff to correctly use the scoring system. This suggests that despite CAP management tools being available to help physicians in their task, these may seldom be used. Moreover, the severity scoring tool is mainly for risk stratification (categorising patients as outpatient, inpatient or intensive care) rather than pathogen directed treatment. Nevertheless, this system appears to help the physician to recommend microbiological diagnosis whenever deemed required.

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Although medical history evidences and information about the geographical and environmental factors could be suggestive of a particular etiological agent of CAP, this approach is neither as sensitive nor specific for guiding antibiotic therapy (Wiersinga et al., 2012). Yu and Stout (2008) expressed their concerns that clinical characteristics are used for diagnosis of Legionnaires disease and therefore recommended the use of laboratory confirmed tests because of the risks for fatal consequences owing to under- diagnosed cases of legionellosis.

2.11 TREATMENT OF CAP AND LEGIONNAIRES’ DISEASE

Despite Legionnaires disease being a treatable disease, like pneumococcal pneumonia, it has the highest mortality rate among severe CAP cases (Yu and Stout, 2008). This can be explained by the different local and national guidelines used to diagnose and treat CAP. A Danish study (St-Martin et al., 2013) provides an example whereby high mortality rate was noted for legionellosis cases. The reason for the fatalities, as explained by the authors, is most likely to be the guidelines for treatment of CAP in that country. As evaluated in the study, penicillin (-lactam agent) which is inefficient against Legionella was the antibiotic of choice for initial treatment of CAP.

The choice of antibiotics for the treatment of legionellosis depends on the severity of the disease. Macrolides have been the first reference treatment for legionellosis in non- severe manifestations. Either a fluoroquinolone monotherapy or a combination therapy, which includes any of the following antibiotic classes; macrolides, fluoroquinolones or rifampicin, is generally used in severe cases (Chidiac et al., 2012). Legionella is an intracellular pathogen, and macrolides and fluoroquinolones, which are able to penetrate cells, have demonstrated good activity. However, fluoroquinolones are able to attain high intracellular levels and have a lower minimum inhibitory concentration against Legionella and also have been reported to exhibit superior clinical activity as compared to macrolides (Zarogoulidis et al., 2011).

An empiric therapy (generally beta-lactams and macrolides combinations or fluoroquinolones alone), commonly used for the treatment of CAP is usually based on a broad range antibiotic treatment that covers both typical and atypical pathogens (Robenshtok et al., 2008). However, the preferred treatment of CAP is still debatable. Several studies and meta-analyses have been carried out but there does not seem to be a consensus about the treatment regimen to follow. 17 | Page Chapter 2 Literature Review

Whilst some studies suggest that there is no notable difference in consequences from using a broad spectrum antibiotic regimen (combination antibiotic therapy) rather than pathogen-directed treatment (monotherapy) (Van Der Eerden et al., 2005, Chalmers et al., 2011), others found that there is a significantly better outcome with decreased mortality rate (Caballero and Rello, 2011, Rodrigo et al., 2013) especially for legionellosis (Eliakim-Raz et al., 2012). Although Van der Eerden et al. (2005) noticed that there was no significant difference comparing combination antibiotic therapy to monotherapy, they noted that infection with Legionella was an exception for the atypical arm as observed by others. It should also be noted that the use of the scoring system for managing CAP, and administration of antibiotics have biases and/or limitations in them. As a matter of fact, the outcomes of most studies are not reproducible (Waterer et al., 2011) and for that reason not completely reliable. Nonetheless, it is recognised that among all the atypical pneumonia pathogens, Legionella is the only one which requires specific therapy (Robenshtok et al., 2008, Bartlett, 2011).

2.12 IMPLICATIONS OF IMPROPER USE OF ANTIBIOTICS

As the etiology of CAP is often unknown at presentation, patients are initially prescribed with an empiric antibiotic regimen (Stralin, 2008). However, while, in terms of its scale of activity and efficacy, this may be suitable, there has always been a growing concern regarding the implications of improper use of antibiotics with such practice (Dryden et al., 2009). An early initial treatment failure can be due to administrations of improper antibiotic or an incorrect diagnosis (Nair and Niederman, 2011). The emergence of antibiotic-resistant pathogens and the paucity in development of new antibiotics is the greatest threat associated with inappropriate use of antibiotics (Charles and Grayson, 2004, Wise et al., 2011). Adverse effects and increased cost are also associated with no pathogen-targeted treatment (Mandell et al., 2007, Sorde et al., 2011). High mortality rate can also be a result of inappropriate empiric antibiotic prescriptions (Lueangarun and Leelarasamee, 2012).

Blasi (2004) suggested that the macrolides, the most commonly used class of antibiotics especially in empiric treatment to cover atypical respiratory pathogens because of their demonstrated efficiency, have to be closely monitored because of the risk of emergence

18 | Page Chapter 2 Literature Review of resistant strains in pathogens which do not necessarily require treatment with macrolides. Chidiac et al. (2012), recommend the use of fluoroquinolones for severe cases only because of the concerns of antibiotic resistance. As evaluated by Welte et al. (2012), antibiotic resistance has significant clinical and economic repercussions. In the event of empiric antibiotic treatment failure owing to resistance, this can result in an increase in the cost of treatment in case a more expensive class of antibiotics, or longer stay at the hospital, is required or may also eventuate into death of patients.

Brown et al., (2012) suggest that although antibiotics and vaccines are readily available for important respiratory pathogens, CAP remains a substantial and prevailing health problem with considerable complications and mortality rate. Therefore, for a potentially improved risk stratification and outcome, the authors propose the use of biomarkers and individualised patient management. Indeed, Sordé et al. (2011) demonstrated beneficial outcomes of knowing the etiological agent by carrying out specific laboratory tests which is helpful in pathogen-directed therapy (narrow spectrum antibiotic).

2.13 EPIDEMIOLOGY OF LEGIONELLOSIS

Epidemiology is of paramount importance since it is considered as the foundation of public health practice. The study of the health of human populations provides valuable scientific information about the agents, hosts and environmental factors that affect human health. This helps in determining the causes and trends of illnesses, identification of population groups at risk from a particular disease, evaluation of efficiency of health programs and establishing priorities for research and action (Brownson, 2011).

Since the initial outbreak in Philadelphia, legionellosis has gained a considerable epidemiological attention and an increasing amount of information is being unraveled about the disease. Legionnaires disease is a notifiable disease in many countries which enables a better understanding of the incidence rate and the prevalence of the disease.

Figure 2.5 shows the yearly notification rate of legionellosis cases in Australia, USA and Europe from 2003 to 2012. The rate of notification was found to be higher in Australia compared to USA and Europe. There could be several reasons for such an observation. Firstly, the disease may be more prevalent in Australia. The other reason

19 | Page Chapter 2 Literature Review could be the fact that Australia is a smaller continent; the surveillance program is possibly more efficient. Yet another explanation for a higher notification rate in Australia could be due to the use of better diagnostic tests and also routine microbiological testing for CAP cases.

Legionellosis notification rate

1.8 1.6 1.4 1.2 1 Australia 0.8 USA 0.6 Europe 0.4 0.2 0

Notification rate per 100,000 population 2002 2004 2006 2008 2010 2012 Year

Figure 2.5: Legionellosis notification rate http://www9.health.gov.au/cda/source/rpt_4.cfm http://www.cdc.gov/mmwr/PDF/wk/mm6053.pdf http://ecdc.europa.eu/en/publications/Publications/legionnaires-disease-in-europe-2011.pdf

The relative importance of L. longbeachae infection versus L. pneumophila in the Australasian region appears to be unique. Although, L. pneumophila has been consistently reported to be the cause for the majority of Legionella infections worldwide, L. longbeachae infection is interestingly more prevalent in countries such as Thailand (Phares et al., 2007), Australia (Milton et al., 2012) and New Zealand (Lau et al., 2013).

Figure 2.6 shows the prevalence of Legionnaires disease per state/territory per species in Australia in 2010. As it can be noticed, the two major species reported to cause infection are L. pneumophila and L. longbeachae. However, the incidence of infection due to the different species is dissimilar in different states/territory. The epidemiological distribution of L. longbeachae indicates that infections are more common in Western and South Australia, compared to other parts of Australia.

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Cases of legionellosis in Australia in 2010 by species and state/territory 60

L. longbeachae 30 L. pneumophila 20 L. bozemanii unknown spp Number of notified cases 10

0 ACT NSW NT Qld SA Tas Vic WA State or Territory

Figure 2.6: cases of legionellosis in Australia in 2010 ACT: Australian Capital Territory, NSW: New South Wales, NT: Northern Territory, Qld: Queensland, SA: South Australia, Tas: Tasmania, Vic: Victoria, WA: Western Australia.

Figure 2.7 shows the percentage incidence of infection cases reported per Legionella species in 2010. As it can be seen, the number of cases reported for infection due to L. longbeachae was slightly higher than L. pneumophila.

Percentage of cases of Legionellosis in Australia in 2010 per species

Unknown spp, 8.72 L. bozemanii, 0.34

L. longbeachae, 46.31 L. pneumophila, 44.63

Figure 2.7: percentage of cases of legionellosis in Australia in 2010 (http://www.health.gov.au/internet/main/publishing.nsf/Content/cda-cdi3601-pdf- cnt.htm/$FILE/cdi3601.pdf)

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2.14 FACTORS CONTRIBUTING TO THE PREVALENCE OF LEGIONELLOSIS

Legionellosis is considered a non-communicable disease as there have not been any reported cases of person-to-person disease transmission (Darby and Buising, 2008). However, the dispersal distance of Legionella contaminated aerosols has been found to be more than 10 kilometers in suspected outbreaks in New-Zealand (White et al., 2013) and Norway (Nygård et al., 2008). Geostatiscal and molecular techniques used during investigation for the outbreak in New-Zealand suggested the anisotropic spreading of aerosol to the prevailing wind in the area encompassing the different clusters of interest where infection cases with L. pneumophila were reported.

Whiley and Bentham (2011) suggested that composting materials could offer a more conducive environment than soil with high moisture content and heat for the multiplication of L. longbeachae. The fact that the L. longbeachae genome encodes proteins that can metabolise plant compounds, may explain its presence in plant-derived compost materials (Cazalet et al., 2010). Studies have shown that the difference in the potting mix components in Australia compared to European countries may be the reason for the occurrence of Legionella longbeachae in Australian potting mixes (Steele et al., 1990b) and hence the high prevalence of L. longbeachae infection in Australia. Lau et al. (2013) mentioned that the high prevalence of L. longbeachae infections in New Zealand was noted during warmer spring and summer months during which most of agricultural and horticultural activities occur. These activities involve the use of soils and composts, and therefore greater potential exposure to contaminating L. longbeachae.

Environmental factors appear to affect the incidence rate of legionellosis which seems to be a seasonal disease with more reported cases around late spring and autumn and greater prevalence in temperate regions (Graham et al., 2012). Herrera-Lara et al. (2013) noticed a higher rate during summer with elevated temperature. Increased rainfall has also been associated with increase in legionellosis cases (Hicks et al., 2007). Conza et al. (2013) identified that higher water vapor pressure and heat were linked with a higher rate of incidence of legionellosis. These meteorological influences are indicative of climatic factors that are involved with legionellosis incidence rate, although they may differ depending on geographical regions (Conza et al., 2013,

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Graham et al., 2012). However, Travis et al. (2012), failed to identify any connection between the environment and incidence of L. longbeachae infections as did Cunha et al. (2010), for the unusually high incidence of L. pneumophila infections during the H1N1 influenza pandemic. Euser et al. (2012) were also not able to determine the reason for the decrease in the number of legionellosis reported cases during 2009 in the Netherlands. The incidence rate of reported legionellosis case appears to be inconsistent and may be dependent on several variables and, therefore, warrants further investigations.

Protozoans are the primary host of the Legionellae, and the latter is an opportunistic pathogen of human (Wang et al., 2012). Legionellae exist as planktonic organisms or biofilms if not replicating in amoebae in water and soil environments. Metazoans have also been demonstrated as a potential natural host of Legionella spp. in the soil environment (Brassinga et al., 2010). These environments are the major colonising and multiplication reservoirs of the organisms until infection occurs in human through inhalation of dispersed Legionella contaminated aerosols (Hilbi et al., 2011). Many countries have set guidelines for Legionella control to minimise risks of infection, such as monitoring and decontamination of cooling towers (Carducci et al., 2010). Cramp et al. (2010) also recommended warning labels of risks of acquiring legionellosis on potting mixes packaging and the use of proper safety apparatuses to minimise the possibility of exposure to contaminating L. longbeachae. Moreover, an earlier study carried out by OConnor et al. (2007), also suggested the warning labels on potting mix packaging. The authors also referred to the lack of epidemiological evidence about the risk factors involved with L. longbeachae infection. Despite set guidelines for preventive measures, legionellosis cases are being increasingly reported (Rota et al., 2013, Neil and Berkelman, 2008).

An understanding of the local Legionella epidemiology is significant in deciding whether to test for Legionella infection, and if so, which test to use. However, the number of confirmed diagnoses of legionellosis outside of a research setting is irregular. In routine practice, this failure in the diagnosis of Legionnaires disease is mainly because of the impracticably difficult distinction of legionellosis from other causes of pneumonia, failure to order diagnostic tests and the inadequacies in the available diagnostic tests for diagnosis of Legionnaires disease (Murdoch, 2003). 23 | Page

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2.15 LEGIONELLOSIS SYMPTOMS, CO-INFECTION AND UNDERLYING CO- MORBIDITIES Legionellosis is a serious disease that can develop into a life-threatening illness. The disease has been identified to be more common in elderly people, immunocompromised patients and smokers (Hilbi et al., 2010). The disease can cause several other complications apart from pulmonary disease, such as encephalomyelitis (De Lau et al., 2010), multi-organ dysfunction (Kassha et al., 2009) and even fetal demise (Vimercati et al., 2000). The common symptoms of Legionnaires disease include high fever, dry cough, dysnopea, confusion, hyponatraemia, hepatic dysfunction and gastrointestinal symptoms especially diarrhea (Darby and Buising, 2008). However, these clinical and radiological symptoms are difficult to use in diagnosis as they are not specific to legionellosis (De Lau et al., 2010). Apart from the non-specific nature of clinical manifestations, Legionnaires disease can be masked by co-infections (Hilbi et al., 2010), underlying comorbidities and consequently result in misdiagnosis (Sopena et al., 2007, Hunter et al., 2013). Therefore for any suspected cases of legionellosis a specific diagnostic test is recommended for a timely appropriate antibiotic therapy and a favorable outcome (Zarogoulidis et al., 2011, St-Martin et al., 2013).

2.16 LEGIONELLA LONGBEACHAE AND LEGIONNAIRES’ DISEASE

Legionellosis due to L. pneumophila has been extensively studied. However, few studies have been carried out on L. longbeachae in spite of its importance in some countries (Amodeo et al., 2010). Although L. longbeachae may adapt to a different ecological niche compared to its congeneric species, the clinical manifestations in human infections appear to be broadly similar. While the mode of transmission of the bacteria is uncertain and believed to be either by ingestion or inhalation, gardening and use of potting mixes seem to be unique factors that have been associated with a greater risk of L. longbeachae infections. Poor hygiene and smoking have been found as host related predictable factors of increased risk of infection by L. longbeachae (O'Connor et al., 2007).

L. longbeachae has been described as an emerging etiological agent of legionellosis because of its high prevalence in certain countries (Whiley and Bentham, 2011). Following both in vivo and in vitro studies, Doyle et al. (2001) suggested that Australian isolates of L. longbeachae were more virulent compared to non-Australian 24 | Page Chapter 2 Literature Review isolates. This may explain the higher incidence of infections with L. longbeachae in the Australasian region compared to other parts of the world. Improving diagnostic techniques with higher sensitivities and specificities may be another reason for the ever increasing number of reported cases of L. longbeachae infection. Despite some limitations of the in-house-PCR technique used during their investigation in Christchurch, New-Zealand, Murdoch et al. (2013) demonstrated that 85% of reported legionellosis cases were due to L. longbeachae with an increase of more than four-fold in the total number of detected cases, as compared to the conventional culture method used.

A plausible explanation for the increase in the number of infections with L. longbeachae being reported in other parts of the world, such as the United Kingdom (Pravinkumar et al., 2010) and the Netherlands (Den Boer et al., 2007), could be the use of improved epidemiological surveillance systems (Joseph et al., 2010, Whiley and Bentham, 2011).

It is now believed that the current status regarding the number of infections with L. longbeachae may actually be under-reported, most likely because of the unavailability of specific diagnostic tests (Kozak et al., 2010). As this is the case with other Legionella species, it is likely that infection with L. pneumophila is being over-reported.

2.17 DIFFERENCES BETWEEN L. PNEUMOPHILA AND L. LONGBEACHAE

The genome sequences of L. longbeachae (NSW150 and D-4968) unraveled significant differences between its congeneric species, L. pneumophila (Philadelphia). The chromosome of L. pneumophila Philadelphia is about 680,000 base pairs smaller than L. longbeachae NSW150 and D-4968. The L. longbeachae NSW150 genome consists of a chromosome and a plasmid of 4,077,332 and 71,826 base pairs in size, respectively. L. longbeachae NSW150 has been predicted to encode 3,512 proteins of which 58.3% have not been assigned any putative function. The L. longbeachae D-4968 genome consists of 3,821 protein coding genes. On the other hand L. pneumophila Philadelphia is reported to encode 2,942 proteins (Kozak et al., 2010, Cazalet et al., 2010). The genes found only in either species of the congeneric species have been found to be associated with their adaptation to a particular niche, lifestyle, locomotion and sensory functions, and virulence (Cazalet et al., 2010, Kozak et al., 2010).

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L. longbeachae is predominantly found in soils and compost materials whereas L. pneumophila is found in cooling towers and other man-made water environments. L. longbeachae has been found to encode homologues of eukaryotic proteins which suggest potential relationships with plants. The latter also encodes proteins that are found in phytopathogenic bacteria such as enzymes that might have the ability to degrade plant materials, especially cellobiohydrolase and pectin lyase which are not found in L. pneumophila. L. longbeachae has been described to possess putative cyanophycin synthase and cyanophycinase genes for cyanophycin metabolism. Except for Acitenobacter baylyi, cyanophycin has so far been found in cyanobacteria only and it serves as a nitrogen, carbon and energy storage compound. The cyanophycin may be used to supply energy when substrate could be limiting. These metabolic features of L. longbeachae are reflective of its soil habitat (Cazalet et al., 2010).

Chemotaxis system related genes have also been found in L. longbeachae but not in L. pneumophila. The presence of a chemotaxis system enables organisms to migrate towards more favorable conditions (Cazalet et al., 2010, Kozak et al., 2010).

L. longbeachae may possibly have an outer capsular envelope as compared to L. pneumophila, but, unlike L. pneumophila, the genome of L. longbeachae does not encode flagella although certain flagellar regulatory genes are present. However, Asare et al. (2007) reported the presence of flagella in certain L. longbeachae strains. Cazalet et al. (2010) and Kozak et al. (2010) suggest that the absence of flagella in L. longbeachae could possibly explain the difference in their susceptibility to murine macrophages. Irrespective of the presence or absence of flagella in L. longbeachae, most mice strains are resistant to L. pneumophila infection but more susceptible to L. longbeachae (Asare et al., 2007). Flagellin has been demonstrated to be responsible for inducing caspase 1 activity mediated proinflammatory macrophage death and also makes the bacteria vulnerable to Naip5 protein of the murine innate immune system response (Molofsky et al., 2006). As genome sequencing data suggests that L. longbeachae most likely does not encode flagella, it could be the reason for its higher infectivity potential in murine models as compared to L. pneumophila (Cazalet et al., 2010, Kozak et al., 2010).

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A comparative analysis, by Cazalet et al. (2010), of the differential expression of genes at two phases of growth, the exponential (replicative/avirulent) and post-exponential (transmissive/virulent) phases in both L. pneumophila and L. longbeachae, indicated a less pronounced biphasic lifestyle of L. longbeachae. As speculated by the authors, the difference in the transcriptome of L. longbeachae at the two different phases of growth is not as strongly marked as in L. pneumophila, it might be able to proliferate intracellularly irrespective of the growth phase (Asare and Abu Kwaik, 2007) and therefore may be ready to infect at an early stage of the life cycle.

The contrasting features of the two Legionella species may be indicative of certain selective pressures to adapt to different ecological niches, and explain the dissimilarity in susceptibility to the host defense system (Kozak et al., 2010, Cazalet et al., 2010).

2.18 INFECTION AND IMMUNE RESPONSE

There exist two aspects of the human immune system; the innate and the adaptive immune systems. The innate immune system is evolutionarily older and is mostly the first line of defense against harmful intruders by identifying and eliminating them. It comprises a range of biologically active molecules such as cytokines and phagocytic cells such as macrophages. Despite the innate system being rapid in responding by inducing an inflammatory response, this aspect of the immune system lacks specificity and memory and therefore subsequent response to the same intruder may not be as efficient. The innate system has therefore further evolved into a sophisticated system which is the adaptive immune system. This system consists of antigen-presenting cells such as dendritic cells, and lymphocytes such as T and B cells. The adaptive immune system mainly recognises proteins which are antigenic and this system offers a remarkable specificity with the ability to distinguish between structural differences even due to one differing amino acid. Unlike the innate immune system, the adaptive immune system can retain memory which enables a highly specific, rapid and enhanced response in the event of a subsequent encounter with the same antigen. Nevertheless, the innate immune system is recognised to function closely with the adaptive immune system by guiding the latter to respond appropriately to a particular kind of intruding microorganism (Lilic, 2009).

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In case cell-mediated immunity is required for efficient protection, whereby T-cytotoxic or phagocytic cells are recruited, the innate immune system will recruit T-helper 1 (Th1) cells, which produce cytokines such as interferon gamma (IFN-), and interleukin 12 (IL-12), responsible for the activation of monocytes and macrophages. If antibodies are needed, the innate immune system triggers the secretion of cytokines such as IL-4, IL-5, IL-10 and IL-13, thereby inducing a Th2-type response that ensures activation of B cells and antibody (IgA, IgG and IgM) production (Lilic, 2009). As suggested by Lilic (2009), an understanding of the mechanism underlying the immune response to infection will be helpful in improving diagnosis and treatment.

Humoral (antibody-mediated) immunity is vital for host in counteracting bacterial pathogens (Twigg, 2005). Legionella is a pathogen of the lower respiratory tract. Apart from a critical strong inflammatory response in limiting the proliferation of Legionella in the lungs, T cells and B cells are also required for clearing the infection (Newton et al., 2010). The relative amount of immunoglobulins found in the lower respiratory tract, with IgG being of prime importance differs radically from that of the upper respiratory tract (Twigg, 2005). Immunoglobulins are the second most abundant class of proteins found in bronchoalveolar lavage after albumin. Approximately the same level of the four IgG (IgG 1 - 4) subclasses is found in the bronchoalveolar lavage as in serum. However, the IgG1 subclass which is involved in response to protein antigens comprises 60 to 70% of the total amount of IgG with the IgG2 subclass which acts against polysaccharide antigens consisting of 20 to 25% (Merrill et al., 1985, Twigg, 2005).

Antibodies have been demonstrated to confer protection from recurrent L. pneumophila infection (Weber et al., 2012). The authors identified several immunogenic proteins, most of which were surface exposed and therefore more accessible to antibodies. This opens the avenue for vaccine development for Legionnaires disease.

2.19 PROTEINS AS ANTIGENS AND IMMUNOGENS

An immunogen is a substance that has the ability to elicit an immune response by stimulating B cells, T cells or both. An antigen on the other hand, is a molecule that reacts with the products (antibodies and/or T lymphocyte receptors) of an immune response triggered by an immunogen. An antigenic determinant, alternatively called an epitope, is the specific domain of an antigenic molecule that stimulates an immune 28 | Page Chapter 2 Literature Review response. An antigen may consist of several epitopes that can be either linear or conformational. An immunogen is normally at least of 10,000 kDa in molecular weight and generally a protein or a polysaccharide. Antigens may cross-react if there are shared domains found on the antigenic determinant of a different antigen (Cruse and Lewis, 2003). Therefore, the specificity of an epitope lies in its conformation and composition.

Approximately 40% of the surface of many bacterial species is covered by polysaccharides, mainly from the capsule. These polysaccharides may be virulence associated and also immunogenic. However, polysaccharides may be poor immunogens and not considered for purposes such as vaccine development (Von Gabain and Klade, 2012) because firstly, most of the polysaccharide induced immune responses are T lymphocyte independent, and therefore lack memory. Secondly, the high structural heterogeneity between the polysaccharides found within and across species is problematic, which is the case with Legionella (Luck and Helbig, 2013). Thirdly, because of their structural similarity to human glycolipids and glycoproteins, some polysaccharides are poor immunogens (Weintraub, 2003). Some species may have no capsule at all which mainly consists of polysaccharides (Von Gabain and Klade, 2012).

Unlike polysaccharides, proteins are normally more antigenic and stimulate antibody classes with high complement-fixing ability (Von Gabain and Klade, 2012). Literature shows that numerous studies have been carried out on bacterial species to identify antigenic proteins from different sub-cellular localizations for diagnostic applications and targeting them as potential vaccine candidates.

2.20 BACTERIAL OUTER-MEMBRANE AND OUTER-MEMBRANE PROTEINS

A schematic representation of the cell envelope of Gram-negative bacteria is shown in Figure 2.8. Gram-negative bacterial cells are surrounded by an outer envelope comprising of an inner and an outer membrane separated by a thin layer of peptidoglycan. The inner membrane is the site for several biochemical reactions and synthesis of structural membrane components. On the other hand, the outer membrane acts as a physical barrier between the inner cellular components and the external environment. Moreover, the outer membrane also contains components that are involved in binding and trans-membrane transportation of components required for bacterial biochemical processes (Silhavy et al., 2010). The bacteria also decorate the 29 | Page Chapter 2 Literature Review cell surface with several proteins which are virulence and pathogenesis related and are of particular interest as they are the first point of pathogen-host interactions.

MAP

MOMP IP P P Outer membrane

Peptidoglycan Inner membrane

IP Inducible proteins P Porin Proteins

MAP Membrane associated proteins

MOMP Major Outer membrane proteins Minor Outer membrane proteins Figure 2.8: Gram-negative outer membrane structure Adapted from Chart, 1995

Proteins play major roles in the structure and function of the outer membrane. Some are considered to be constitutive components of the outer membrane, as they are always present irrespective of the bacterial environment. Pore-forming proteins involved in the passage of molecules across the outer membrane and lipoproteins that act as an anchorage of the outer membrane to the peptidoglycan layer are such examples of the constitutively expressed proteins. In contrast, other proteins are found in the outer membrane only under certain environmental conditions and are called inducible proteins (Chart, 1995).

2.21 ANTIGENICITY OF OUTER-MEMBRANE PROTEINS

The surface exposed proteins of the outer membrane are of high importance as they harbour domains that are vital for the survival of the organism (Confer and Ayalew, 2013) and responsible for their pathogenicity (Khemiri et al., 2008). Some of the outer membrane proteins possess antigenic epitopes that elicit the production of antibodies and identification of the latter may be useful in the development of diagnostic tests.

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The major constitutive outer membrane porin protein F of Pseudomonas aeruginosa (Peluso et al., 2010) and inducible heat shock proteins from many different bacterial species, in certain cases even without the need to be induced (Chowdhury et al., 1996), have been reported as antigenic proteins.

2.22 ANTIGENIC PROTEINS OF LEGIONELLA

Several outer membrane proteins of Legionella have been identified and reported to be immunogenic. These proteins have been subjects of further investigations for their applications as biomarkers in the diagnosis of legionellosis.

2.22.1 Major outer membrane protein (24kDa)

Butler et al. (1985) identified a 24 kDa major outer membrane protein to be antigenic in Legionella. This is believed to be the same protein reported by other groups to be in the range 24 to 29 kDa (Nolte and Conlin, 1986). This difference in molecular weight could be due to different extraction methods (Butler et al., 1985). However, out of nine other species included in their study (Butler et al., 1985), L. bozemanii serogroup 1 did not show any cross-reactivity to the polyclonal antibody produced against the protein, whilst Nolte and Conlin (1986) found a species-specific epitope to L. pneumophila with a monoclonal antibody.

2.22.2 Macrophage Infectivity Potentiator (MIP)

The Macrophage Infectivity Potentiator (MIP) protein exhibiting petidyl-prolyl-cis- trans-isomerase activity has been identified as a surface exposed protein which possesses a genus-specific epitope (Helbig et al., 1995). Even though Legionella- specific reaction was noticed on immunoblot, an ELISA test demonstrated that six of the Legionella species exhibited a weak reaction. This could possibly be because of the difference in the accessibility of the epitope on intact cells as explained by the authors (Helbig et al., 1995).

MIP appears to be an inducible protein of the outer membrane. Although cross- reactivity was not observed with other genera, this study did not include other pneumonia causing species such as Coxiella burnetti and Rickettsia, which appear to express protein of about 50% homology to the MIP protein. Moreover, no further 31 | Page Chapter 2 Literature Review characterisation of the genus-specific epitope was carried out to detect similar epitope- specific antibody in serum samples from humans infected with Legionella. Neither has it been reported in the literature that a monoclonal antibody was used to detect the antigen in human body fluids. This may explain why no diagnostic test appears to be commercially available based on this antibody.

2.22.3 Peptidoglycan associated lipoprotein (PAL, 19 kDa)

The 19 kDa peptidoglycan associated lipoprotein (PAL) is another immunogenic outer membrane protein of Legionella. This protein was analysed for its specificity and sensitivity by Kim et al. (2003). Although the authors claim the characterised protein to be highly specific to Legionella, and their test to be rather sensitive, it does not appear to be commercially available. This may be due to certain limitations in their study. Firstly, from the myriad of Legionella species, only six were tested to demonstrate genus-wide cross-reactivity.

Secondly, as demonstrated by Engleberg et al. (1991), the protein appears to have a structural homology to lipoproteins found in other gram-negative bacteria with over 50% of amino acid similarity to E. coli and Haemophilus influenza PAL. Even though the authors mentioned that control urine samples were used to detect presence of the antigen and determine any cross-reactivity from non-Legionella pneumonia patients, the etiological agents were not specified. Thirdly, no soluble antigen was prepared from other pneumonia causing bacteria to determine the specificity of the antibody raised against the antigen.

Moreover, the urine samples obtained from guinea pigs infected with Legionella and controls, showed some ambiguous results.Whilst, urine samples from two out of nine non-pneumophila species infected guinea pigs showed no reactivity to anti-PAL antibody, some control urine samples showed slight reactivity. The authors also mentioned that for some of the Legionella species, the PAL was not very distinctive on the SDS-PAGE gel even though there was strong reaction to anti-PAL IgG in the immunoblot. Therefore, this antigen may not be appropriate to use in diagnosis unless a Legionella genus-specific epitope is determined for the PAL or further cross-reactivity confirmatory tests are carried out.

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2.22.4 Heat shock protein (60 kDa)

Sampson et al. (1986), identified a 58 kDa protein as a genus-wide antigenic protein. This protein was further characterised in several other studies. The protein was identified to possess a Legionella genus-specific epitope as well as cross-reactive epitopes with other bacterial species (Plikaytis et al., 1987, Pau et al., 1988). This led to advanced investigations whereby Helsel et al. (1988) identified it as a cytoplasmic membrane protein, varying from 57.2 to 62.1 kDa in molecular weight across 23 Legionella species tested, and raised a monoclonal antibody against the protein. However, the monoclonal antibody showed cross-reactivity to homologous proteins in other genera. The protein was later described as the 60 kDa Legionella Heat Shock Protein (HSP) and further characterised by Steinmetz et al. (1991), who identified a monoclonal antibody that was Legionella-specific. However, the antibody was not tested for its activity against human body fluid samples to detect the antigen. All of the studies seem to have some limitations.

2.23 CROSS-REACTIVITY OF IMMUNOGENIC PROTEINS

Immunogenic reaction with antigen from L. micdadei, and sometimes L. dumoffi, seemed to be very low in the studies carried out by Sampson et al. (1986), Plikaytis et al. (1987), and Pau et al. (1988). Steinmetz et al. (1991) pointed out that the reaction was stronger with the antigen when the cells were disrupted and they noticed no reaction with L. micdadei, L. cherrii and L. anisa when whole cells were used in their immunoassay. Moreover, certain pneumonia causing Gram-negative bacterial species, such as Francisella tularensis, Coxiella burnetti and Rickettsia typhi, of which some are known to cross-react with Legionella in immunoassays, were not included in the studies.

Also, no further study was carried out to determine the sequence of the genus-specific epitope on the antigen which could be used to design peptide and detect whether the same monoclonal antibody could be detected from human serum sample for the antigen. The monoclonal antibody or the synthetic peptide could have then been used to determine the specificity and sensitivity with a view to developing a test. This may explain why antibodies are not commercially available and no diagnostic test has been developed. 33 | Page Chapter 2 Literature Review

2.24 PROTECTIVE IMMUNITY OF ANTIGENIC PROTEINS

As infection with Legionella has been demonstrated to confer adaptive immunity (Joller et al., 2007), a study carried out by Weber et al. (2012) to identify protective B cell antigens for vaccine development, found 21 antigenic proteins using a proteomic approach. Although the authors did not mention the cellular localisation of the identified proteins, a prediction carried out using PSORTb shows that the localisation varies from cytoplasmic to extracellular with five proteins from the outer membrane.

A BLAST analysis revealed that all of the five outer membrane proteins showed varied degree of specificity to Legionella.

(1) The major outer membrane protein (lpg2961) which has a molecular weight of approximately 31kDa is most probably an isoform of the 24-29kDa major outer membrane protein described earlier in section 2.22.1. A study carried out by Krinos et al. (1999), demonstrated the existence of the two isoforms with a 25kDa isoform being present in the virulent L. pneumophila strain.

(2) The outer membrane protein Tolc (gene: lpg0699) does not appear to have been previously reported as an antigenic protein, neither does it appear to be very specific to Legionella through a BLAST analysis.

(3) The hypothetical protein (lpg2959) has also not been previously reported as an antigenic protein. However, a BLAST analysis suggests that the protein may be specific to Legionella.

(4) The long-chain fatty acid transporter protein (gene: lpg1862) appears specific to Legionella and reported as antigenic for the first time.

(5) The intracellular multiplication protein (IcmX) (gene: lpg2689) as predicted by PSORTb appears to be an extracellular protein and very much specific to Legionella, especially conserved to L. pneumophila as determined by BLAST analysis. However, this protein was found to be a surface exposed protein by Khemiri et al. (2008).

These proteins were not characterised as potential biomarkers. The other proteins as analysed by BLAST and PSORTb did not appear to be specific to Legionella and they were also mostly cytoplasmic. One of those, the 60kDa heat shock protein (HSP) has multiple cellular localisations.

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2.25 ANTIGENICITY OF SECRETED PROTEINS AND PROTEINS FROM OTHER CELLULAR LOCALISATIONS

Apart from the outer membrane proteins, secreted proteins and proteins from other cellular localisations have been demonstrated to be antigenic in Legionella. The Zn- metalloendopeptidase, a 38kDa cytotoxic and haemolytic exoprotease of L. pneumophila is an antigenic protein (Keen and Hoffman, 1989). However, this protein does not appear to have been further characterised as a potential marker for the diagnosis of Legionellosis.

Major histocompatibility complex II (MHC-II) molecules are involved in presenting processed exogenous (extracellular) antigens to CD4 T-lymphocytes and are therefore crucial in the initiation of antigen-specific immune responses (Holling et al., 2004). Neild et al. (2005) were unable to identify the antigens responsible for presentation on major histocompatibility complex II. Nevertheless, they suggested that the Dot/Icm secretion system may be involved in proteins synthesized by the pathogen being translocated to the lysosome which could trigger the MHC-II dependent pathway of the immune response.

2.26 DIAGNOSTICS

2.26.1 Microbiological diagnosis of community-acquired pneumonia

The indistinguishable nature of symptoms associated with CAP due to different organisms renders it impracticable to separate etiological agents based on clinical manifestations alone (Bewick and Lim, 2009). Therefore, Bewick and Lim (2009) suggest that misdiagnosis can regularly occur in CAP diagnosis based on clinical history and examination, and therefore believe that more specific investigation is necessary to support such a diagnostic approach. There are a variety of tests available for diagnosis of CAP, however, only some of them are most commonly used and are preferred over the rest.

Culture of the microorganism, one of the methods used in identification of the etiological agent in CAP, can be particularly useful for clinicians when challenged with antibiotic resistance. Expectorated sputum in the majority of the cases is the most

35 | Page Chapter 2 Literature Review readily available material and can therefore be used to try growing the infectious organism. Although culture is considered the gold standrad for legionellosis, this method appears to have several drawbacks. Relatively fastidious contaminating oropharyngeal flora may outgrow the organism being investigated and can thus be particularly problematic for identification purposes (Bartlett, 2011). The rate of success in identifying the etiological agent of CAP is generally quite low and the study carried out by Mueller et al. (2007), is an example whereby only 21.5% of cases had the etiological agent identified by culture of the respiratory secretion. Moreover, the incubation period can be quite long for a timely treatment. Also, the sensitivity of sputum culture is reduced if antibiotic treatment has already started (Stralin et al., 2006, Nolte, 2008). It has been identified that less than 50% of patients with Legionella infection produce sputum (Murdoch, 2003, Diederen et al., 2007).

Blood culture is another method of recovering the infectious agent and is only possible in bacteremic pneumonia. As Legionella has only rarely been reported to cause bacteremic pneumonia (Lai et al., 2010) this method is not suitable for all types of pneumonia.

Other than culture, Polymerase Chain Reaction (PCR) has been applied to blood and respiratory secretion samples and has shown promising results in identifying etiological agents with high sensitivity and specificity (Murdoch et al., 2013, Bartlett, 2011). There is the concern of false positive reaction with S. pneumoniae as some healthy individuals have been identified to carry the organism in the nasopharyngeal passages. Nevertheless, this problem may be overcome by using quantitative PCR which can evaluate the actual bacterial load indicative of a positive infection or not (Bewick and Lim, 2009). However, this technique appears to be limited to the institution willing to adopt this approach (in-house developed test) of diagnosis as it needs to be standardised (Bewick and Lim, 2009) and approved by certain authorities before implementation and commercialisation (Burd, 2010, Halling et al., 2012), which could be the reason for a lack in commercial availability of PCR based assays.

Several biomarkers are used in the management of CAP. Procalcitonin, a protein which supports CAP diagnosis, is generally present at low levels in healthy individuals, but an increase is stimulated by lesions caused by inflammation and infections. Severe bacterial infections, sepsis and multiple organ dysfunction syndrome induce the 36 | Page

Chapter 2 Literature Review production of this protein (Seligman et al., 2012). The turnaround time for a procalcitonin result can however be lengthy, which may limit its clinical effectiveness (Watkins and Lemonovich, 2011). C-reactive protein is yet another biomarker commonly used in the diagnosis of CAP (Bewick and Lim, 2009). However, this protein demonstrates low specificity as it can also be expressed due to other underlying comorbidities (Seligman et al., 2012). Although these two proteins appear to be appropriate biomarkers and the ones most widely used in diagnosis of CAP, the etiological agent remains unidentified without further investigation (Bewick and Lim, 2009).

Serology is used to diagnose CAP based on the identification of circulating antibodies against infectious agents. This test has the ability to identify the etiological agent and is still practiced in many places. However, the main limitation of this test is that seroconversion can take around 10 to 14 days from the onset of the disease. As acute phase serum may not be as sensitive and specific, a paired test with convalescent phase serum is required to perform a confirmatory test (Bewick and Lim, 2009). Therefore, seroconversion test is time dependent and may not be suitable for prompt initiation of treatment.

Antigen tests for S. pneumoniae and L. pneumophila serogroup 1 appear to be the most popular test used in diagnosis of CAP. These tests have been demonstrated to have rather high sensitivity and specificity with a very rapid turnaround time for the tests. Antigens can be detected in body fluids and respiratory samples for S. pneumoniae and in urine for L. pneumophila serogroup 1 (Bewick and Lim, 2009, Watkins and Lemonovich, 2011).

Although the assumption exists that empiricism generally works and that microbiological diagnoses are impracticable as antibiotic treatment has to be initiated rapidly (Bartlett, 2011), the specific causative agent has to be determined if there is a suspicion that this will alter the antibiotic treatment regimen (Mandell et al., 2007, Watkins and Lemonovich, 2011). In a study carried out by Masia et al. (2007), the etiological agent was identified in only about 50% of the suspected CAP cases by combining clinical, radiological and epidemiological features, biomarkers, culture, serology and urinary antigen test.

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Could the inadequacies of current tests and lack of specific diagnostic tests be accountable for fatalities from CAP? If so, this suggests that novel and improved diagnostic tests are required for identification of etiological agents.

2.26.2 Diagnosis of legionellosis

Table 2.2 shows the tests that are used in the diagnosis of Legionnaires disease and their limitations.

Table 2.2: Comparison of methods for laboratory diagnosis of Legionnaires' disease

Method Sensitivity Specificity Comments (%) (%)

Culture • Gold standard” Sputum 5-70 100 • Requires 2-4 days, sometimes 14 days Bronchoalveolar lavage (BAL) 30-90 100 • Highest specificity or transtracheal aspirate • Seroconversion may require 3-9 weeks Lung biopsy 90-99 100 • Only for L. pneumophila Blood 10-30 100 Serogroup1, limited data for other serogroups or species

Serology • Seroconversion may require 3-9 weeks Seroconversion 70-90 95-99 Single specimen (unknown) 50-70 Urinary Antigen 75-99 99-100 • Only for L. pneumophila serogroup1 • Very rapid (15min-3h), may remain positive for several weeks/months

Direct Immunofluorescence assay testing • Very rapid (2-4h) Sputum or BAL 25-75 95-99 • Limited sensitivity • Experience needed • No validated reagents for non- Lung biopsy 80-90 99 pneumophila species

Polymerase Chain Reaction • Rapid Respiratory tract specimen 85-92 94-99 • Diagnostic validity of positive results without confirmation by other methods remains unclear • Detects all Legionella species • Not commercially available Urine, serum 33-70 98

Source: Legionella and the prevention of legionellosis (WHO) (Bartram J. Y., 2007)

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Chapter 2 Literature Review

The four major diagnostic methods are the determination of the antibody titre, immunofluorescent microscopic observation of organism in tissue or body fluids, isolation of the organism on culture medium and detection of antigen in urine (Edelstein, 2002). Of the four methods described, the culture method is still considered the gold standard method for identification of the organism (Lueck, 2010). Although this method has the highest specificity, the time required for the confirmation of a positive test is quite considerable with an incubation period of 2 10 days. The rapid antigenuria test available is only effective for the identification of L. pneumophila serogroup 1. PCR has also been identified as a powerful tool in the diagnosis of legionellosis but may have certain limitations.

2.26.2.1 Culture method

Legionella culture is carried out on BCYE (Buffered charcoal yeast extract)agar ketoglutarate, with or without antimicrobial agents. The most-س supplemented with commonly used antibiotics in the culture media are polymixin against Gram-negative bacteria, anisomycin to inhibit growth of yeast, and cefamandole and vancomycin for control of Gram-positive bacterial growth (Diederen, 2008). A variety of clinical sample types (respiratory samples, blood and lung biopsies) can be used for isolation of Legionellae, although lower respiratory tract secretions are preferred (Diederen, 2008). This technique has 100% specificity but does not have very high sensitivity (Maiwald et al., 1998, Diederen, 2008). Cultures require ample processing of the samples and technical expertise. Moreover, a positive result may be obtained only after several days of incubation (Diederen, 2008). This technique, while considered the Gold Standard method, has several limitations, such as low sensitivity, expertise required to perform the task, long incubation time before a confirmatory positive result and invasive procedures may be involved to obtain samples.

2.26.2.2 Serology

Serology is another method which is still used in the diagnosis of Legionnaires disease. Tests such as indirect immunofluorescent assay (IFA) and Enzyme-linked immunosorbent assay (ELISA) are the most frequently used (Diederen, 2008, Blyth et al., 2009). This diagnostic test depends on the seroconversion to detect antibodies (IgG and IgM) due to Legionella infection. It has a rather high specificity and reasonable

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Chapter 2 Literature Review sensitivity. A four-fold increase in antibody titre can be considered as a positive test for Legionella infection. However, seroconversion may take a long time, which is not desirable in diagnosis (Diederen, 2008, Blyth et al., 2009). IgM detection is commonly performed to test for infections with Legionella as it can be detected earlier, at the onset of the disease. However, high IgM levels may persist for several years (Blyth et al., 2009) and therefore may give false positive results.

Direct immunofluorescent assay can also be carried out for diagnosing legionellosis. This test is carried out to identify the organism in respiratory secretion and tissue samples using microscopy. However, this technique is seldom used as due to its low specificity and sensitivity it requires a high bacterial load in the sample being analysed. Furthermore, it is technically demanding (Blyth et al., 2009).

Biological samples are generally complex mixtures consisting of a large variety of components over a wide range of molecular masses. Varying concentrations of some thousand different proteins and peptides may be present in the samples. In addition to blood or serum, urine is an appropriate source for the detection of proteins, peptides and their metabolised fragments (Heine et al., 1997). Human urine is known to contain trace amounts of different proteins and the evaluation of some can be diagnostically valuable (Anderson et al., 1979). Comparative analyses of proteins profiles in biological fluids from infected and healthy individuals are being used ever more for biomarker discovery, and also for the identification of biochemical processes key to disease pathogenesis.

Most of the currently available tests, measure either the level of expression of a single protein or the total level of urine proteins. The emerging proteomic technologies such as protein microarrays allow qualitative and quantitative examination of the patterns of multiple urinary proteins simultaneously as well as their correlation with diagnosis, response to treatment or prognosis. Analysis of the numerous proteinaceous constituents found in urine may lead to the development of novel non-invasive diagnostic tests and may also be helpful in therapeutic guidance and prognosis information for clinicians (Barratt and Topham, 2007).

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Chapter 2 Literature Review

The urinary antigen for detection of legionellosis is widely used. The antigen is now believed to be a lipopolysaccharide (Williams and Lever, 1995). As it is believed that about 90% of Legionella infection occurs due to L. pneumophila, this may explain why this test is commonly used. The test is highly sensitive and specific, and has a very quick turnaround time, making it a more desirable test to perform. However, the antigenuria test is only specific for L. pneumophila serogroup 1. Besides this, time and again comparative analyses have been carried out to determine the specificity and sensitivity of the tests which are commercially available, such as Binax (ICT, EIA), BinaxNow (ICT), Biotest (EIA), SD Bioline (ICT) and Bartel Legionella urinary antigen test (ELISA). A meta-analysis carried out by Shimada et al. (2009) demonstrated that the antigenuria test actually has high specificity but a modest sensitivity. Twenty six percent of patients were found to have a false negative result with this test and as a result the authors suggested that a negative urinary antigen test should not be used to rule out suspected cases of L. pneumophila serogroup 1 infections. Another comparative analysis carried out by Svarrer et al. (2012) suggested that the commercially available urinary antigen detection kits for Legionella infection may not be very sensitive and therefore do not recommend the use of these test alone for diagnosis. As urinary antigen test is used as the first diagnostic test and sometimes the only test performed in many health institutions to identify infection with Legionella, misdiagnosis may not be uncommon. The investigation carried out by Chien et al. (2010) regarding a patient who was finally diagnosed with Legionella infection after being admitted to three different hospitals is a good example of how legionellosis cases can be missed if all available tests are not carried out to investigate a negative urinary antigen test. Another case study by Zarogoulidis et al. (2011) revealed that the urinary antigen test was misleading in identifying infection with Legionella at the beginning and infection was confirmed by PCR which helped in the recovery of the patient with alteration of the antibiotic treatment regimen.

2.26.2.3 Polymerase Chain Reaction (PCR)

PCR has proven to be a particularly useful tool in the diagnosis of Legionnaires disease, as it has the ability to differentiate between species. 16S rRNA gene, 23S-5S spacer region, 5S rDNA and MIP gene are some of the targeted regions for identifying Legionella infection by PCR. The study carried out by Mentasti et al. (2012) 41 | Page

Chapter 2 Literature Review demonstrated a greater sensitivity of using qPCR in diagnosis of legionellosis as compared to the culture method. However, their study was specific to L. pneumophila only. The investigation carried out by Murdoch et al. (2013) using PCR as diagnostic tool for identification of Legionella infection from respiratory samples was remarkable. With the introduction of PCR as routine diagnostic test, they were able to notice a four- fold increase in the identification of Legionella as a CAP. Their investigation about the incidence rate of legionellosis suggests that the disease burden is indeed higher than documented. More interestingly, 85% of the identified cases were due to infection with L. longbeachae. However, as discussed earlier, about 50% of Legionnaires disease patients do not produce respiratory secretions and therefore this may be a limiting factor unless expectorate can be induced to obtain samples as postulated by the authors.

Therefore it appears that there is a need for improved/novel diagnostic tests for the diagnosis of Legionnaires disease.

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Chapter 3 Materials & Methods

MATERIALS & METHODS

3.1 MATERIALS

3.1.1 Sterilisation of hard goods

Hard goods such as glassware, micropipette tips (in autoclavable tip boxes) and microcentrifuge tubes (in glass jars) were sterilised by autoclaving at 121°C for 20 minutes, followed by a drying time of 10 minutes at 100°C.

3.1.2 Culture media

3.1.2.1 BCYE (Buffered Charcoal Yeast Extract) agar culture plates

The BCYE agar culture plates were prepared according to the manufacturers specifications (Oxoid) by using the CYE agar base (CM0655) and Legionella growth culture supplement (SR0110). 2.5 g of CYE agar base powder was weighed and transferred to a beaker to which about 80 mL of distilled water was added and warmed to dissolve the powder. The mixture was then transferred to a clean Schott bottle and autoclaved at 121°C for 30 minutes using a gentle heating cycle. After sterilisation the mixture was allowed to cool down to about 50°C before one vial of the Legionella culture supplement suspended in sterile distilled water was added and swirled to mix properly. The final volume was brought up to 100 mL before pouring in sterile culture plates. About 15 mL of the mixture were poured in each culture plate aseptically and placed in a biosafety cabinet. After the medium had set, the culture plates were sealed in zip lock plastic bags, wrapped with aluminium foil and stored at 4°C.

3.1.2.2 ACES-buffered yeast extract (AYE) broth

The AYE broth was prepared using a modified protocol based on the one developed by Eylert et al. (2010). 1g of yeast extract was dissolved in approximately 80 mL of distilled water in a Schott bottle. The solution was then autoclaved at 121°C for 30 43 | Page Chapter 3 Materials & Methods minutes using a gentle heating cycle. The solution was allowed to cool down before 1g of ACES, 0.04g of L-cysteine HCl and 0.025 g of iron pyrophosphate were added and mixed on a magnetic stirrer. The pH of the solution was adjusted to 6.8 using potassium hydroxide pellets. The final volume was then brought up to 100 mL by adding sterile distilled water. The solution was then aseptically filtered using a 0.22 µm filter and syringe in a biosafety cabinet. The resulting broth was stored at 4°C.

3.1.2.3 Luria-Bertani (LB) agar culture plates

For 100 mL of LB agar culture medium 1 g of tryptone, 0.5 g of yeast extract and 1 g of sodium chloride were dissolved in approximately 80 mL of distilled water in a Schott bottle. 1.5 g of agar was then added to the solution and autoclaved at 121°C for 30 minutes using a gentle heating cycle. After sterilisation, approximately 15 mL of the medium was poured in sterile culture plates aseptically and placed in a biosafety cabinet. The medium was allowed to set, after which, the culture plates were placed in a labelled zip-lock bag and stored at 4°C.

3.1.3 Common Solutions

3.1.3.1 1.5M Tris-HCl pH 8.8

One litre of this solution was prepared by first weighing 181.71 g of Tris (Trisaminomethane) in a clean beaker. 900 mL of Millipore water were added to completely dissolve the chemical under magnetic stirring. A pH meter was used to monitor the pH of the solution, adjusted to pH 8.8 by carefully adding dropwise 1M hydrochloric acid (HCl). The solution was then transferred to a 1 litre standard flask and the final volume adjusted with Millipore water. The solution was thoroughly mixed and transferred into a reagent bottle and was autoclaved at 121°C for 30 minutes using a gentle heating cycle to sterilise the solution. The solution was stored at room temperature.

3.1.3.2 0.5M Tris-HCl pH 6.8

500 mL of this solution was prepared by first weighing 30.3 g of Tris in a clean beaker and then adding approximately 400 mL of Millipore water to completely dissolve the chemical under magnetic stirring. A pH meter was used to monitor the pH of the solution, adjusted to pH 8.8 by carefully adding dropwise 1M HCl. The solution was

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Chapter 3 Materials & Methods then transferred to a 500 mL standard flask and the final volume adjusted with Millipore water. The solution was thoroughly mixed and transferred into a Schott bottle and was autoclaved at 121°C for 30 minutes using a gentle heating cycle to sterilise the solution. The solution was stored at room temperature.

3.1.3.3 10% SDS

100 mL of the solution was prepared by dissolving in a standard flask 10 g of SDS in sterile distilled water in a final volume of 100 mL. The solution was then transferred to a sterile labelled reagent bottle and stored at room temperature.

3.1.4 Protein isolation buffers

3.1.4.1 Cell wash buffer

To prepare 100 mL of this buffer, 0.238 g of Tris and 0.071 g of magnesium acetate were transferred into a clean beaker. Approximately 90 mL of distilled water was added to dissolve the mixture under magnetic stirring. A pH meter was used to monitor the pH of the solution, adjusted to pH 8 by carefully adding dropwise 1M hydrochloric acid. The solution was then transferred to a 100 mL standard flask and the final volume was adjusted. The solution was mixed properly and aseptically filter-sterilised using a 0.22 µm filter and syringe in a sterile labelled reagent bottle. The solution was allowed to cool down and stored at 4°C.

3.1.4.2 0.05% bromophenol blue

10 mL of this solution was prepared by dissolving in a standard flask 0.005 g of bromophenol blue in sterile distilled water to a final volume of 10 mL. The solution was then transferred to a sterile reagent bottle and stored at room temperature.

3.1.4.3 10mM HEPES pH 7

100 mL of this solution was prepared by first weighing 0.238 g of HEPES (2-[4-(2- hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) in a clean beaker. Approximately 80 mL of distilled water was added to dissolve the chemical under magnetic stirring. A pH meter was used to monitor the pH of the solution, adjusted to pH 7 by careful addition of 1M NaOH solution (sodium hydroxide).

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The solution was then transferred to a 100 mL standard flask, the final volume adjusted by adding distilled water and was thoroughly mixed. The solution was then transferred to a reagent bottle and then sterilised by autoclaving at 121°C for 30 minutes using a gentle heating cycle. The solution was stored at room temperature.

3.1.5 2D sample buffers

3.1.5.1 Whole cell lysate proteome/2D Lysis buffer

(7 M urea, 2 M thiourea, 30 mM Tris, 4% 3-[(cholamidopropyl)dimethylammonio]- propanesulfonate (CHAPS) and sterile Millipore water)

To prepare 50 mL of the buffer, 21 g of urea, 7.6 g of thiourea, 2 g of CHAPS and 0.182 g of Tris were poured in a clean beaker, to which approximately 25 mL of sterile Millipore water was added and left to dissolve under magnetic stirring, after which the solution was transferred to a 50 mL standard flask. The final volume of the solution was then adjusted to 50 mL by carefully adding sterile Millipore water and was thoroughly mixed. The buffer was filter-sterilised using a 0.22 µm filter and syringe, labelled and stored at -20°C in 10 mL aliquots in sterile corning tubes.

3.1.5.2 Secretome

(8 M urea, 50 mM dithiothreitol (DTT), 4% CHAPS and sterile Millipore water)

For preparing 50 mL of the buffer, 24 g of urea, 0.385 g of DTT and 2 g of CHAPS were poured in a clean beaker and approximately 25 mL of sterile distilled were added. The mixture was left to dissolve under magnetic stirring, after which it was transferred to a 50 mL standard flask. The final volume of the solution was brought up to 50 mL and was mixed thoroughly. The solution was then filter-sterilised using a 0.22 µm filter and syringe before dispensing in labelled sterile corning tubes in 10mL aliquots. The buffer was then stored at -20°C.

3.1.5.3 2D base equilibration buffer

(6 M urea, 2% sodium dodecyl sulphate (SDS), 0.05 M Tris-HCl pH 8.8, 20% glycerol)

100 mL of the buffer was prepared by pouring 36 g of urea, 2 g of SDS, 3.3 mL of 1.5 M Tris-HCl pH 8.8 and 20 mL of 100% glycerol into a clean beaker and sterile

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Millipore water was added not exceeding a total volume of 90 mL. The mixture was left to dissolve on a magnetic stirrer after which the solution was transferred to a 100 mL standard flask. The final volume was brought up to 100 mL by carefully adding sterile Millipore water and then thoroughly mixed. The final solution was filter-sterilised using a 0.22 µm filter and syringe, labelled and stored at -20°C in 10 mL aliquots.

3.1.5.4 Outer-membrane sub-proteome

(5 M urea, 2 M thiourea, 2% CHAPS, 2% amidosulfobetaine-14 (ASB-14), 30 mM Tris and sterile distilled water)

To prepare 50 mL of the buffer, 15 g of urea, 7.6 g of thiourea, 1 g of CHAPS, 1 g of ASB-14 and 0.182 g of Tris were poured in a clean beaker and to which approximately 25 mL of sterile Millipore water was added. The mixture was left to dissolve on a magnetic stirrer after which it was transferred to a 50 mL standard flask. The final volume of the solution was adjusted to 50 mL by carefully adding sterile Millipore water and was then thoroughly mixed.

The final solution was then filter-sterilised by using a 0.22 µm filter and syringe before dispensing in labelled sterile corning tubes in 10 mL aliquots. The buffer was then stored at -20°C.

3.1.5.5 SDS sample loading buffer/sample reducing buffer (SRB)

(0.125 M Tris-HCl pH 6.8, 4% SDS, 5% 2-mercaptoethanol, 20% glycerol, 0.1% bromophenol blue, sterile distilled water)

Ten millilitres of 2X SRB was prepared by mixing 2.5 mL of 0.5 M Tris-HCl pH 6.8, 0.4 g of SDS, 500 µL of 2-mercaptoethanol, 2 mL of 100% glycerol and 0.01g of bromophenol blue in a 10 mL standard flask.

The final volume was adjusted to 10 mL by carefully adding sterile distilled water and the solution was mixed thoroughly. The solution was then dispensed in sterile labelled microcentrifuge tubes in 1mL aliquots and stored at -20°C.

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3.1.6 Electrophoresis buffer

Tris/glycine/SDS (TGS) gel electrophoresis running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS and Millipore water)

For 1 litre of a 10X TGS stock solution, 30.3 g of Tris, 144 g of glycine and 10 g of SDS were weighed and transferred in a clean beaker. Approximately 900 mL of Millipore water was added to the mixture and was left under magnetic stirring to dissolve. The solution was then transferred to a 1 litre standard flask and the final volume was adjusted with Millipore water and mixed thoroughly. The buffer was stored at room temperature in a clean labelled reagent bottle.

3.1.7 Gel fixing and staining solutions

3.1.7.1 Gel fixing solution

(40% ethanol, 10% glacial acetic acid and distilled water)

For 1 litre of gel fixing solution 400 mL of 100% ethanol and 100mL of 100% glacial acetic acid were transferred in a 1 litre standard flask. The final volume of the solution was adjusted to 1 litre by carefully adding distilled water. The solution was properly mixed by inversion and was then transferred to a clean labelled reagent bottle. The solution was stored at room temperature.

3.1.7.2 Colloidal coomassie blue stock staining solution

(0.1% coomassie brilliant blue G250, 2% ortho-phosphoric acid, 10% ammonium sulphate and distilled water)

One litre of the stock colloidal coomassie blue staining solution was prepared by transferring 1 g of coomassie brilliant blue G250, 23.5 mL of 85% ortho-phosphoric acid and 100 g of ammonium sulphate to a 1 litre standard flask. Distilled water was then carefully added to adjust the volume to 1 litre. The solution was then thoroughly mixed and was transferred to a clean labelled reagent bottle. The solution was stored at room temperature and was only used after 24 hours from the time it was prepared. Prior to gel staining the stock solution was mixed with methanol in a ratio of 4:1 according to the desired volume.

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3.1.8 Polyacrylamide gel casting

3.1.8.1 Hand casting gels

The gels were prepared according to the formulations given in Table 3.1 and Table 3.2. Clean long and short glass plates were wiped off with 70% ethanol with kimwipe. The glass plates were then assembled into gel casting cassettes according to the Mini- PROTEAN® Tetra Cell kit (Bio-rad cat#:165-8000) instructions. 10% Ammonium persulfate solution was freshly prepared before casting gels by weighing 0.1 g of ammonium persulfate in a sterile microcentrifuge tube and adding 1 mL of sterile distilled water and was briefly vortexed to mix. The 40% Bis-acrylamide (40% acrylamide and biscacrylamide solution 37.5:1) solution was degassed before use by using the suction method. The gels were casted by pouring 3.3 mL of the resolving gel solution in the assembled cassette per gel first. The solution was overlayed with distilled water saturated butanol.

The resolving gels were allowed to set after which the butanol solution was decanted and the gels were washed with distilled water several times. Then stacking gel solution was poured onto the resolving gels until the cassette was full and a comb was inserted carefully to avoid air bubbles being trapped in the gels. The gels were allowed to set. The combs were carefully removed to avoid damaging the wells. The assembled gels were then rinsed with distilled water before use.

3.1.8.2 Mini-Protean gels for 2-Dimensional gel electrophoresis

These were commercially purchased from Bio-rad (MINI-PROTEAN TGX 4-20% IPG/10 cat#:4561091). These were 7 cm in length and 1 mm in thickness.

3.1.8.3 Mini-Protean gels for 1-Dimensional SDS-PAGE

These were hand-casted according to the table below. These were 7 cm in length and 0.75 mm in thickness.

3.1.8.4 Criterion gels for 2-dimensional gel electrophoresis

These were ready-made gels commercially purchased from Bio-rad (CRTGEL12%BIS- TRIS IPG+1 cat#:3450121). The gels were 11 cm in length and 1 mm in thickness.

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Table 3.1: 12% Resolving gel solution (Total volume 3.5mL)

Reagents Amount per gel

Sterile filtered distilled water 1.505 mL

1.5 M Tris HCl pH 8.8 solution 875 µL

40% Bis-acrylamide solution 1.05 mL

10% SDS solution 35 µL

10% Ammonium persulfate solution 35 µL

TEMED solution 1.4 µL

Table 3.2: 4% Stacking gel solution (Total volume 1.5mL)

Reagents Amount per gel

Sterile filtered distilled water 928.5 µL

0.5 M Tris HCl pH 6.8 solution 150 µL

40% Bis-acrylamide solution 390 µL

10% SDS solution 15 µL

10% Ammonium persulfate solution 15 µL

TEMED solution 1.5 µL

3.1.8.5 Protean gels (18 cm by length and 1 mm in thickness)

These were hand-casted according to the Table 3.3. Clean casting glass plates were wiped off with 70% ethanol using kimwipes before casting the gels. The short and long glass plates were assembled into a casting cassette according to the instructions for IPG Protean II XL system kit (Bio-rad 165-3183). However, before the glasses were clamped, the edges were sealed with bluetack to prevent leakage. 10% Ammonium persulfate solution was freshly prepared before casting gels by weighing 0.1 g of ammonium persulfate in a sterile microcentrifuge tube and adding 1mL of sterile distilled water and was briefly vortexed to mix. Prior to use the Bis-acrylamide solution was degassed by the suction method. The gel solution was poured in the cassettes until 50 | Page

Chapter 3 Materials & Methods they were full. The IPG+1 well comb were then carefully placed to avoid trapping air bubbles. After the gels were set, the cassettes were disassembled to remove the bluetack used for sealing and then were clamped back again. The assembled gels were then rinsed with distilled water before use.

Table 3.3: 12% Resolving gel solution for Protean gels (total volume 40mL)

Reagents Amount per gel

Sterile filtered distilled water 17.2 mL

1.5 M Tris HCl pH 8.8 solution 10 mL

40% Bis-acrylamide solution 12 mL

10% SDS solution 400 µL

10% Ammonium persulfate solution 400 µL

TEMED solution 16 µL

3.1.9 Mass Spectrometry solutions

3.1.9.1 50 mM Tetraethylammonium bromide (TEAB)

A 50 mM TEAB solution from 1 M stock solution was prepared by diluting 5 mL of the stock solution in sterile Millipore water to a final volume of 100 mL. The solution was mixed properly and stored in a labelled reagent bottle at room temperature.

3.1.9.2 Gel plug destaining solution for Mass Spectrometry

(50% Acetonitrile in 50 mM TEAB)

For making 50 mL of the gel plug destaining solution 25 mL of acetonitrile and 25 mL of 50 mM TEAB solution were mixed and stored at room temperature in a labelled reagent bottle.

3.1.9.3 Gel plug reduction solution

(50 mM Tris(2-carboxyethyl)phosphine (TCEP) in 50 mM TEAB)

The gel plug reduction solution was always freshly prepared. For making 10 mL of the gel plug reduction solution, 1 mL of a 0.5 M stock solution of TCEP was mixed with 50 mM TEAB solution to a final volume of 10 mL.

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3.1.9.4 Gel plug alkylation solution

(100 mM iodoacetamide in 50 mM TEAB)

The alkylation solution was always freshly prepared before use. To one vial of the lyophilised iodoacetamide 3 mL of 50 mM TEAB was added and mixed properly.

3.1.9.5 Trypsin solution

20 ng/µL Trypsin in 50mM TEAB

A 20 ng/µL trypsin solution was always freshly prepared before use by dissolving the content of one vial (Pierce trypsin Protease, MS Grade cat#90057) in 1 mL of 50mM TEAB. The solution was mixed properly before use.

3.1.9.6 Mass Spectrometry Eluents

Solvent A: 5% (v/v) acetonitrile in 0.1% formic acid

Solvent B: 100% (v/v) acetonitrile in 0.1% formic acid

3.1.10 Immunoassays (ELISA, Dot-blot and Western blot) solutions

3.1.10.1 Transfer buffer (Towbin buffer)

25 mM Tris, 192 mM glycine, 20% (v/v) methanol, pH 8.3

3.03 g of Tris base and 14.4 g of glycine were dissolved in 500 mL of distilled water. 200 mL of methanol was then added and the volume was adjusted to 1 litre with distilled water. The resulting solution was mixed properly and then filter sterilised.

3.1.10.2 Towbin buffer with Sodium dodecyl sulfate (SDS)

To the above buffer, SDS was added from a 10% stock solution to a final concentration of 0.05% when required.

3.1.10.3 Tris-buffered saline buffer (TBS): 20 mM Tris-HCl, 500 mM sodium chloride, pH 7.5

4.84 g of Tris base and 58.48 g of sodium chloride were dissolved in 1.5 litre of distilled water and the pH was adjusted to 7.5 by carefully adding 1M HCl. The volume adjusted to 2 litres and the solution was mixed properly and autoclaved. 52 | Page

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3.1.10.4 Tris-bufferd saline buffer-Tween20 (TBS-T)

To the above buffer, Tween20 was added from a 100% stock solution to a final concentration of 0.005% when required.

3.1.10.5 Blocking solution (5% skim milk-TBS)

To 100 mL of TBS, 5 g of skim milk was added and mixed properly to dissolve. This solution was prepared fresh when required.

3.1.10.6 Antibody buffer

To make the antibody buffer 5 g of skim milk was added to the TBS-T buffer and mixed properly. This solution was prepared fresh when required.

3.1.11 Trypsin shaving

3.1.11.1 Shaving buffer: 50 mM Tris, 20mM calcium chloride, pH 7.5 (autoclaved)

3.1.11.2 Trypsin (P8101S, New England BioLabs) was prepared according to the manufacturers specifications.

3.1.12 Affinity purification solutions

3.1.12.1 Coupling/Binding/Wash buffer: Phosphate buffered saline (PBS) solution

0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2 (autoclaved)

3.1.12.2 Quenching buffer

1 M Tris-HCl, pH 7.4 (autoclaved)

3.1.12.3 Elution buffer

0.1 M glycine-HCl, pH 2.5 (filtered)

3.1.12.4 Neutralisation buffer

1 M Tris-HCl, pH 9 (autoclaved)

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3.2 METHODS

3.2.1 Determination of post-exponential growth phase of Legionella species The post-exponential phase of growth was pre-determined to be after 24 hours of incubation, starting with an optical density of 0.05 at 600 nm. For the DIGE (Difference Gel Electrophoresis) experiments, the Legionella species were cultured as described hereafter.

Legionella pneumophila ATCC33152 (kindly donated by Dr Ratcliff, IMVS, Australia), Legionella longbeachae NSW150 (kindly donated by Dr Kozak, CDC, USA) and Legionella longbeachae D4968 (kindly donated by Prof Hartland, The University of Melbourne, Australia) cryopreserved on Cryocare beads at -70°C were suspended in sterile distilled water. The suspension was plated on BCYE agar and incubated at 37°C. Four single colonies per species were picked after 3 days of incubation and inoculated in 4 different flasks (100 mL sterile conical flasks) containing 25 mL of AYE broth. The four replicates for L. pneumophila ATCC33152 were labelled as L1 L4, for L. longbeachae NSW150 were labelled as N1 N4 and for L. longbeachae D4968 were labelled as D1 D4. The culture flasks were incubated at 37°C with shaking at 220 rpm. After 24 hours of incubation, aliquots of the culture suspensions were taken and inoculated in fresh broth with the optical density (OD) adjusted to ~0.05 for all the cultures, resulting in 4 replicate cultures for each species. The cultures reached post- exponential growth phase after about 25 hours of inoculation and 30 hours post- inoculation was the point at which bacterial cells were harvested for protein extraction. This was controlled by measuring the optical densities of the cultures at different time points to determine the post-exponential growth phase of the bacteria. The time intervals were: 0 h, 3 h, 6 h, 10 h, 24 h and 27 h. A mixture of 100 µL of clear AYE broth and 900 µL of sterile distilled water was used for blanking the spectrophotometer at a wavelength of 600 nm. 100 µL of culture suspension was taken for each replicate and diluted in 900 µL of sterile distilled water and mixed properly by inversion in a cuvette separately. The absorbance was then read at 600 nm and the values recorded. The readings were carried out in duplicates. Cultures were also checked for contaminating microorganisms by plating aliquots of the culture suspension on LB agar. No bacterial contaminations were found after incubation for more than 3 days at 37°C.

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3.2.2 Protein extraction

The culture suspensions were centrifuged at 10,000 g for 10 minutes at 4°C. The supernatants were filtered using a syringe with a 0.22 µm filter and were used for secretome isolation.

3.2.2.1 Total cell soluble proteins isolation

The cells were then washed by suspending in 10 mL of cell wash buffer. The suspension was centrifuged at 10,000 g for 10 minutes at 4°C to pellet the cells. The cells were then resuspended in 1 mL of sterile de-ionised water for a second wash. The cells were spun at 10,000 g for 5 minutes and the washings discarded. 500 µL of 2D lysis buffer was added to the micro-centrifuge tubes containing the cell pellets and vortexed to resuspend the cells. The cells were disrupted by sonication at 50% amplitude with 10 seconds of pulse on and 60 seconds of pulse off for a total of 2.5 minutes on ice slurry. The lysates were incubated on ice for an hour with intermittent vortexing to help protein solubilisation. Thereafter, the lysates were centrifuged at 12,500 g for 15 minutes to remove the debris and any insoluble materials. The supernatant which contained the total cellular soluble proteins were then transferred in fresh labelled tubes and stored at -70°C.

3.2.2.2 Secretome isolation

For secretome isolation, to the collected supernatants, 10% (w/v) of Trichloroacetic acid (TCA) was added and incubated overnight (~ 12 hours) at 4°C. 50% (v/v) of ice cold acetone was then added to the tubes and incubated at -20°C for an hour. The precipitated proteins were then pelleted by centrifuging at 10,000 g for 15 minutes at 4°C. The supernatants were discarded and the pellet air-dried for no longer than 5 minutes. The dry pellets were then solubilised in 100 µL of 2D buffer for secretome.

3.2.2.3 Outer membrane proteins isolation

Bacteria from post-exponential phase were harvested by centrifuging the cultures at 10,000 g for 20 minutes at 4°C. The bacterial pellet was then suspended in 10 mL of 10 mM HEPES pH 7.0 solution with 0.5% (w/v) lysozyme and incubated at 37°C for 3 hours. The resulting spheroplasts were collected by centrifuging at 10,000 g for 20 minutes at 4°C. The pellet was then suspended in 5 mL of 10 mM HEPES with 1%

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CHAPS (w/v) solution by vortexing. The cells were then disrupted by sonication with cycles of 10 seconds pulse on and 60 seconds pulse off at 50% amplitude for a total of 2.5 minutes on ice slurry. The lysate was incubated on ice for approximately 30 minutes after which it was centrifuged at 10,000 g for 20 minutes at 4°C. The supernatant was collected in a fresh labelled tube and the pellet was discarded. 1% (w/v) of N- laurylsarcosine was added to the supernatant and was incubated overnight with end over end mixing at 4°C. After the overnight incubation, 5 mL of 0.1 M sodium carbonate pH 11 solution was added and incubated for 1 hour at 4°C with end over end mixing. The outer membrane sub-proteome was then isolated from the mixture by ultracentrifugation at 100,000 g for 1 hour at 4°C. The resulting pellet was air dried for no longer than 5 minutes and then solubilised in the outer membrane 2D buffer. The samples were stored at -70°C until required.

3.2.2.4 Surfaceome/trypsin shaving

Approximately 0.05 g of cells was collected from post-exponential phase of growth by centrifuging the culture at 10,000 g at 4°C and then discarding the supernatant. The cells were first washed with cell wash buffer once and then with trypsin shaving buffer. The cells were then suspended in 500 µL of trypsin shaving buffer to which 2 µg of trypsin (v/v) was added. The suspension was mixed by vortexing briefly, and incubated at 37°C for 1 hour. The cells were then pelleted by centrifuging at 10,000 g for 10 minutes. The supernatant was then carefully aspired and transferred to a fresh sterile microcentrifuge tube. The liquid was then evaporated by using the rotary evaporator at 37°C. The concentrated peptides were then dissolved in 15 µL of TEAB solution. The peptides were stored at 4°C until analysed by mass spectrometry.

3.2.3 Bradford Assay for total protein quantification

3.2.3.1 Total soluble proteins

Total protein quantification for the total cellular soluble protein was carried out prior to 2D-PAGE (2 Dimensional- Polyacrylamide Gel Electrophoresis) by using the Quick Start Bradford Protein Assay (Bio-rad cat# 500-0201). The 2D cell lysis buffer was diluted twenty times to be used as blank and so were the samples to meet the compatible concentration for the microplate assay using the kit.

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To remove contaminating nucleic acid material which interfered in the 2D-PAGE analysis, 300 µg of protein from each sample was transferred in fresh labelled tubes. The samples were subjected to endonuclease treatment by adding 50 units (2 µL) of Benzonase® Nuclease (Novagen cat#70746-4) to each tubes and incubating on ice for 1 hour. The total protein was then re-quantified using the above-mentioned method before proceeding to the DIGE experiment and 2D-PAGE.

3.2.3.2 Secretome sample clean-up and protein quantification

As the secretome samples appeared contaminated with materials from the broth, and were problematic in protein quantification, the 2-D Clean-Up Kit (GE Healthcare cat# 80-6484-51) was used for cleaning the samples according to the manufacturers specifications. The samples were then finally solubilised in 50 µL of secretome 2D buffer. The amount of total protein in the secretome was then quantified using the Quick Start Bradford Protein Assay (Bio-rad cat# 500-0201) following the manufacturers protocol for the microplate assay.

3.2.3.3 Outer membrane sub-proteome proteins quantification

The outer membrane sub-proteome protein quantification was carried out by using the Quick Start Bradford Protein Assay (Bio-rad cat# 500-0201). The 2D outer membrane sub-proteome buffer was diluted twenty times to be used as blank and so were the samples to meet the compatible concentration for the microplate assay using the kit.

3.2.4 2D Gel Electrophoresis – Criterion gels

3.2.4.1 11cm IPG strip rehydration

The 3-10 linear 11 cm IPG strips (Bio-rad) were used to separate the proteins of the outer membrane sub-proteome fraction. 200 µg of sample in a total volume of 200 µL was used for IPG strip rehydration. Before rehydration, 2 µL each of TBP (Tributylphosphine), carrier ampholytes and 2 µL of bromophenol blue were added to the samples and the final volume brought up to 200 µL using the 2D outer membrane sub-proteome buffer. The samples were dispensed in the respective labelled wells and the IPG strips laid on the solution carefully with the gel side of the strip facing down. Any air bubbles trapped under the IPG strips were removed by carefully and gently 57 | Page

Chapter 3 Materials & Methods pressing on the plastic backing of the IPG strips. The samples were left to be absorbed by the gel matrix of the IPG strips for an hour before they were overlayed with mineral oil. Just enough mineral oil was poured to cover the IPG strips to prevent them from drying during the rehydration process. IPG strips were rehydrated for 12 hours at room temperature.

3.2.4.2 Iso-electric focussing (IEF) – 1st dimension separation

Paper wicks were placed on both ends of the wells to be used. The paper wicks were moistened with sterile distilled water (9 µL per paper wick). Excess mineral oil from the IPG strips was first drained. The IPG strips were then placed on the labelled focussing tray wells containing moistened paper wicks. The IPG strips were placed with the gels side down and in the correct orientation according to the electrodes as mentioned on the IPG strips. The IPG strips were then covered with mineral oil. Any trapped air bubbles were removed. The temperature was set to 20°C.

Step1: 250 V, linear ramp, 20 minutes

Step2: 8000 V, linear ramp, 150 minutes

Step3: 8000 V, rapid ramp, 20000 Volt Hours

After the IEF was complete the excess oil was blotted using filter papers. The IPG strips were kept on clean labelled rehydration tray with the gel side up. The tray was covered with aluminium foil to protect the DIGE IPG strips from exposure to light and was stored at -20°C until further required.

3.2.4.3 IPG strip equilibration

The gel electrophoresis was carried out immediately after IEF. The IPG strips were equilibrated as following: Reduction was first carried out by incubating the IPG strips with gentle shaking for 15 mins in equilibration buffer containing 2% (w/v) DTT. This process required 2.5 mL of the reduction equilibration buffer per IPG strip. After 15 minutes the solution was drained to carry out the alkylation process. 2.5 mL of alkylation equilibration buffer containing 2.5% (w/v) of iodoacetamide was added per IPG strip and the incubated for 15 minutes in dark without shaking.

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3.2.4.4 2nd Dimension SDS-PAGE

The equilibrated IPG strips were placed on the ready-made 12% criterion gels and molten overlay agarose was poured to fill the empty space and stabilise the strip. Trapped air bubbles were carefully removed. The gel electrophoresis was performed at 150 V for one hour after which the gels were carefully removed from the cassettes. The gels were briefly rinsed in distilled water and were fixed for 1 hour by submerging the gels in the fixing solution. The gels were stained overnight using the colloidal coomassie blue staining solution.

3.2.5 2D Gel Electrophoresis - Protean gels

3.2.5.1 17cm IPG (Immobilised pH Gradient) strip rehydration

150 µg (analytical gel: DIGE) or 300 µg (preparative gels) of protein in a final volume of 300 µL: was used for IPG strip rehydration and protein separation via IEF (Iso- electric Focussing) and SDS-PAGE. DTT (Dithiothreitol) was added to the samples at a final concentration of 50mM (15 µL from a freshly prepared 1 M solution) and carrier ampholytes (3 10) was added to a final concentration of 0.5%. 3µL of bromophenol blue from a 0.05% stock solution was also added to the samples. The final volume was brought to 300 µL by adding the required amount of the 2D whole cell lysate proteome/Lysis buffer. IPG strips were non-linear of pH gradient 3 10 and 17 cm in length. The samples were dispensed in the respective labelled wells and the IPG strips laid on the solution carefully with the gel side of the strip facing down. Any air bubbles trapped under the IPG strips were removed by carefully and gently pressing on the plastic backing of the IPG strips. The samples were left to be absorbed by the gel matrix of the IPG strips for an hour before they were overlayed with mineral oil. Just enough mineral oil was poured to cover the IPG strips to prevent them from drying during the rehydration process. IPG strips were rehydrated for 12 hours at room temperature (in dark for DIGE).

3.2.5.2 DIGE sample preparation

Samples for DIGE analysis were prepared according to the manufacturers specifications (Lumiprobe) using the 3Dye 2D DIGE kit. The pH of the samples was checked using the narrow range pH paper to ascertain that the pH was within 8.5 9.0. Aliquots of 50 µg of total proteins were taken per sample and dispensed in tubes for 59 | Page

Chapter 3 Materials & Methods labelling with either Cy3 or Cy5 dye. For the pooled internal standard 25 µg of total protein from each sample was taken and dispensed in one tube. The protein mixture was then briefly vortexed to mix properly. The resulting mixture was divided in six equal volumes and dispensed in 6 labelled tubes (S1 S6). These samples were used for labelling with Cy2 dye. The labelling was carried out according to Table 3.4. The lyophilised Cy dyes were first reconstituted in DMF to a final concentration of 1 mM (10 µL of DMF added to 10 nmol of dye per tube). Aliquots of the different dyes were taken and further diluted in DMF to a final concentration of 0.4 mM (4 µL of the reconstituted dye stock + 6 µL of DMF). 1 µL (400 picomoles) of respective dye was added to each tube according to the table. The tubes were then briefly vortexed and then a pulse centrifuge was carried out to collect the mixture at the bottom of the tube. The samples were then incubated on ice in dark for 30 minutes. The labelling reaction was then quenched by adding 1 µL of 10 mM of freshly prepared lysine solution to each tube. This was mixed by briefly vortexing the tubes and a pulse centrifuge carried out to collect the mixture at the bottom of the tube. The samples were then further incubated on ice in dark for 10 minutes. The samples were then mixed according to the table below to finally have a total of six tubes consisting of one internal pooled standard sample, a Cy3 labelled sample form one species/isolate replicate and a Cy5 labelled sample from a different species/isolate replicate. The final volume of the mixture was then brought up to 300 µL by adding DTT and carrier ampholytes at a concentration of 50 mM (15 µL from freshly made 1M stock: 0.0077 g in 50 µL of sterile Millipore water) and 0.5% respectively and the required amount of 2D lysis buffer.

Table 3.4: Total cellular soluble protein samples labelling for DIGE

Tube No. Cy Dye used for labelling Gel No. Cy2 Cy3 Cy5

1 S1 L1 N4 2 S2 L2 D2 3 S3 N3 L3 4 S4 D1 L4 5 S5 N2 D4 6 S6 D3 N1

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3.2.5.3 Iso-electric Focussing (IEF)

Iso-electric focussing was carried out using the Bio-rad Protean IEF machine. The focussing conditions were as follows (carried out in dark for DIGE the light exposed lid was covered with aluminium foil): Paper wicks were placed on both ends of the wells to be used. The paper wicks were moistened with sterile distilled water (9 µL per paper wick). Excess mineral oil from the IPG strips was first drained. The IPG strips were then placed on the labelled focussing tray wells containing moistened paper wicks. The IPG strips were placed with the gels side down and in the correct orientation according to the electrodes as mentioned on the IPG strips. The IPG strips were then covered with mineral oil. Any trapped air bubbles were removed. The temperature was set to 20°C.

Step1: 250 V, linear ramp, 20 minutes

Step2: 10,000 V, linear ramp, 150 minutes

Step3: 10,000 V, rapid ramp, 66,000 Volt Hours

After the IEF was complete the excess oil was blotted using filter papers. The IPG strips were kept on clean rehydration tray with the gel side up. The tray was covered with aluminium foil to protect the DIGE IPG strips from exposure to light and was stored at - 20°C until further required.

3.2.5.4 IPG strip equilibration

IPG strips were removed and thawed to room temperature before the second dimension. The IPG strips were then equilibrated to solubilise focussed proteins and to allow SDS binding by reduction and alkylation of cysteine residues as follows (carried out in dark for DIGE):

Reduction was first carried out by incubating the IPG strips with gentle shaking for 15 mins in equilibration buffer containing 2% (w/v) DTT. This process required 5 mL of the reduction equilibration buffer per IPG strip. After 15 minutes the solution was drained to carry out the alkylation process. 5 mL of alkylation equilibration buffer containing 2.5% (w/v) of iodoacetamide was added per IPG strip and the incubated for 15 minutes in dark without shaking.

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3.2.5.5 2nd Dimension SDS-PAGE

The second dimension was carried on 12% hand-cast modified Lamelli gels. The gels were prepared according to Table 3.3. The gel mixture was degassed before addition of the polymerising agents. Strips were briefly dipped in SDS-PAGE running buffer and laid on top of the gel. The strips were then overlayed with molten agarose and any visible air bubbles were removed. The agarose was allowed to set before the gels were run. The gel running conditions were as follows: 16 A per gel at 150 V for 30 minutes and the 24 A per gel at 150 V for 8 hours when the bromophenol blue dye front reached the end of the gel (The whole process was carried out in dark or with limited light exposure for DIGE).

3.2.5.6 Gel staining

After electrophoresis the preparative gels were carefully taken out of the glass-plate assembly and rinsed twice with distilled water for 5 minutes each time on a platform shaker. The gels were then fixed in the gel fixing solution (10% acetic acid (v/v), 40% methanol (v/v)) for 1 hour. The gels were then stained in 50 mL of colloidal coomassie blue stain overnight. The excess of stain was then removed by washing the gels in distilled water several times. To obtain a clearer background, the gels were again rinsed in gel fixing solution for 1 hour on a platform shaker with the solution changed at least one time within that period. The gels were then finally rinsed with distilled water a couple of times to wash away the acetic acid and methanol.

3.2.5.7 Gel imaging

Both preparative gels and analytical gels were scanned using a Typhoon Imager. However, the parameters were different. The scanning parameters for the filters for DIGE gels (Analytical) were set as following:

1. 526 SP blue (488 nm) for Cy2 dye 2. 580 BP30 Green (532 nm) for Cy3 dye 3. 670 BP30 Red (633 nm) for Cy5 dye

A 100 micron scanning was carried out at the following photomultiplier tube (PMT) according to Table 3.5.

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Table 3.5: Parameters used for DIGE gel imaging using Typhoon Trio

Gel No. PMT Voltage (V) Cy2 Cy3 Cy5 1 550 500 450 2 550 500 450 3 550 500 460 4 550 500 460 5 550 500 460 6 550 500 460

For preparative gels, a 100 micron scanning was carried out using fluorescence. No filter was used and the laser red 633 nm was selected. The PMT voltage was set at 600 V. If saturation of spots was observed, the PMT voltage was adjusted.

3.2.6 DIGE gel analysis

DIGE gel analysis was performed by using the ImageQuant (GE Healthcare) software and DeCyder version 6.5 (GE Healthcare) software. The DIGE gel images were first cropped according to the region of interest using the ImageQuant software. The cropped images were then loaded on the DeCyder software for Difference In-gel Analysis (DIA) and Biological Variation Analaysis (BVA).

3.2.6.1 Difference in-gel Analysis (DIA)

The gel images were processed as following: DIA was performed individually for each gel. The Cy2 labelled gel image was set as the primary image and the Cy3 labelled gel as the secondary image for each gel during the analysis. The estimated number of protein spots was set to 2500 and the threshold was set at 2.0 fold. To define exclusion criteria to be able to remove non-proteinaceous materials identified as potential protein spots, the data from the protein table panel were sorted according to the largest to the smallest maximum slope and from the smallest to the largest area, volume and peak height one at a time for each of the parameters. After each individual sorting according to the different parameters, scrolling down the table, each spot identified as a potential protein spot was confirmed according to the manufacturers specifications. When a potential protein spot was identified as a real protein, the value for the parameter was noted to set the exclusion criteria, as summarised in Table 3.6.

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Table 3.6: Non-proteinaceous spots exclusion criteria parameters

Gel Maximum Area Volume Peak 1 1.10 297 25350 101 2 1.19 164 18513 99 3 0.97 243 22959 127 4 1.02 169 11332 158 5 0.87 172 11277 160 6 1.12 184 18847 99

As a result of DIA Gel 3 was identified as the Master Gel with the greater number of protein spots identified across the 6 gels. The Cy2 labelled image of Gel 3 was therefore used as the primary image for further analysis.

3.2.6.2 Biological Variation Analysis (BVA)

For the BVA, three groups were created from the unassigned folder (gels not assigned to any particular labelling dye) in addition to the standard (Cy2 labelled image of all 6 gels). The three groups were labelled as L (L. pneumophila), N (L. longbeachae NSW150) and D (L. longbeachae D4968). Protein spot matching was carried out on each gel by first setting landmarks on the Cy2 labelled image of all the 6 gels. The brightness and contrast of the gel was adjusted to facilitate landmarking. In all, 16 spots were used as landmarks including as many difficult spots as possible across the 6 gels. The gels were then processed by the software for matching. A total of 21 spots from level 1 (5 spots suggested by manufacturer) and level 2 (10 sposts suggested by manufacturer) were manually checked and confirmed as real protein (for confirmatory purpose of proper landmarking and spot matching). After matching the spots on the gels, two sets of statistical analysis were carried out, with a comparison between the groups L & N and N & D. A Students T-test was carried out with the p-value set at 0.001 and the average ratio -2 or 2. The resulting differentially expressed proteins were then individually checked and assigned as proteins of interest.

3.2.7 Protein identification 3.2.7.1 In-gel tryptic digestion

Coommassie stained gels were used for protein identification. Protein spots of interest were excised from the gel with a scalpel and transferred into 1.5 mL low-binding microcentrifuge tubes. The excised gel plugs were further sliced into smaller pieces if required. 64 | Page

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The gel plugs were first destained in 200 µL of gel plug destaining solution by incubating at 37°C for 15 minutes with shaking. The gel plug destaining solution was then removed and the process was repeated one more time. To the tubes, 500 µL of 100% acetonitrile was added and left for 10 minutes until the gel pieces became opaque and hard. The acetonitrile was then carefully removed. 50 µL of freshly prepared gel plug reduction solution was added to the tubes and incubated at 60°C for 1 hour in a heating block. The tubes were then pulse centrifuged at 12,000 g and the solution was removed. The tubes were allowed to cool down to room temperature before 50 µL of freshly prepared gel plug alkylation solution was added to each tube. The tubes were incubated in dark at room temperature for 30 minutes. The solution was then removed and the gel pieces were rinsed twice in 900 µL of destaining solution for 2 minutes each time. The gel pieces were then dehydrated by adding 500 µL of 100% acetonitrile and incubating for 10 minutes. Once the gel pieces turned opaque, acetonitrile was removed and the gel pieces were allowed to briefly air dry. 10 µL of the trypsin solution was added to each sample. The samples were kept in the fridge for 30 minutes for the gel matrix to absorb the trypsin solution. After the gels were swollen, 20-30 µL of 50 mM TEAB solution was added to each tube, enough to cover the gel plugs depending on the size. The tubes were incubated overnight (~14 hours) at 37°C. The tubes were then briefly centrifuged to collect all the solution at the bottom of the tube. 15 µL of solution from each sample was transferred to a separate labelled tube compatible with the mass spectrometry machine. The samples were kept at 4°C until they were ready to be analysed.

3.2.7.2 Mass spectrometry

Peptide-containing samples were concentrated and loaded onto a microfluidic trap column packed with ChromXP C18-CL 3-m particles (300Å nominal pore size; equilibrated in Mass Spectrometry Eluent Solvent A) at 7Lmin 1 using an Eksigent NanoUltra cHiPLC system. An analytical (15cm×75m ChromXP C18-CL 3) microfluidic column was then switched in line and peptides were separated by using the Mass Spectrometry Eluent Solvent B at the following gradient: 2% to 15% Solvent B for 0.1 min, 15% to 80% Solvent B in 10 min and maintained at 80% Solvent B for 0.5 min followed by equilibration at 2% Solvent B for 6 min before the next sample injection where an additional 5 minutes of equilibration was carried out. Separated

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Chapter 3 Materials & Methods peptides were analysed with an AB SCIEX 5600 TripleTOF mass spectrometer equipped with a Nanospray III ion source and accumulating up to 10 MS/MS spectra per second. The MS was scanning at 200-1250 Da and selecting 2+ to 5+ charged peptides for data dependent MSMS. Repeated peptide exclusion was enabled after 3 occurrences for 90 seconds.

3.2.7.3 MASCOT® Search for protein identity (MSILE)

The data files generated by the mass spectrometer for the samples analysed were first converted into MASCOT® Generic Format (MGF) files and these were then used for protein identification with the MSILE software (Bio21 Institute). The search parameters were set according to Table 3.7.

Table 3.7: Parameters for protein identification using mass spectrometry data

Search Specifications Database/Parameters Database UniProt Enzyme Trypsin Missed Cleavages 2 Precursor Tolerance 10 ppm Fragment Tolerance 0.2 Da Charge State(s) 1+, 2+ and 3+ Fixed Modifications Carbamidomethyl (C) Variable Modifications Oxidation (M)

The False Discovery Rate (FDR) was adjusted to zero for all positive protein identification hits returned. This was done by changing the p-value until the FDR reached zero. As such, single hit wonder (protein hit based on only one peptide) protein hits belonging to Legionella were also reported as positively identified proteins.

3.2.8 Basic Local Alignment Search Tool (BLAST)

A BLAST analysis was carried out on National Centre for Biotechnology Information (NCBI) database to identify potentially specific proteins to L. longbeachae, L. pneumophila and the Legionella genus. The results were recorded.

3.2.9 Prediction of subcellular localisation of proteins

PSORTb version 3.0.2 database was used to predict the subcellular localisation of the identified proteins. Protein sequences were input in the FASTA format in the database and the results were recorded. 66 | Page

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3.2.10 Immunoassays

3.2.10.1 Dot blot

Grids (1 cm2) were drawn on nitrocellulose membrane and 2 µL of protein sample (0.05 µg/µL) or peptide (0.025 µg/µL) were carefully spotted on the respective labelled grids. The samples were allowed to air-dry and the membrane was then blocked by incubating the membrane immersed in blocking buffer with gentle shaking at room temperature for three hours. The blocking buffer was then decanted and rinsed once with TBST-T solution. The membrane was then incubated at room temperature for three hours with gentle shaking with the desired serum sample applying it as the primary antibody, diluted in antibody buffer (1:4000). The solution was then discarded and the membrane was washed thrice with TBST-T for five minutes each time. The membrane was then incubated with HRP conjugated anti-human IgG in antibody buffer (1:10000) with gentle shaking at room temperature for two hours. The membrane was then washed thrice with TBS-T for five minutes each time. The membrane was finally washed with TBS once and was then incubated in a substrate solution containing 3,3- Diaminobenzidine (DAB) and hydrogen peroxide until the colour development had reached the desired intensity to prevent background staining. The substrate solution was prepared according to the manufacturers specifications using the Sigmafast tablets (cat#: D4168, Sigma). The membrane was then washed with sterile distilled water for ten minutes and was transferred on a filter paper in a petri-dish and was left to dry.

3.2.10.2 Western blotting

A semi-dry transfer method was employed for transferring proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membrane. After gel electrophoresis, the gel was briefly rinsed in distilled water and then equilibrated in transfer buffer for 10 minutes. In the meantime, the bottom layer of the sandwich was assembled. First, an appropriate-sized (depending on gel size) extra-thick filter paper pre-soaked in transfer buffer was placed on the platinum anode of the trans-blot machine. A roller was rolled over to remove any trapped air bubbles under the filter paper. The PVDF membrane which was briefly soaked in methanol and then pre-soaked in the transfer buffer for ten minutes was placed on top of the filter paper. A roller was carefully rolled over the membrane to remove any trapped air bubbles. The pre-

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Chapter 3 Materials & Methods equilibrated gel was then placed on top of the membrane and a roller was gently rolled over to remove any trapped air bubbles to ensure proper contact between the gel and the membrane. An extra thick filter paper soaked in transfer buffer was finally placed on top and a roller was rolled over to remove any trapped air bubbles. After assembling the whole sandwich, the cathode was placed on the stack. Western transfer was the performed at 15 Volts for 30 minutes, 45 minutes and 60 minutes for the mini-protean, criterion and protean gels respectively.

After the transfer, the sandwich was carefully disassembled and the membrane was immediately placed in TBS solution to prevent it from drying. The membrane was rinsed in the solution for five minutes. The solution was discarded and the membrane was then immersed in blocking buffer and kept at 4°C overnight. Following the blocking step, the blocking buffer was discarded and the membrane was rinsed twice in TBS-T for 5 minutes each time with gentle shaking at room temperature. Serum sample which was used as primary antibody was diluted in antibody buffer (1:4000) and the membrane was incubated in this solution for three hours at room temperature with gentle shaking. The solution was then discarded and the membrane was rinsed in TBS-T thrice for 5 minutes each time. The HRP conjugated anti-human IgG which was used as the secondary antibody was diluted in the antibody buffer (1:10000). The membrane was then incubated with the secondary antibody solution for two hours at room temperature with gentle shaking. The membrane was then washed thrice in TBS-T for five minutes each time. A final washing was performed with TBS before detection of reaction with DAB.

3.2.10.3 Indirect ELISA

Five micrograms of proteins in final volume of 100 µL of TBS buffer from cell lysates of different Legionella species were coated in the ELISA plate wells. For synthetic peptides, 0.25 µg in a final volume of 100 µL of carbonate buffer were coated in ELISA plate wells. For the protein samples, the plates were then incubated at room temperature for three hours, whereas the peptides were allowed to coat the wells overnight at 4°C. The wells were then emptied by flicking the plate over the sink and the remaining drops were removed by patting the plate on a paper towel. The remaining protein binding sites in the wells were then blocked by adding 200 µL of blocking solution and incubated for three hours at room temperature. The wells were then emptied as mentioned above and 68 | Page

Chapter 3 Materials & Methods rinsed once with TBS-T for five minutes. 100 µL of serum sample used as primary antibody diluted in antibody buffer (1:4000) was added to the wells and incubated at room temperature for three hours. The wells were then emptied as described earlier and were washed thrice with TBS-T for five minutes each time. The HRP conjugated anti- human IgG used as secondary antibody was diluted in antibody buffer (1:10000) and 100 µL of the solution was added to the wells and incubated for two hours at room temperature. Thereafter, the wells were emptied as mentioned previously and washed thrice with TBS-T for five minutes each time. A final washing step was performed with TBS before 100 µL of TMB substrate was added to the wells. The colour was allowed to develop and 50 µL of 0.2 M sulphuric acid was added to stop the reaction after 15 minutes of incubation and the optical density was read at 450 nm with a plate reader. The experiment was done in triplicates.

3.2.11 Affinity purification

The NHS-activated agarose dry resin columns were used for this purpose.

3.2.11.1 Affinity purification of IgG

A 200 µL of the goat anti-human IgG (Fab specific) aliquot was first mixed with 200 µL of Coupling/Wash buffer and added to NHS-Activated Agarose spin Column (Cat# 26198, Pierce, Thermoscientific). The anti-human IgG was immobilised on the dry resin by incubating at room temperature for two hours with end over end mixing. The bottom seal of the column was snapped off and the column was placed on a 1.5 mL microcentrifuge tube (collection tube). The column was then centrifuged at 1000 g for 1 minute. The column was washed twice by adding 400 µL of Coupling/Wash buffer and centrifuging at 1000 g for 1 minute using a fresh collection tube every time. The washings were stored. To block the remaining free active sites, Quenching buffer was added to the resin and incubated at room temperature for 30 minutes with end over end mixing. The column was placed on a new collection tube and spun at 1000 g for 1 minute. The column was then washed twice by adding 400 µL of Coupling/Washing buffer and centrifuging at 1000 g using fresh collecting tubes for every washing. The washings were stored. To 150 µL of Binding/Wash buffer, 50 µL of serum sample was added and mixed properly and was added to the column. This was incubated for two hours at room temperature with end over end mixing. The column was centrifuged at 69 | Page

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1000 g for 1 minute and the solution collected in the microcentrifuge tube was stored. The column was then washed thrice with Binding/Washing buffer and the washings were collected in fresh collection tubes each time by centrifuging at 1000 g for 1 minute. The IgG was then eluted in 6 fractions by adding 180 µL of Elution buffer and collecting approximately 30 µL of the eluted IgG by centrifuging at 1000 g for 10 seconds each time. About 10 µL of sample was saved from each fraction. 20 µL of the eluted IgG was pooled from fractions 2 6 and 400 µL of neutralising buffer was added and mixed properly. The buffer was then exchanged and the affinity purified IgG was concentrated using amicon centrifugal units. The volume of the solution was brought down to 100 µL by centrifuging at 5000 g before it was topped up with PBS. This step was repeated once again and the final volume was brought down to approximately 50 µL. The affinity purified IgG fractions were stored at -20°C.

3.2.11.2 Affinity purification of antigen

The affinity purification of antigen was performed using a similar technique as affinity purification for IgG. The only differences were that firstly, 50 µL of purified IgG from serum samples was used to immobilise on the NHS activated agarose column. Secondly, 400 µL of total soluble protein from L. longbeachae was used for the affinity purification.

3.2.12 In silico prediction of epitopes

Linear epitopes were predicted by using the protein sequences of interest in FASTA format and analysing them on the IEDB online software (http://tools.immuneepitope.org/tools/bcell/BepipredDisplayServlet).

3.2.13 Peptide design

Peptides design was carried out by first aligning the homologous protein sequences from different Legionella species on the UniProt database. The conserved regions were targeted for peptide design. BLAST was then carried out to determine the specificity of the selected regions. The amino acid sequences were then analysed to determine the hydrophilicity by using the grand average hydropathy (GRAVY) value. The regions which were found hydrophilic were matched to the list of predicted epitopes and then peptide sequences were selected from flanking regions. 70 | Page

Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

COMPARATIVE PROTEOMICS ANALYSIS OF L. PNEUMOPHILA VS. L. LONGBEACHAE

4.1 INTRODUCTION

Genome mining is becoming ever more accessible with the development of high- throughput sequencing technologies. This in turn has revolutionised the field of clinical microbiology. Other than for evolutionary studies, genome sequencing of many bacterial pathogens is being carried out because of its ability to reveal unprecedented information regarding pathogenesis (virulence) and epidemiology which can successively be translated into improved methods of diagnosis and treatment (Figure 4.1) (Fournier et al., 2007, Wilson, 2012). Recently, the genomes of two L. longbeachae isolates were sequenced (Cazalet et al., 2010, Kozak et al., 2010) and with the genome sequence data available for L. pneumophila, this opens new avenues for exploration.

Virulence-associated proteins are immunologically valuable as they can be targeted as potential vaccine candidates and new markers for diagnosis. A few studies have demonstrated pronounced differential expression of virulence-associated traits of L. pneumophila at the post-exponential phase in in vitro cultures (Byrne and Swanson, 1998, Hayashi et al., 2010, Cazalet et al., 2010). A comparative analysis of L. pneumophila and L. micdadei carried out by Joshi and Swanson (1999) to determine virulence-associated characteristics suggested that the two Legionella species differ in their virulence, reflected by their different phenotypic features they exhibit and may therefore use different strategies to infect human macrophages. The study carried out by Doyle et al. (2001) suggested that Australian isolates of L. longbeachae might be more virulent as compared to isolates from other parts of the world. Therefore, it appears that susceptibility to infection is related to virulence traits of Legionella.

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Figure 4.1 shows the different applications of genomic data and how it can consequently be used in the development of detection methods and vaccines.

Figure 4.1: Downstream applications of genomic data

Adapted from Fournier et al. (2007)

Based on the genome sequences available for L. pneumophila and L. longbeachae, a transcriptomics study was carried out by Cazalet et al. (2010) to identify differentially expressed genes at different stages of the bacterial life cycle. Compared to genomics and transcriptomics, proteomics has the advantageous ability to define differentially expressed proteins beyond the transcriptional level (Wu et al., 2008) whereby protein expression can be affected by the rapid decay of mRNAs due to various stimuli (Hegde et al., 2004). Proteins can also undergo several post-translational modifications such as glycosylation and phosphorylation (Cain et al., 2014) which may be the case with the immunogenic 60kDa chaperonin of Legionella as suggested by Garduno et al. (2011). Post-translational modifications have also been demonstrated to play a crucial role in virulence factors (Wu et al., 2008). 72 | Page Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

Difference gel electrophoresis (DIGE) has proven to be a powerful tool for comparative proteomic analysis. DIGE has the ability to circumvent many problems such as reproducibility and the inaccuracy and insensitivity of quantitative proteomic analyses normally encountered in classical 2 Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) (Lilley and Friedman, 2004). As no two gels (same sample) behave in an identical manner during electrophoresis, this can result in experimental variation. DIGE substantially reduces gel-to-gel variation because of its ability for multiplexing. It allows multiple samples (typically 3) to be analysed on the same gel, including an internal standard. The internal standard is a pooled mixture of all the different samples and their replicates which is conventionally labelled with the Cy2 fluorescent dye and is included in every gel. This allows for matching of similar spots on different gels and therefore enhances the confidence in the biological variation data by considerably decreasing differences owing to experimental dissimilarities (Marouga et al., 2005).

4.2 EXPERIMENTAL DESIGN

4.2.1 Aims and objectives

In this study, an intra-specific comparative analysis at the proteome level of two L. longbeachae clinical isolates, NSW150 (Australian isolate) and D4968 (American isolate), and an inter-specific comparison with the type strain, L. pneumophila Philadelphia ATCC 33152, was conducted to determine differentially expressed proteins at the post-exponential phase. This study was carried out to identify proteins which could be novel virulence determinants either specific to L. longbeachae or to the Legionella genus to provide greater insights into pathogenesis and cellular physiology of these bacteria. Moreover, this study was performed as the groundwork for further investigations with the view to identifying immunogenic proteins, which could be applied in the development of improved diagnostic tests.

4.2.2 Experimental procedures

Briefly, the Legionella strains were grown to the post-exponential phase under similar conditions (temperature, length of incubation, volume of culture broth and shaking in incubator) using the same level of inoculum. The total cell soluble proteome and the secretome were then isolated from four biological replicates of the species and isolates under study. 2D gel electrophoresis was carried out for the secretome samples whereas 73 | Page Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae the total cell soluble proteomes were subjected to DIGE analysis. Gel images were acquired and then analysed either by the DeCyder or imageJ software. Coomassie stained preparative gels were used to manually pick proteins of interest (POI: Proteins of interest - most abundant and differentially expressed proteins). Tryptic digests of the POI were analysed by mass spectrometry for protein identification. Bioinformatics analyses were then carried out to predict the sub-cellular localisation, specificity based on sequence homology and function(s) of the proteins. A comparative analysis was also carried out for the differentially expressed proteins between the two species and the two isolates. A detailed description of the materials and methodology for this chapter can be found in the Materials and Methods chapter 3.

4.3 RESULTS

4.3.1 Determination of post-exponential growth phase of Legionella

Figure 4.2 shows the growth curves of the four replicates of L. pneumophila ATCC33152, L. longbeachae NSW150 and L. longbeachae D4968 used to confirm that the cultures had reached the post-exponential phase of growth before protein extraction was carried out.

Legionella growth curve 3 Post-exponential phase L1 L2 2.5 L3 L4 2

Absorbance at N1

- 1.5 N2

600nm N3 1 N4 Lag-phase D1 0.5 D2 Optical Density (OD) Optical Density D3 0 D4 0 5 10 15 20 25 30 Incubation time (Hours)

Figure 4.2: Determination of post-exponential phase of growth of Legionella spp.

L1-L4, N1-N4 and D1-D4 denote 4 different biological replicates of L. pneumophila ATCC33152, L. longbeachae NSW150 and L. longbeachae D4968, respectively. The OD for the bacterial cultures were measured at 600nm at different time points of incubation to construct the growth curve and determine the time at which the cultures reached the post-exponential phase. 74 | Page Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

The bacteria reached the beginning of the stationary phase (post-exponential) after approximately 25 hours of incubation. The bacterial cells were harvested 30 hours post- inoculation as it was noticed that the broth started browning after more than 30 hours of incubation. This was particularly problematic in the study of the secretome (Appendix 1 and Appendix 2) by 2D gel electrophoresis.

4.3.2 Protein quantitation (Bradford Assay)

Figure 4.3 shows the standard curve that was generated to determine the protein concentration of the samples from the 12 replicates.

Protein quantitation-Bradford Assay standard curve

0.9 pre-endonuclease 0.8 y = 0.6971x + 0.096 R² = 0.9926 treatment at 595nm 0.7 0.6 post-endonuclease treatment 0.5

Absorbance y = 0.7141x + 0.0643 - 0.4 R² = 0.9937 Linear (pre- endonuclease treatment) 0.3 0.2 Linear (post- 0.1 endonuclease treatment) 0 Optical Density (OD) Optical Density 0 0.2 0.4 0.6 0.8 1 1.2 Bovine Serum Albumin (BSA) concentration (µg/µL)

Figure 4.3: BSA standard curve for protein quantitation of total soluble proteome samples pre and postendonuclease treatment

Solutions of known concentrations of BSA protein were used to generate a standard curve using the Bradford Assay. The optical density (OD) of diluted whole cell soluble proteome samples was measured to determine the concentration from the standard curve.

Protein quantitation is an important initial step before proceeding to 2-Dimensional gel based analysis of complex mixtures for successful separation of proteins. As the DIGE experiment is primarily based on quantitative analysis of differentially expressed proteins, determining the protein concentration of the samples is crucial. Table 4.1 represents the protein concentration of the 12 samples used in this study. After any type of sample treatment, in this case endonuclease treatment used to remove contaminating nucleic acid, protein concentrations have to be re-determined. The endonuclease treatment was carried out as part of the optimisation process for 2D gel electrophoresis. 75 | Page

Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

Table 4.1: Protein concentration of samples pre and post endonuclease treatment

Pre-endonuclease treatment Post-endonuclease treatment Sample concentration (µg/µL) concentration (µg/µL) replicates OD Diluted Original OD Diluted Original L1 0.63 0.76 15.19 0.77 0.99 19.76 L2 0.66 0.81 16.18 0.73 0.93 18.64 L3 0.54 0.64 12.80 0.52 0.64 12.73 L4 0.62 0.76 15.16 0.79 1.01 20.30 N1 0.52 0.61 12.14 0.51 0.63 12.59 N2 0.40 0.43 8.65 0.37 0.42 8.48 N3 0.52 0.61 12.15 0.51 0.63 12.54 N4 0.59 0.71 14.13 0.59 0.74 14.84 D1 0.55 0.65 13.04 0.63 0.80 15.93 D2 0.58 0.70 13.93 0.59 0.74 14.84 D3 0.52 0.61 12.22 0.52 0.64 12.71 D4 0.48 0.55 11.05 0.48 0.58 11.70 L1-L4, N1-N4 and D1-D4 denote 4 different biological replicates each of L. pneumophila ATCC33152, L. longbeachae NSW150 and L. longbeachae D4968, respectively. These are whole cell soluble proteome samples. The dilution factor was 20.

As evident from the table, the concentrations of the pre-treated protein samples were found to be slightly different compared to the endonuclease treated samples. The result also demonstrates that the method used for protein extraction was adequate as evidenced by the yield of satisfactory amount of protein. Therefore, this allowed the elimination of further sample processing to concentrate the protein mixture in the samples. Moreover, after the endonuclease treatment, no viscosity was observed in the samples.

4.3.3 Differential in-gel analysis (DIA) DIA analysis shows the direct inter-specific (L. pneumophila vs. L. longbeachae) and intra-specific (L. longbeachae NSW150 vs. L. longbeachae D4968) comparisons based on the protein abundance from each biological sample on the same gel (Figures 4.4 and 4.5). The decrease and increase in protein abundance in this case represent the differentially expressed proteins in either of the biological samples based on the one selected as the primary sample for comparative analysis. As expected, the result demonstrates higher inter-specific variation and lower intra-specific variation. Although these data showed predicted variations (differences in genomes), DIA addresses differences above system variations, but biological variations may not be represented as no biological replicates are involved in the analysis. Hence, DIA may not be statistically significant and, therefore, Biological Variation Analysis (BVA) was carried out.

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Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

Biological No. of % of sample protein total No. spots of spots

L. longbeachae 553 26 (secondary gel)

L. pneumophila 944 44.3 & L. longbeachae

L. pneumophila 633 29.7 (primary gel)

Figure 4.4: Histogram of differentially expressed proteins of L. pneumophila vs. L. longbeachae

The red and blue dots on the histogram represent the differentially expressed proteins spots on the gel for L. pneumophila and L. longbeachae, respectively. The green dots represent similar protein expression patterns in both. The table represents the number of spots for each type of expression.

Biological sample No. of % of protein total No. spots of spots

LLB D4968 19 0.9 (Secondary gel)

LLB D4968 & 2039 98.3 LLB NSW150

LLB NSW150 17 0.8 (Primary gel)

Figure 4.5: Histogram of differentially expressed proteins of L. longbeachae D4968 vs. NSW150 LLB: Legionella longbeachae

The red and blue dots on the histogram represent the differentially expressed proteins spots on the gel for L. longbeachae D4968 and NSW150, respectively. The green dots represent similar protein expression patterns in both. The table represents the number of spots for each type of expression.

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4.3.4 Results of Biological Variation Analysis (BVA) analysis

4.3.4.1 L. pneumophila vs. L. longbeachae differential expression of proteins

As illustrated in Figures 4.6 and 4.7, after carrying out BVA analysis, a total of 270 protein spots were found to be differentially expressed (Appendix 8 and Appendix 9), 125 of which were from L. pneumophila and 145 from L. longbeachae (Appendix 6 and Appendix 7).

Figure 4.6: DIGE gel3 annotation for L. pneumophila differentially expressed proteins

The gel image represents the spots (101 annotations) that were successfully identified as differentially expressed proteins by BVA analysis using DeCyder and imageJ software. The amber spots are from L. pneumophila and green from L. longbeachae. The yellow spots are similar expression.

One hundred and one unique spots out of the 125 differentially expressed proteins identified by BVA analysis for L. pneumophila were successfully matched from the preparative gel on the DIGE gel. Thirteen spots remained unmatched, while 11 spots were repeats of some of the 101 unique spots as they were identified as two spots on the 78 | Page Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

DIGE gel but were resolved as one spot on the preparative gel. Therefore, approximately 89% of the differentially expressed protein spots were successfully matched from the preparative gel onto the DIGE gel.

Figure 4.7: DIGE gel 3 annotation for L. longbeachae differentially expressed proteins

The gel image represents the spots (83 annotations) that were successfully identified as differentially expressed proteins by BVA analysis using DeCyder and imageJ software. The green spots belong to L. longbeachae, amber spots are from L. pneumophila and yellow spots show similar expression.

Eighty-three unique spots out of the 145 differentially expressed proteins identified by BVA analysis for L. longbeachae were successfully matched from the preparative gel (Appendix 3) on the DIGE gel. Thirty-five spots remained unmatched, while 15 spots were repeats of some of the 83 unique spots as they were identified as two spots on DIGE gel but were resolved as one spot on the preparative gel. Moreover, annotations of 12 of the differentially expressed proteins were missing on the DIGE gel after BVA 79 | Page Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae analysis and were not matched on the preparative gel. In this analysis, approximately 70% of the differentially expressed protein spots were successfully matched from the preparative gel onto DIGE gel. The unmatched proteins were most likely because of their low abundance and therefore might not have stained with the coomassie blue stain which is less sensitive (~4 folds) than Cy dyes.

Figure 4.8 shows graphical views and the captured 3D image views as an example of the differentially expressed proteins for each corresponding spot. The graphical view shows the relative abundance of the particular protein for all the 12 replicates against the standard in the gel (0).

Figure 4.8: Graphical representation and spot image of (A) spot 284 from Figure 4.6 and (B) spot 137 from Figure 4.7

On X-axis: L: L. pneumophila, N: L. longbeachae NSW150, D: L. longbeachae D4968

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4.3.4.2 L. longbeachae NSW150 vs. L. longbeachae D4968 differential expression

Figure 4.9 shows the DIGE gel of a comparative analysis between the proteomes of the two L. longbeachae isolates, NSW150 and D4968. There was no significant difference found between the isolates in terms of number of proteins as can be depicted from the image and also confirmed by BVA analysis (Appendix 5).

Figure 4.9: DIGE gel 6 annotations for L. longbeachae NSW150 and D4968 differentially expressed proteins

The gel image shows four annotated spots that were successfully identified as differentially expressed proteins by BVA analysis using DeCyder and imageJ software. The yellow spots show similar expression. The amber spots belong to L. longbeachae NSW150 and the green ones are from L. longbeachae D4968.

Four out of the seven proteins found to be differentially expressed by BVA analysis comparing L. longbeachae NSW150 to L. longbeachae D4968 were successfully matched on the DIGE gel from the preparative gel.

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The graphical view and 3D image of spot 5 (Figure 4.10) shows the differential expression of the protein. As can be seen, the protein was expressed in L. longbeachae NSW150 only. The result was also consistent throughout the four replicates indicating that the protein peak demarcated with the magenta border is a unique protein.

Figure 4.10: Graphical representation and spot image of spot 5 from Figure 10

On x-axis: L: L. pneumophila, N: L. longbeachae NSW150, D: L. longbeachae D4968

4.3.5 Mass spectrometry

As an example, Figure 4.11 shows the MS/MS spectrum for the precursor ion 664.4 Da from sample spot 138 for L. longbeachae. 92% of the total number of spots analysed by mass spectrometry generated spectra that successfully matched to protein(s) belonging to the Legionella genus using the search parameters as described in Chapter 3 - Materials and Methods.

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Figure 4.11: Mass spectrometry data for spot 138 of L. longbeachae

Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

Table 4.2 shows the number of excised spots from the preparative gels per sample type that were analysed by mass spectrometry, and the number of spots which were successfully identified as known Legionella proteins based on the MSILE search engine (Bio21 Institute).

Table 4.2: Results of mass spectrometry analysis of protein spots

No. of No. of No. of spots No. of spots proteins proteins (POI) returning identified identified Sample Type analysed by successful (including (excluding mass protein potential potential spectrometry identification isoforms & isoforms & PTMs) PTMs) DIGE (whole cell soluble proteome) L. pneumophila ATCC33152 300 280 1123 419 L. longbeachae NSW150 221 208 589 288 309 (LLB) 140 (LLB) Pooled standard (12 replicates) 151 134

222 (LP) 119 (LP) Pooled L. longbeachae NSW150 16 16 51 36 & L. longbeachae D4968 Secretome L. pneumophila ATCC 33152 69 57 261 114

Pooled L. longbeachae NSW150 12 10 19 8 and L. longbeachae D4968 (LLB): L. longbeachae. (LP): L. pneumophila. PTMs: Post-translational modifications. POI: proteins of interest.

A total of 769 spots of the whole cell soluble proteomes and secretomes from L. pneumophila and L. longbeachae were analysed by mass spectrometry. Table 4.2 includes the total number of proteins per sample type, including various isoforms migrating at different spots on the gel with different molecular weight and/or isoelectric point of certain proteins and unique proteins, excluding their isoforms. The isoforms could be a result of post-translational modifications as there were a few proteins such as phosphorylating enzymes and proteases that were identified in this study. A total of 652 proteins were identified for L. pneumophila from all of the samples types of which 496 had unique UniProt accession numbers. On the other hand, a total of 472 proteins were identified for L. longbeachae from all the sample types, of which 350 had unique

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Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

UniProt accession numbers. The number of proteins identified does not correspond to the number of proteins of interest as for some spots, more than one protein were identified as they may have similar molecular weight and isoelectric point, and therefore migrated to the same spot on the gel.

Figure 4.12 illustrates the number of proteins that were either equally expressed or differentially expressed for the species and/or the isolates for the whole cell soluble proteome, secretome and pooled standards.

L. pneumophila ATCC 33152 345 + 16 homologs

0 0 135 L. longbeachae L. longbeachae D4968 NSW150 213 1 1

Figure 4.12: Venn diagram of intra and inter-specific differentially expressed proteins

A total of 135 proteins were identified to have significant homology across the 3 groups. L. pneumophila had 345 unique proteins, and 16 proteins formed a subset with different accession numbers but significant homology to either one of the 345 proteins which were differentially expressed. L. longbeachae NSW150 and L. longbeachae D4968 were found to have 213 proteins in common and 1 protein differentially expressed in each of them. Although there were a few proteins that were found to be differentially expressed between the two L. longbeachae isolates, homologs of the proteins were subsequently identified as proteins which had resolved as different spots on the gels. The homologs were of different molecular weight and/or isoelectric point mainly due to amino acid substitution, addition or deletion. One differentially expressed protein for L. longbeachae D4968 was spot 1 from the secretome. The differentially expressed protein from L. longbeachae NSW150 was from spot 5 of the whole cell soluble proteome.

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4.3.6 Bioinformatics prediction of cellular localisation and specificity of identified proteins

Figure 4.13 represents the predicted sub-cellular localisation of the identified proteins and their specificity to the Legionella genus or the species. The prediction of cellular localisation was carried out via PSORTb and the specificity of the proteins was predicted by BLAST on the NCBI database.

A B

Predicted cellular localisation of Predicted cellular localisation of proteins: PSORTb Version3.0 proteins: PSORTb Version3.0 Legionella pneumophila Legionella longbeachae

5.24% cytoplasmic 2.00% 2.57% 2.82% 2.42% 2.57% 6.00% cytoplasmic 3.23% cytoplasmic 1.43% cytoplasmic 2.42% membrane extracellular 2.86% membrane 4.64% extracellular multiple localisation multiple outer localisation membrane outer periplasmic membrane 79.23% 82.57% periplasmic unknown unknown

C D

Predicted specificity of proteins: Predicted specificity of proteins: BLAST-NCBI BLAST-NCBI Legionella pneumophila Legionella longbeachae

2.02% 4.29% 10.48% Potential 8.00% homologs in Potential other organisms homologs in other organisms Potentially specific to Potentially Legionella specific to Legionella Potentially 87.50% 87.71% specific to L. Potentially longbeachae specific to L. pneumophila

Figure 4.133: Pie charts of predicted cellular localisation of proteins ((A & B) and of predicted specificity of proteins identified (C & D) for L. pneumophila (A & C) and L. longbeachae (B & D)

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Most of the proteins (approximately 80%) were predicted to be localised to the cytoplasm (Figure 4.14 A & C). The proteins were also investigated to determine whether they shared any sequence homology with any other congeneric species or other organisms. The majority of the proteins (approximately 88%) were found to share significant sequence homology ( 35%) with proteins from organisms other than Legionella (Figure 4.14 B and D). About 12% of the identified proteins for each L. pneumophila and L. longbeachae were found specific to the genus Legionella. However, L. longbeachae was found to have at least four times as many species-specific proteins as compared to L. pneumophila. These predictions were carried out to assist the downstream analyses and help to set filters and assign proteins of interest for further investigations.

Figure 4.14 illustrates the most probable cellular localisation of some of the proteins identified in this study and which have been reported to be associated with virulence and pathogenesis. Surface exposed proteins are likely to be immunogenic.

Figure 4.144: Most probable cellular localisation of identified virulence and pathogenesis related proteins

(?) denotes that the final cellular localisation of the protein is unknown (*) denotes that the protein is translocated into the host cell cytoplasm during infection Hsp60: Heat shock protein 60 kDa. LvgA: Legionella virulence gene A protein. MOMP: Major Outer Membrane Protein. Mip: Major infectivity potentiator. Dot(H, N, O, B): Defect in organelle trafficking (H, N, O, B) proteins. Enh, EnhC: Enhanced entry proteins. IcmX: Intracellular multiplication X protein. SidC and SidA: Dot/Icm substrates. SdhA: succinate dehydrogenaseflavoprotein subunit A. VipF: N- terminal acyltransferase. IspE: Type II secretory protein E. Lgt: Prolipoprotein diacylglyceryl transferase. WipC: Dot/Icm related protein. RpoS: RNA polymerase Sigma factor. LidA: Rab effector. The diagram represents the cellular localisation of proteins that have been identified from the KEGG PATHWAY and PATRIC (Virulence Factors of Pathogenic Bacteria (VFDB)) databases.

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4.3.7 Potential virulence and pathogenesis related proteins

Tables 4.3 and 4.4 demonstrate that there are several proteins that were not annotated as proteins related to pathogenesis and virulence in the VBDF database. References were obtained from literature to support the hypothesis that some proteins may actually be homologs of proteins found in different bacteria. Fifty-two out of eighty proteins are or are hypothesised to be potentially related to L. pneumophila virulence and pathogenesis, as supported by literature. On the other hand, 26 out of 67 proteins from L. longbeachae are supported by literature as participating or hypothesised to participate in virulence and pathogenicity.

The roles of other proteins in virulence and pathogenesis, but not supported by literature, are predicted based on their function, cellular localisation and/or specificity. A majority of these proteins are uncharacterised and therefore do not have any function assigned, nor were they found to have significant homology to any characterised proteins in other organisms. At least twice as many uncharacterised proteins were found in L. longbeachae as compared to L. pneumophila. Moreover, only four virulence and pathogenesis proteins were found to be specific to L. pneumophila compared to 14 from L. longbeachae.

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Table 4.3: Potential virulence and pathogenesis related proteins for L. pneumophila

S. UniProt Accession Protein Most probable function Cellular Specificity Reference No. No. localisation

1 CH60_LEGPH 60 kDa chaperonin (Hsp60) chaperone - stress response/protein folding ML NS PATRIC & KEGG pathway

2 MIP_LEGPH Outer membrane protein (MIP) virulence factor pathogenesis OM NS PATRIC & KEGG pathway

3 Q5ZRC1_LEGPH Major outer membrane protein (MOMP) unknown OM SLG PATRIC & KEGG pathway

4 Q5ZS30_LEGPH IcmX (IcmY) transport/binding and pathogenesis EX SLG PATRIC & KEGG pathway

5 Q5ZSK6_LEGPH SidC, interaptin toxin production and pathogenesis ML SLG PATRIC & KEGG pathway

6 Q5ZYB6_LEGPH IcmB (DotO) unknown UN NS PATRIC & KEGG pathway 7 Q5ZYB7_LEGPH IcmJ (DotN) unknown UN NS PATRIC & KEGG pathway

8 Q5ZYC2_LEGPH IcmK (DotH) transport/binding and pathogen function PP NS PATRIC & KEGG pathway

9 Q5ZY48_LEGPH Hypothetical virulence protein (LvgA) toxin production CY SLG PATRIC & KEGG pathway 10 Q5ZVU0_LEGPH L-lysine dehydrogenase (Enh) amino acid metabolism- oxidoreductase CY NS PATRIC & KEGG pathway

11 Q5ZS43_LEGPH DotB transport and binding/pathogenesis CY NS PATRIC & KEGG pathway

12 Q5ZVW9_LEGPH Type II secretory pathway protein E (IspE) protein transport activity CY NS PATRIC & KEGG pathway

13 Q5ZTF6_LEGPH Putative uncharacterized protein (WipC) unknown CY SLG PATRIC & KEGG pathway

14 Q5ZZA8_LEGPH N-terminal acetyltransferase, GNAT family (VipF) protein fate/hydrolysis/secretion-transferase CY SLG PATRIC & KEGG pathway

15 Q5ZY43_LEGPH Succinate dehydrogenase flavoprotein subunit A TCA cycle- oxidoreductase CM NS PATRIC & KEGG pathway (SdhA)

16 Q5ZVS2_LEGPH Putative uncharacterized protein (Lgt) unknown CY SLP PATRIC & KEGG pathway

17 Q5ZSK7_LEGPH SdcA unknown CY SLG PATRIC & KEGG pathway

18 ENO_LEGPH Enolase glycolysis: carbohydrate metabolism-lyase ML NS (Sha et al., 2009) 19 G0Y714_9GAMM Peptidyl-prolyl cis-trans isomerase protein folding- isomerase OM NS (Fischer et al., 1992)

20 KATG2_LEGPH Catalase-peroxidase 2 hydrogen peroxide metabolism- oxidoreductase ML NS (Bandyopadhyay et al., 2003) 8 9

21 LLY_LEGPH 4-hydroxyphenylpyruvate dioxygenase cytolysis haemolytic activity CY NS (Wintermeyer et al., 1994)

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22 Q5F2T9_LEGPH Peptidyl-prolyl cis-trans isomerase protein folding- isomerase OM NS (Fischer et al., 1992)

23 Q5ZR84_LEGPH Astacin protease proteolysis(processing of extracellular proteins) ML NS (Galka et al., 2008) 24 Q5ZRN2_LEGPH Ribonuclease, T2 family DNA/RNA degradation ML NS (Rossier et al., 2009)

Table 4.3: Potential virulence and pathogenesis related proteins for L. pneumophila (continued)

S. UniProt Accession Protein Most probable function Cellular Specificity Reference No. No. localisation 25 Q5ZRR6_LEGPH Aminopeptidase hydrolase protease EX NS (Rossier et al., 2008) 26 Q5ZRZ4_LEGPH Peptidyl-prolyl cis-trans isomerase cell envelope/toxin synthesis- hydrolase CY NS (Fischer et al., 1992) 27 Q5ZS47_LEGPH Zinc protease (Peptidase, M16 family) hydrolase protein fate UN NS (Rechnitzer and Kharazmi, 1992) 28 Q5ZS48_LEGPH Zinc protease (Peptidase, M16 family) protease PP NS (Rechnitzer and Kharazmi, 1992) 29 Q5ZSD0_LEGPH D-alanyl-D-alanine carboxypeptidase, fraction B proteolysis PP NS (Jameson-Lee et al., 2011) 30 Q5ZSD1_LEGPH Acid sphingomyelinase-like phosphodiesterase DNA/RNA degradation EX SLG (Lurie-Weinberger et al., 2010) 31 Q5ZSD3_LEGPH Cysteine protease, papain C1 family hydrolase proteolysis ML SLG (Choy et al., 2012) 32 Q5ZSW5_LEGPH Putative secreted esterase beta lactamase like domain UN SLG (Aragon et al., 2000) 33 Q5ZTE5_LEGPH Chitinase domain carbohydrate metabolic process PP NS (Debroy et al., 2006) 34 Q5ZTW5_LEGPH Peptidoglycan associated lipoprotein integral to outer membrane OM NS (Shevchuk et al., 2011) 35 Q5ZTY9_LEGPH Serine metalloprotease hydrolase proteolysis EX NS (Huston, 2010) 36 Q5ZU46_LEGPH Peptidyl-prolyl cis-trans isomerase toxin production and pathogenesis-isomerase ML NS (Fischer et al., 1992) 37 Q5ZU63_LEGPH 3',5'-cyclic nucleotide phosphodiesterase signal transduction ML NS (Levi et al., 2011) 38 Q5ZU69_LEGPH Polyketide synthase, type I virulence/pathogenesis secondary metabolites CM NS (Nosanchuk and Casadevall, 2006) biosynthesis- transferase 39 Q5ZU97_LEGPH D-alanyl-D-alanine carboxypeptidase protein hydrolysis and secretion PP SLG (Jameson-Lee et al., 2011) 40 Q5ZUF7_LEGPH 27 kDa outer membrane protein integral to outer membrane OM NS (Khemiri et al., 2008) 41 Q5ZUI8_LEGPH Long chain fatty acid transporter trasnport and binding OM SLG (Shevchuk et al., 2011) 42 Q5ZW43_LEGPH LvrE transport and binding EX NS (Cianciotto, 2009) 43 Q5ZWG6_LEGPH Major acid phosphatase hydrolase EX NS (Aragon et al., 2001)

90 44 Q5ZWG9_LEGPH Chitinase carbohydrate metabolic process- hydrolase EX SLG (Debroy et al., 2006)

I Page 45 Q5ZX37_LEGPH Putative uncharacterized protein- Sel1 repeat unknown UN SLG (Newton et al., 2007) 46 Q5ZX67_LEGPH Cytochrome c type biogenesis protein CycH generation of precursor metabolites and energy EX NS (Yip et al., 2011)

47 Q5ZXD4_LEGPH Putative uncharacterized protein (TolC) unknown UN SLG (Stewart et al., 2011) 48 Q5ZYA5_LEGPH Zinc metalloprotease proteolysis EX NS (Rechnitzer and Kharazmi, 1992)

Table 4.3: Potential virulence and pathogenesis related proteins for L. pneumophila (continued)

S. UniProt Accession Protein Most probable function Cellular Specificity Reference No. No. localisation 49 Q5ZZ24_LEGPH Zinc metalloprotein hydrolase proteolysis ML NS (Rechnitzer and Kharazmi, 1992) 50 Q5ZZ87_LEGPH Cytochrome c4 electron carrier energy metabolism PP NS (Yip et al., 2011) 51 Q5ZZH8_LEGPH Leucine aminopeptidase hydrolase proteolysis EX NS (Rossier et al., 2008) 52 SODF_LEGPH Superoxide dismutase [Fe] antioxidant-oxidoreductase PP NS (Sadosky et al., 1994) 53 Q5ZTW6_LEGPH Outer membrane protein unknown OM SLG N/A 54 Q5ZX25_LEGPH Flagella basal body P-ring formation protein FlgA cell projection OM SLG N/A 55 Q5ZXA5_LEGPH Agglutination protein protein transport OM NS N/A 56 Q5ZXJ9_LEGPH Putative uncharacterized protein (putative antigen) unknown OM SLG N/A 57 Q5ZXU7_LEGPH Putative uncharacterized protein (substrate of dot/icm) unknown UN SLP N/A 58 Q5WTS7_LEGPL Putative uncharacterized protein unknown UN NS N/A 59 Q5ZRE0_LEGPH Putative uncharacterized protein unknown OM SLG N/A 60 Q5ZRF5_LEGPH Putative uncharacterized protein unknown UN SLG N/A 61 Q5ZRK7_LEGPH Putative uncharacterized protein unknown ML SLP N/A 62 Q5ZSJ8_LEGPH Putative uncharacterized protein unknown UN SLG N/A 63 Q5ZSU3_LEGPH Putative uncharacterized protein unknown UN SLP N/A 64 Q5ZT45_LEGPH Putative uncharacterized protein unknown UN SLG N/A 65 Q5ZT87_LEGPH Putative uncharacterized protein unknown UN SLG N/A 66 Q5ZTA5_LEGPH Putative uncharacterized protein unknown UN SLG N/A 67 Q5ZTD3_LEGPH Saframycin Mx1 synthetase B potential virulence factor-catalytic activity CY NS N/A 91 68 Q5ZSJ1_LEGPH Putative uncharacterized protein unknown UN NS N/A

I Page 69 Q5ZUI1_LEGPH Putative uncharacterized protein unknown UN SLG N/A 70 Q5ZUM2_LEGPH Peptide maturation protein PmbA protein and peptide secretion and trafficking CY NS N/A

71 Q5ZV15_LEGPH Putative secreted protein unknown UN NS N/A

Table 4.3: Potential virulence and pathogenesis related proteins for L. pneumophila (continued)

S. UniProt Accession Protein Most probable function Cellular Specificity Reference No. No. localisation

72 Q5ZV98_LEGPH PQQ (Pyrrolo quinoline) WD40-like repeat, enzyme protein insertion OM SLG N/A repeat domain protein 73 Q5ZVK9_LEGPH Putative uncharacterized protein unknown UN NS N/A

74 Q5ZY32_LEGPH Probable membrane protein YdgA-like unknown UN SLG N/A

75 Q5ZYR1_LEGPH Putative uncharacterized protein unknown UN SLG N/A

76 Q5ZZC0_LEGPH Conserved domain protein unknown UN SLG N/A

77 Q5ZZC6_LEGPH Putative uncharacterized protein unknown UN NS N/A

78 Q5ZZG9_LEGPH Putative uncharacterized protein proteolysis UN NS N/A

79 Q5ZZK2_LEGPH Putative uncharacterized protein unknown UN SLG N/A

80 Y2959_LEGPH UPF0422 protein lpg2959 unknown OM NS N/A

Cellular localisation: (EX=Extracellular, OM=Outer membrane, UN=unknown, CY=cytoplasmic, CM=cytoplasmic membrane, ML=multiple localisations, PP=periplasmic).

92 Specificity: (SLG=specific to Legionella genus, SLP=specific to L. pneumophila, NS denotes: potential homologs (orthologs, paralogs) found in other organisms based on

I Page BLASTp (Basic Local Alignment Sequence Tool protein) sequence homology (>35%)). References: N/A not available (references were either for proteins identified for Legionella or homologs in other bacteria).

Table 4.4: Potential virulence and pathogenesis related proteins for L. longbeachae

S. UniProt Accession Protein Most probable function Cellular Specificity References No. No. localisation

1 D3HIV8_LEGLN Coiled coil protein, some similarities with (LidA) similar to LidA effector PP SLB PATRIC & KEGG pathway

2 D3HJ70_LEGLN Peptidyl-prolyl cis-trans isomerase (MIP) interconversion of peptidyl prolyl bond OM NS PATRIC & KEGG pathway

3 D3HKW1_LEGLN Enhanced entry protein (EnhC) beta-lactamase activity/hydrolase OM SLG PATRIC & KEGG pathway

4 D3HL94_LEGLN Putative uncharacterized protein (DotO) unknown CM NS PATRIC & KEGG pathway

5 D3HM60_LEGLN SidC protein (SidC & SidA) dot/icm secretion system substrate CY SLG PATRIC & KEGG pathway

6 D3HPY7_LEGLN 60 kDa chaperonin (Hsp60) protein refolding / response to stress CY NS PATRIC & KEGG pathway

7 D3HJZ0_LEGLN Putative dehydrogenase (Enh) degradation of toxic compounds/regeneration CY NS PATRIC & KEGG pathway of NAD+

8 D3HRI0_LEGLN RNA polymerase sigma factor (Rpos) transcription regulation CY NS PATRIC & KEGG pathway

9 D3HPB2_LEGLN Succinate dehydrogenase flavoprotein subunit (SdhA) carbohydrate metabolism/Electron transport CY NS PATRIC & KEGG pathway

10 D1RM17_LEGLO Protein tyrosine phosphatase catalytic domain- signal transduction- hydrolase UN SLG (Salomon and Orth, 2013) containing protein 11 D3HK14_LEGLN Chitin-binding protein CbpD pathogen-host interaction/viral capsid-like EX NS (Debroy et al., 2006)

12 D3HKH5_LEGLN Enolase glycolysis EX NS (Sha et al., 2009) 13 D3HL32_LEGLN Zinc metalloproteinase ProA/Msp proteolysis- virulence and pathogenesis EX NS (Rechnitzer and Kharazmi, 1992)

14 D3HMQ0_LEGLN Thiol:disulfide interchange protein DsbA required in pilus biogenesis PP NS (Jameson-Lee et al., 2011)

15 D3HN33_LEGLN ATP synthase subunit beta ATP synthesis ML NS (Shinoy et al., 2013)

16 D3HNC2_LEGLN Superoxide dismutase oxidoreductase PP NS (Sadosky et al., 1994)

17 D3HNJ9_LEGLN Cytochrome c Hsc electron carrier PP NS (Yip et al., 2011)

18 D3HNX4_LEGLN Aminopeptidase N aminopeptidase activity ML NS (Rossier et al., 2008)

19 D3HP47_LEGLN Peptidyl-prolyl cis-trans isomerase isomerase activity OM NS (Fischer et al., 1992) 93

20 D3HP66_LEGLN Putative stringent starvation protein B starvation protein UN NS (Abu-Zant et al., 2006)

I Page 21 D3HQ84_LEGLN Protein with a bacterial immunoglobulin-like domain potential invasin/intimin ML NS (Matsunaga et al., 2003) 22 D3HP86_LEGLN Putative zinc protease protease UN NS (Rechnitzer and Kharazmi, 1992)

23 D3HRD0_LEGLN Putative long-chain fatty acid transporter porin protein OM SLG (Shevchuk et al., 2011)

Table 4.4: Potential virulence and pathogenesis related proteins for L. longbeachae (continued)

S. UniProt Accession Protein Most probable function Cellular Specificity References No. No. localisation 24 D3HRJ7_LEGLN Ankyrin repeat protein unknown CY SLB (Al-Khodor et al., 2010) 25 D3HNH8_LEGLN Putative ankyrin repeat protein unknown CY SLB (Al-Khodor et al., 2010) 26 Q5ZTP9_LEGPH 24 kDa macrophage-induced major protein toxin production and pathogenesis OM NS (Miyamoto et al., 1993) 27 D3HRL3_LEGLN Putative uncharacterized protein unknown CY SLB N/A 28 D3HS61_LEGLN Putative uncharacterized protein unknown UN NS N/A 29 D3HSC0_LEGLN Putative uncharacterized protein pmbA unknown CY NS N/A 30 D3HSK4_LEGLN Putative uncharacterized protein unknown PP SLG N/A 31 D3HSK6_LEGLN Putative uncharacterized protein unknown CY SLG N/A 32 D3HSW5_LEGLN Putative coiled coil protein, weakly similar to unknown CY SLB N/A eukaryotic protein 33 D3HSY2_LEGLN Putative uncharacterized protein unknown UN NS N/A 34 D3HT11_LEGLN Putative uncharacterized protein unknown UN SLG N/A 35 D3HTL7_LEGLN Putative uncharacterized protein unknown UN SLG N/A 36 D3HTR4_LEGLN Putative uncharacterized protein unknown CY NS N/A 37 D1RCR6_LEGLO Putative uncharacterized protein Unknown (potentially Dot/Icm substrate) PP SLG N/A 38 D1REZ3_LEGLO Putative uncharacterized protein unknown UN NS N/A 39 D1RGM8_LEGLO BNR/Asp-box repeat domain protein complex sugar degradation/anti-bacterial EX SLB N/A defense strategy/pathogenesis (neuraminidase) 40 D3HK57_LEGLN Putative uncharacterized protein unknown CY SLG N/A 41 D3HK81_LEGLN Putative uncharacterized protein unknown UN SLB N/A 42 D3HK82_LEGLN Putative uncharacterized protein unknown CY SLB N/A 94 43 D3HKN1_LEGLN Putative uncharacterized protein Unknown (substrate of Dot/Icm secretion) CY SLG N/A

I Page 44 D3HKV6_LEGLN Putative uncharacterized protein unknown CY SLG N/A 45 D3HLF3_LEGLN Putative uncharacterized protein unknown UN SLB N/A 46 D3HLV2_LEGLN Putative uncharacterized protein unknown CY NS N/A

Table 4.4: Potential virulence and pathogenesis related proteins for L. longbeachae (continued)

S. UniProt Accession Protein Most probable function Cellular Specificity References No. No. localisation 47 D3HLW3_LEGLN Putative uncharacterized protein unknown CY NS N/A 48 D3HLW4_LEGLN Putative uncharacterized protein unknown CY SLG N/A 49 D3HM66_LEGLN Putative uncharacterized protein unknown CY NS N/A 50 D3HM79_LEGLN Putative uncharacterized protein unknown UN SLB N/A 51 D3HM80_LEGLN Putative coiled-coil protein unknown CY SLB N/A 52 D3HM81_LEGLN Putative uncharacterized protein unknown UN SLB N/A 53 D3HM82_LEGLN Putative coiled-coil protein unknown CY SLB N/A 54 D3HMI8_LEGLN Putative uncharacterized protein stress response similarity to heat shock UN SLG N/A 55 D3HN80_LEGLN Putative uncharacterized protein unknown CY SLG N/A 56 D3HNR9_LEGLN Putative coiled-coil protein unknown ML SLB N/A 57 D3HPI1_LEGLN Putative uncharacterized protein Unknown (Dot/Icm substrate) CY SLG N/A 58 D3HPX5_LEGLN Putative uncharacterized protein unknown UN SLG N/A 59 D3HQ67_LEGLN Putative uncharacterized protein unknown SLG SLG N/A 60 D3HQI7_LEGLN IcmL-like protein potentially macrophage killing EX SLG N/A 61 D3HQP8_LEGLN Putative uncharacterized protein unknown CY SLG N/A 62 D3HQX7_LEGLN Putative uncharacterized protein unknown CY NS N/A 63 D3HR60_LEGLN UPF0234 protein LLO_1035 unknown CY NS N/A 64 D3HR67_LEGLN Some similarity with eukaryotic proteins unknown CY SLG N/A 65 D3HR81_LEGLN Putative outer membrane protein oxidoreductase OM NS N/A 66 D3HR93_LEGLN Weak similarity to eukaryotic proteins unknown CY SLG N/A 95 67 D3HRL1_LEGLN Putative uncharacterized protein unknown UN SLG N/A

I Page Cellular localisation: (EX=Extracellular, OM=Outer membrane, UN=unknown, CY=cytoplasmic, CM=cytoplasmic membrane, ML=multiple localisations, PP=periplasmic). Specificity: (SLG=specific to Legionella genus, SLB=specific to L. longbeachae, NS denotes: potential homologs (orthologs, paralogs) found in other organisms based on

BLASTp (Basic Local Alignment Sequence Tool protein) sequence homology (>35%)). References: N/A not available (references were either for proteins identified for Legionella or homologs in other bacteria).

Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

4.3.8 Classifying identified proteins based on function

4.3.8.1 Grouping of proteins based on most probable cellular function

Figure 4.15 shows all the identified proteins for L. pneumophila and L. longbeachae grouped according to their most probable function in the cell. In this study, proteins involved in a variety of functions have been identified.

Protein grouping according to most probable cellular function

Post-translational modification L. longbeachae L. pneumophila Fatty acid & Lipid metabolism

Carbohydrate metabolism

DNA and nucleotide metabolism

Cell division

Cell homeostasis/chaperone

Virulence and pathogenesis

physiological processes Transport

Transcription and translation…

Amino acid & protein metabolism

Others

Unknown

0 20 40 60 80 100 120 Number of identified proteins

Figure 4.15: Grouping of identified proteins according to most probable cellular function

Proteins that do not belong to any of the 10 major groups, excluding those classified as unknown, are grouped under others. Although the total number of proteins identified for L. pneumophila was higher compared to L. longbeachae, it can be seen that L. pneumophila had a higher number of proteins identified compared to L. longbeachae for most of the cellular metabolic processes. As inferred from the bar chart, it is possible that cellular metabolic processes may be more pronounced in L. pneumophila than in L. longbeachae. To investigate this hypothesis, carbohydrate metabolism pathways were chosen for further analysis by data mining as L. pneumophila has been reported to preferably use amino acids as its carbon source and that the availability of glucose as a

96 | Page

Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae substrate may not affect its growth (Harada et al., 2010). Therefore, four major pathways involved in energy metabolism, glycolysis/gluconeogenesis, the citric acid cycle and the Entner-Doudoroff and Pentose Phosphate pathway were studied to determine any differences by comparing the data available for all the identified proteins for L. pnuemophila and L. longbeachae.

4.3.8.2 Investigation of the glycolysis/gluconeogenesis pathway

Overall, there appears to be a fully functional glycolytic/gluconeogenetic pathway present in both Legionella species. Figure 4.16 gives a schematic representation of the enzymes involved in glycolysis/gluconeogenesis and those which were identified in this study.

Figure 4.166: Proposed glycolysis/gluconeogenesis pathway The pathway was constructed with metabolic pathway data available from PATRIC and KEGG pathways. The arrow in red indicates that Legionella does not seem to possess the enzyme for conversion of fructose 1, 6 bi-phosphate to fructose 6-phosphate as mentioned in literature. The genes for the highlighted enzymes are known to be present in Legionella but were not identified in this study. 97 | Page Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

Most of the proteins involved in the pathway were identified. Aldehyde dehydrogenase, fructose-biphosphate aldolase and phosphofructokinase identified for L. longbeachae are annotated on the database as putative proteins of the corresponding assigned functions. This result shows that the putative proteins may possibly be carrying out the assigned function.

4.3.8.3 Investigation of the citric acid cycle

Figure 4.17 shows the pathway constructed with metabolic pathway data available from PATRIC and KEGG pathways, to illustrate possible links with identified proteins.

Figure 4.177: Proposed citric acid cycle pathway

The pathway was constructed with metabolic pathway data available from PATRIC and KEGG pathways. 98 | Page Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

The citric acid cycle, also known as the tricarboxylic acid cycle or Krebs cycle, is a very important pathway to generate energy in the form of ATP for cellular processes by the oxidation of acetate derived from carbohydrate, fats and proteins/amino acids metabolism. It also provides intermediate substrates for the metabolism of fatty acids and amino acids. The citric acid cycle appears to be complete with the identification of all the enzymes required in the pathway.

4.3.8.4 Investigation of the Entner-Doudoroff and Pentose Phosphate pathway

Figure 4.18 shows the proposed Entner-Doudoroff and Pentose Phosphate pathway that may be used by the bacteria for glucose metabolism.

Figure 4.188: Proposed Entner-Doudoroff and Pentose Phosphate pathway The pathway was constructed with metabolic pathway data available from PATRIC and Kegg pathways. The arrow in red indicates that Legionella does not seem to possess the enzyme for conversion of fructose 1, 6 bi-phosphate to fructose 6-phosphate, as mentioned in literature. The genes for the highlighted enzymes are known to be present in Legionella but were not identified in this study. 99 | Page Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

The Entner-Doudoroff pathway is an alternative glucose metabolic pathway found in a few organisms. Legionella appears to use this pathway together with the glycolysis/gluconeogenesis pathway. The phosphogluconate dehydratase identified for L. longbeachae is annotated in the database as putative protein of the corresponding assigned function, suggesting that the putative protein may possibly be carrying out the assigned role.

4.4 DISCUSSION

4.4.1 Optimisation of growth conditions Legionella bacterial cells were harvested 30 hours post-inoculation as it was observed in preliminary investigations to determine the time for cultures to reach the post- exponential phase that the broth started browning after this time. This browning of the broth, which was found to be problematic for analysis of the secretome by 2D gel electrophoresis, has been reported in the literature (Steinert et al., 1995) and was suggested to be the result of melanin production, mediated by the protein legiolysin, at the late stationary phase of growth as a survival response to light stress. While this did not seem to be associated with virulence, Nosanchuk and Casadevall (2006) suggested that not only does melanin protect microorganisms from light but may also confer the ability to escape host defense mechanisms. The authors also suggested that melanins which are synthesised through the polyketide pathway from acetate are typically black or brown. In this study, 4-hydroxyphenylpyruvate dioxygenase, which converts p- hydroxyphenylpyruvate into homogentisic acid and subsequently oxidises and polymerises to form melanin-like pigment (Steinert et al., 2001), was one of the proteins identified in L. longbeachae. Polyketide synthase Type I was identified from L. pneumophila cultures. These proteins may be responsible for the browning of the broth and are potentially virulence factors. Moreover, both bacterial species and isolates were found to show a similar trend of growth. Therefore, 30 hours post-inoculation was deemed suitable for protein isolation.

4.4.2 Optimisation of 2D gel electrophoresis 2D gel electrophoresis required optimisation as either there were no protein spots visible on the gels or streaks were present. A change in the parameters of isoelectric focussing and an endonuclease treatment were found to improve the quality of the gels.

100 | Page Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

The final step of the isoelectric focussing was increased from 40,000 to 66,000 volt- hours for proper focussing of the proteins on the immobilized pH gradient strip (IPG) strip. However, this resulted in separation of only a few distinct spots and streaks were present when the second dimension separation was performed.

There are several reasons for streaking and, as the samples were slightly viscous, it was believed that the protein mixture was contaminated with nucleic acid. An endonuclease treatment to remove contaminating nucleic acids and the use of 66,000 volt-hours as the final step of the isoelectric focussing resulted in improved gels with distinctly separated protein spots.

4.4.3 Comparative analysis of the two Legionella longbeachae isolates The comparative proteomic analysis to determine differentially expressed proteins between the two L. longbeachae isolates did not show any significant difference. As such, no protein which is potentially related to virulence and specific to L .longbeachae was identified in this study. Therefore, the observation made by Doyle et al. (2001), that the Australian clinical isolates of L. longbeachae were more virulent, could not be verified. In this study, only about 10% of the predicted proteins of L. longbeachae were identified. As the bacteria were grown in vitro, it is possible that the protein(s) which may be responsible for the differential ability to cause infection amongst the L. longbeachae geographical isolates might only be expressed in vivo when a host is encountered. Therefore, it appears that any proteins involved in the differential virulence of the two isolates are not constitutively expressed. Hence, the reason for the higher prevalence of infection with L. longbeachae in the Australasia region remains inexplicable.

4.4.4 Comparative analysis between L. pneumophila and L. longbeachae The comparative proteomic analysis revealed that numerous proteins involved in a variety of functions are equally expressed in both species, and it was not surprising that the majority of proteins isolated were identified as cytoplasmic proteins. Although most of the proteins were cytoplasmic, it was interesting to note that proteins of different physiological functions involved in different cellular metabolic pathways were also identified, of which some were commonly found expressed in both the species, whilst others were probably not expressed in one or the other species. 101 | Page

Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

The proteins equally expressed are the housekeeping proteins , which are mostly enzymes required for functions such as energy metabolism, and protein, fatty acids and amino acids synthesis found ubiquitously in almost all organisms. This was demonstrated in Section 4.3.8.3 where the citric acid cycle is provided as an example. However, as L. longbeachae has not been largely studied, several proteins remain uncharacterised. Four such proteins, which were assigned putative functions, may in fact be carrying out the assigned role as was shown in Sections 4.3.8.4 and 4.3.8.4 for the glycolysis/gluconeogenesis and pentose phosphate pathways, respectively.

L. pneumophila is a fresh-water dwelling microbe whereas L. longbeachae is adapted to soil environments. It was hypothesised that the two species may have different mechanisms to acquire nutrients from their surroundings and probably show differences in the metabolic pathways. In this study, it was found that L. pneumophila may have secreted a considerably larger amount of proteins, including many enzymes which are involved in the carbohydrate and amino acid metabolic pathways that are used to generate energy, as compared to L. longbeachae. As several replicates and similar growth conditions were used in this study and comparable results were obtained for 2D gel electrophoresis, it appears that L. pneumophila has a larger secretome than L. longbeachae. This suggests that either L. pneumophila is metabolically more active than L. longbeachae or the latter may have a different mechanism of acquiring and metabolising nutrients, as it is still able to grow and multiply in the CYE culture medium. However, as this study has some limitations, such as only the most abundant proteins were chosen for identification and not all the proteins were successfully identified, this inference remains inconclusive. An important point to note, though, is that many Legionella species are difficult to culture in vitro (Lee et al., 1993). One of the reasons for many bacterial species being unculturable is that the inappropriate source of carbon is incorporated in the culture media used to grow the organisms (Stewart, 2012).

The inability to isolate several other Legionella species through culturing in the commonly used culture medium to confirm a positive diagnosis/detection may be suggestive that infection with certain species is over-reported. Likewise, the fact that L. pneumophila may require less complex culture medium for growth as compared to its congeneric species may be suggestive of its better survival abilities in different environments. Therefore, an understanding of the metabolic pathways for generating 102 | Page

Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae energy involved in growth, replication and repair may help in the formulation of better culture medium, especially where culture is necessary for reliable diagnosis of Legionella infection. Moreover, since metabolism is crucial for virulence and pathogenic bacteria are becoming increasingly resistant to currently available antibiotics, characterising such pathways may prove to be very valuable in identifying potential targets for drug development (Rohmer et al., 2011).

L. longbeachae has been reported to possibly use different strategies of infection (Pereira et al., 2011). The comparative analysis in this study showed that several uncharacterised proteins were expressed in L. longbeachae, many of which may not be present in L. pneumophila. Given these proteins have not been assigned any function, share no sequence homology with any other proteins and are expressed under similar growth conditions as L. pneumophila, it is suggestive of a different lifestyle for this Legionella species, as was identified by Cazalet et al. (2010) in their comparative genomic analysis. Therefore, these proteins appear interesting for further investigation to demonstrate whether they might be implicated in any differences in pathogenesis.

One such protein which was differentially expressed in L. longbeachae was the bacterial immunoglobulin domain containing protein which is of particular interest as it was found to have homology to invasin/intimin proteins reported in Escherichia coli and Yersiniae (Oberhettinger et al., 2012). This protein has been identified to possess five immunoglobulin domains by bioinformatics analysis, with significant homology to the intimin protein of E. coli. The protein has been reported to be related to virulence and pathogenesis, has been demonstrated as an autotransporter designated to the Type Ve secretion system and was found to mediate an intimate bacterial-host cell interaction via cell adhesion in E. coli (Kelly et al., 1999). A Type V secretion system is absent in L. pneumophila suggesting that L. longbeachae may be using different infection strategies.

It may be anticipated that proteins from two different species to show significant variations due to the differences in the homologous proteins and the proteins to be resolved as different spots on 2D gels. However, this study showed that DIGE can be used for comparative analysis of congeneric species as several proteins were found to be differentially expressed in terms of abundance and certain proteins which were not found from one species or the other under similar growth conditions were also detected.

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Chapter 4 Comparative analysis of L. pneumophila vs. L. longbeachae

4.4.5 Virulence and pathogenesis related proteins of Legionella In this study, virulence and pathogenesis related proteins, such as the effectors and substrates of the Dot/Icm secretion system generally expected to be expressed when Legionella is inside the host cell, have been identified, demonstrating the ability of the pathogen to synthesise these under in vitro culture conditions. It may also suggest that these proteins may actually accumulate in the bacterial cells even before they invade the host cells and may therefore either have multiple functions in the cell or are in the repertoire ready for infection when a host is encountered.

4.5 CONCLUDING REMARKS

Several proteins were identified to be equally and differentially expressed in L. pneumophila and L. longbeachae under similar in vitro growth conditions. Thus, the comparative analysis revealed that there exists a large variation in the proteomes of the two Legionella species at the constitutive level. This is significant in the study of pathogenesis and virulence as the two Legionella species have been reported to have different infectivity potential. However, no such protein was found to be differentially expressed in the two L. longbeachae isolates which could explain the difference in the virulence between the geographical strains. Nevertheless, the genus-specific and species-specific proteins are of particular interest as they can be targeted as potential candidates for biomarker identification and vaccines. In the next chapter, an immunoproteomics approach will be used to identify whether any of the identified proteins are immunogenic and whether they could be targeted as potential biomarkers.

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Chapter 5 Identification of immunogenic proteins

IDENTIFICATION OF IMMUNOGENIC PROTEINS

5.1 INTRODUCTION

The identification of immunogenic proteins is of great importance as it allows their use in numerous applications, such as biomarker discovery for infection diagnosis, potential vaccine candidates, drug targets and the study of pathogenesis. During infection, certain proteins, mainly those exposed on the surface of pathogens, are identified as foreign bodies (antigens) by the host and trigger an immune response, which in turn induce the production of antibodies against the antigens as a defence mechanism. Immunogenic proteins can be identified through several immunoassays, for example, enzyme-linked immunosorbent assay (ELISA) and Western blot. These assays require the use of antibodies acting as probes that recognise specific binding sites on the target antigens. Serum samples collected after an infectious episode represent a good source of such antibodies for the identification of immunogenic proteins.

Legionella pneumophila gained a lot of research attention since it was first discovered and has therefore been well characterised. However, little information is available for other Legionella species, especially L. longbeachae which is being increasingly reported as one of the main causes of Legionella infections in some parts of the world. One possible reason for the scarcity of studies being carried out on non-pneumophila species is because the incidence of infections with those species is believed to be significantly lower compared to L. pneumophila. However, this may be due to a lack of proper diagnostic tests available for non-pneumophila species and therefore it is likely that infection with non-pneumophila species are being under-reported. To date, several antigens have been proposed as potential candidates for the development of a genus- wide diagnostic test, but no such test is currently commercially available.

105 | Page Chapter 5 Identification of immunogenic proteins

5.2 EXPERIMENTAL DESIGN

5.2.1 Aims and objectives

Of the several reported Legionella antigens and other potential virulence and pathogenesis related proteins, many are either genus-specific or species-specific. A summary of these was described in the previous chapter in section 4.3.7.

This study aims at developing a methodology based on an indirect ELISA protocol to detect immunogenic reactions with isolated proteins from several pathogenic Legionella species. The study further aim at determining those proteins that are most likely to elicit an immune response during infections and can therefore be targeted as potential biomarkers. The identification of such proteins which are commonly immunoreactive across different species and may be targeted as genus-specific and the ones which are differentially immunoreactive as species-specific biomarkers is proposed for this study. These proteins may alternatively be targeted as vaccine candidates and drug targets.

5.2.2 Experimental procedures

The first step in the detection of immunogenic proteins was to identify human serum samples from a set of blind-samples (some of which were known to contain antibodies to Legionella by the source laboratory) which were immunoreactive to Legionella total cell soluble proteins by ELISA. The samples that showed immunoreactivity were then subjected to further analysis by a Dot-blot to confirm the result obtained from ELISA.

The whole cell total soluble proteins, secretome and outer-membrane sub-proteome, either from one or several Legionella species, were subjected to Western blot analysis. The immunoreactive proteins detected in the Western blots were identified from the corresponding preparative gels from Chapter 4 for the whole cell total soluble protein and the secretome, and also by SDS-PAGE and peptide mass fingerprinting carried out in this chapter. An analysis of the outer-membrane sub-proteome was also performed as not many outer membrane proteins were identified in the previous study. As L. pneumophila has been well characterised, this section will focus on L. longbeachae.

Serum samples for this study were obtained from two different sources, denoted as Source A and Source B hereafter. The serum samples from Source A were identified as containing antibodies to Legionella species through serology, using an in-house

106 | Page Chapter 5 Identification of immunogenic proteins developed indirect fluorescence assay test (IFAT), whilst antibodies to Legionella from Source B were detected using the LEGIONELLA SP IFA IgG kit from Vircell. Ethics approval was obtained from the Swinburne University of Technology Research Ethics Committee to enable the use of de-identified serum samples with a no consent waiver (Appendix 10).

5.3 RESULTS 5.3.1 ELISA of human sera against Legionella proteins

Figure 5.1 shows the result of an indirect ELISA carried out to determine which serum samples may have antibodies present against Legionella species. This was performed as the serum samples were blinded with respect to samples containing antibodies to which etiological agents were unknown or unconfirmed. A total of 34 serum samples were used.

Two cut-off values were applied in this experiment to be able to select the serum samples most suitable for further analysis. A total of 8 wells of the ELISA plate were not coated with any antigen and were used as negative control. Blank wells were preferred because the use of other genera as negative controls (antigens) may show cross-reaction and affect the selection process of serum samples showing immunoreaction. As genus-specific and species-specific epitopes were to be determined at a later stage, cross-reactivity with other genera at this stage was not deemed to be an issue. The second cut-off was introduced to create a region of suspects between the first and the second threshold values.

The first cut-off value was set at the optical density 0.2 by applying a commonly used formula (Baldi et al., 1999), as mentioned in the footnote of Figure 5.1. As there were no known control serum samples available for this study, the serum samples could not be distinguished as positive or negative because they all showed immunogenic reaction with either one of the species above this cut-off filter. Therefore, another more rigid cut- off value was set using the standard deviation values from the means of each set (set 1 and set 2 from Figure 5.1) in order to minimise inclusion of any serum samples that might have cross-reacted or showed non-specific binding to proteins found in Legionella. As ELISA is mainly based on quantitative analysis, the total soluble protein samples were adjusted to the same concentration for all the species and all serum samples were diluted to the same ratio (1:500) for testing. 107 | Page

Indirect ELISA of L. longbeachae and L. pneumophila antigens against human serum samples 3.5

2.5 L. longbeachae

2 L. pneumophila

cut-off 1.5 St. dev. for OD at 450nm L. pneumophila 1 St. dev. for L. longbeachae 0.5

0 I8F F8L J9V S6L H5J T9S L9R T3E Y4K A4S E1R K9P L7D P4K V2Y A2X A8K R2B U1S Z6D F1N T8D C5D K5H B1G P3N O7K N5A Q3C W7L M7Y X2M P6W D3W Serum samples

108 Figure 5.1: Indirect ELISA for selection of Legionella antibodies containing serum samples

I Page 25 serum samples were from Source A and 9 were from Source B. Whole cell lysate extracts of L. pneumophila (Set 1) and L .longbeachae (Set 2) were tested against the 34 serum samples in this experiment. The error bar on each column represents the average deviation between the 2 replicates used per serum sample. OD: Optical density.

Cut-off filter: (Average optical density of 8 negative control + 3(standard deviation of 8 blank controls)) St. Dev.: standard deviation. This was calculated separately for L. pneumophila (0.72) and L. longbeachae (0.77) based on the OD values for all of the 34 serum samples.

Chapter 5 Identification of immunogenic proteins

In this experiment, eight serum samples were excluded after the first cut-off filter (0.2 OD value) and another sixteen samples were excluded after the second cut-off filter (standard deviation) was applied for L. pneumophila. Similarly, for L. longbeachae, eight samples were excluded after the first cut-off filter and another fourteen were excluded after the second cut-off filter. This resulted in ten serum samples (A2X, R2B, U1S, J9V, Z6D, E1R, C5D, X2M, Y4K and L7D) identified to most likely contain antibodies against L. pneumophila. On the other hand, twelve serum samples (A2X, B1G, T3E, R2B, H5J, U1S, J9V, K5H, Z6D, C5D, X2M and Y4K) were found to most probably have antibodies against L. longbeachae.

These results were used to develop an ELISA protocol for the selection of serum samples for this study that were most likely to contain antibodies against Legionella and were later found to be consistent with the results of other immunoassays used in discriminating between potentially positive and negative samples. This is demonstrated in the next set of results. However, the sensitivity and specificity of the assay could not be determined as there were no confirmed positive and negative controls because none of the serum samples were known to be culture confirmed for Legionella infection.

After applying the standard deviation as a second cut-off filter, serum samples B1G and H5J were found to potentially have antibodies that strongly reacted against L. longbeachae proteins but the reaction was almost negligible for L. pneumophila. As Sample B1G showed a slightly lower OD value for L. pneumophila compared to Sample H5J, the former was chosen for further analysis. Sample C5D showed the closest OD value for both the species and was therefore also selected for subsequent experiments.

5.3.2 ELISA for serum samples B1G and C5D against Legionella proteins

Figure 5.2 shows the results of an indirect ELISA against the total soluble proteins isolated from sonicated cells of 20 Legionella species including two isolates of L. longbeachae. There were no replicates included in this particular experiment as only a small volume of serum samples was available.

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Chapter 5 Identification of immunogenic proteins

Figure 5.2: Indirect ELISA to determine cross-reactivity of serum samples across Legionella species All the Legionella species included in this set of experiment have been reported to be implicated in causing infection in human. The Legionella species were denoted a number from 1 20; 1: L. anisa, 2: L. birminghamensis, 3: L. bozemanii, 4: L. cincinatiensis, 5: L. dumoffi, 6: L. erythra, 7: L. feelei, 8: L. gormanii, 9: L. hackeliae, 10: L. jordanis, 11: L. longbeachae D4968, 12: L. longbeachae NSW150, 13: L. maceachernii, 14: L. micdadei, 15: L. oakridgensis, 16: L. parisiensis, 17: L. pneumophila, 18: L. sainthelensi, 19: L. tucsonensis, 20: L. wadsworthii. St. Dev: standard deviation; serum sample B1G (1.27) and serum sample C5D (0.45)

The results illustrate that whilst the serum samples cross-reacted with several Legionella species, applying the standard deviation from the mean as a cut-off filter excluded several species that could be potentially showing insignificant reaction. As a result, eight species were found to have OD value below the standard deviation threshold or marginally above. This might have been due to no reaction or very weak reaction to any antibodies found in the serum and thus could not be detected. Moreover, as noted from the results of Figure 5.1, L. pneumophila was well under the threshold of the cut-off value of 1.27 for Sample B1G. This indicates that Sample B1G may fall in the suspected region and may not contain antibodies L. pneumophila proteins.

Interestingly, Sample C5D was found to have a much lower standard deviation (0.45) and therefore all the species were well above the standard deviation cut-off threshold. Legionella sainthelensi was found to have the highest OD value against Sample B1G, whereas L. feelei was found to have the highest OD value against Sample C5D. However, it is inconclusive that a stronger immunogenic reaction in this case is indicative of infection with that particular species. Further confirmatory tests were carried out to determine the accuracy and extrapolation of the ELISA results.

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5.3.3 Dot-blot analysis with selected serum samples

Figure 5.3 shows the results of a dot-blot analysis to support the results obatined from ELISA. As can be observed, there was a very strong immunogenic reaction with all the Legionella species against Sample C5D. This suggests that the antibodies present in this serum sample may recognise homologous protein(s) in all Legionella species. Sample T9S, which was used as a control, showed very weak immunogenic reactions detected from the dot-blot. Compared to Sample U1S, Sample A2X showed a weaker reaction with the Legionella proteins as was the case with ELISA, whereby a lower OD value was also recorded (Figure 5.1). Remarkably, the results obtained for the dot-blot with Sample B1G also shows similarity to the results for ELISA (Figure 5.2) whereby some of the species showed much stronger immunoreaction compared to others.

Figure 5.3: Dot-blot of protein from Legionella species against human sera

L1 (Lane 1); 1: L. anisa, 2: L. birminghamensis, 3: L. bozemanii, 4: L. cincinatiensis, 5: L. dumoffi, 6: L. erythra, 7: L. feelei, 8: L. gormanii, 9: L. hackeliae, 10: L. jordanis, 11: Blank. L2 (Lane 2); 1: L. longbeachae D4968, 2: L. longbeachae NSW150, 3: L. maceachernii, 4: L. micdadei, 5: L. oakridgensis, 6: L. parisiensis, 7: L. pneumophila, 8: L. sainthelensi, 9: L. tucsonensis, 10: L. wadsworthii, 11: Negative control

Another dot-blot was performed to validate these results. Sample D3W was chosen as negative control and Sample H5J, which appeared to give similar results to Sample B1G, was tested against L. pneumophila and L. longbeachae. There were no dots visible for Sample D3W. However, for Sample H5J, whilst L. longbeachae showed a very strong reaction, L. pneumophila showed a very weak response, similar to Sample B1G.

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These results were also observed to correlate to the application of the second cut-off filter acquired from the standard deviation. It also suggests that the type of immune response from infection with different Legionella species may be different.

5.3.4 Western blot analyses

Several Western blot analyses were carried out with different serum samples, Legionella species and proteome types to investigate immunoreactivity and identify immunogenic proteins.

5.3.4.1 Total soluble proteins with serum sample B1G

As depicted in Figure 5.4, a Western blot analysis with Sample B1G against the total soluble proteins from 19 Legionella species showed a similar reaction pattern as observed in the ELISA and dot-blot experiments.

Figure 5.4: Western blot analysis of Legionella species with Sample B1G 1: L. cincinatiensis, 2: L. maceachernii, 3: L. wadsworthii, 4: L. oakridgensis, 5: L. birminghamensis, 6: L. sainthelensi, 7: L. longbeachae, 8: L. pneumophila, 9: L. hackeliae, 10: L. parisiensis, 11: L. feelei, 12: L. anisa, 13: L. jordanis, 14: L. micdadei, 15: L. bozemanii, 16: L. gormanii, 17: L. dumoffi, 18: L. erythra and 19: L. tucsonensis. kDa: kiloDalton. M: Marker - Precision Plus Protein Kaleidoscope protein ladder

All the eight species (L. anisa, L. bozemanii, L. cincinatiensis, L. dumoffi, L. longbeachae, L. parisiensis, L. sainthelensi and L. tucsonensis) that showed immunoreaction with Sample B1G in ELISA and dot-blot exhibited similar reaction in Western blot. These results indicate that the ELISA protocol developed was suitable for downstream analyses of the selected serum samples.

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5.3.4.2 Differentially reacting serum samples

Figure 5.5 demonstrates an anticipated immunogenic reaction with serum samples B1G and C5D against the total soluble proteins during a comparative analysis between L. pneumophila and L. longbeachae.

Figure 5.5: Comparative Western blot analysis with serum samples B1G and C5D A: Western blot with serum sample C5D. B: western blot with serum sample B1G. Lanes 1 8 represent total soluble proteins (duplicates were run on the same gel). 1&5: L. pneumophila, 2&6: L. longbeachae NSW150, 3&7: L. longbeachae D4968, 4: empty lane and M: Marker Fermentas PageRuler Plus Prestained protein ladder

The comparative Western blot analysis demonstrated that L. pneumophila was not immunoreactive to antibodies in Sample B1G, whereas Sample C5D most likely contained antibodies which could be showing immunogenic reaction with homologous proteins across the Legionella species. Also, it can be noticed that Sample B1G had antibodies that recognised more proteins from L. longbeachae as compared to Sample C5D.

Based on the previous observations and due to the similarity in the patterns of immunoreaction with both Legionella species with Sample C5D, it can be suggested that Sample C5D originated from a patient infected either with a Legionella species other than L. pneumophila and L. longbeachae or with an another bacterium which is cross-reactive with Legionella species. Two-dimensional gel analyses were also carried out to identify the immunogenic proteins. The results are shown later in this section.

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5.3.4.3 Secretome of Legionella species against serum sample B1G

Figure 5.6 indicates that the secretome also showed similar results to the total soluble proteins with Sample B1G.

Figure 5.6: Western blot of secretomes of L. pneumophila and L. longbeachae with Sample B1G 1 3 represent the secretomes from Legionella species. 1: L. pneumophila. 2: L. longbeachae NSW150, 3 L. longbeachae D4968 and M: Marker Fermentas PageRuler Plus Prestained protein ladder

The secretomes of both L. longbeachae isolates showed immunoreaction with Sample B1G, whereas no reaction was observed with L. pneumophila. The reactive portion from the Western blot was matched on a preparative SDS-PAGE gel for L. longbeachae and identified by peptide mass fingerprinting. The region of interest was excised from the gel and the resulting gel portions were subjected to mass spectrometry for protein identification.

Table 5.1 lists the proteins identified from the secretome but not identified from the total soluble protein and outer-membrane sub-proteome. Two of the proteins were found to be potentially specific to L. longbeachae. The putative chitin-binding protein showed homology to proteins in other organisms but not with any other Legionella species. The zinc metalloproteinase was the most abundant protein found in the secretome fraction. Except for the zinc metalloproteinase proteins, the other three proteins are reported here for the first time as immunogenic and can be targeted as potential biomarkers.

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Table 5.1: Immunogenic proteins belonging to the secretome fraction only

S. Accession No. Protein's name Function MW PI Cellular Specificity No. localisation

1 D3HSB6_LEGLN Putative uncharacterized unknown 30981 4.63 Multiple specific to protein localisation L. longbeachae 2 D3HS87_LEGLN Endonuclease/Exonuclease/ nucleic acid 30563 7.04 unknown specific to phosphatase family protein hydrolysis L. longbeachae

3 D3HK14_LEGLN Putative chitin-binding adhesion/ viral27886 5.81 extracellular Not specific protein capsid

4 D3HL32_LEGLN Zinc metalloproteinase proteolysis 58960 5.71 extracellular Not specific

5.3.4.4 Total soluble proteins from L. longbeachae

Figure 5.7 shows the detection and identification of immunogenic proteins by 2D gel electrophoresis and Western blotting. The analysis revealed that numerous proteins from L. longbeachae reacted to the antibodies found in Sample B1G. These proteins were subsequently matched onto the preparative gels for identification.

Figure 5.7: Western blot of total soluble protein from L. longbeachae against serum sample B1G pI: Isoelectric point. kDa: kiloDalton. A and B are replicates of the Western blot. M: Marker - Precision Plus Protein Kaleidoscope protein ladder

In this study, non-linear 3 10 immobilised pH-gradient (IPG) strips were used. Western blotting demonstrated that most of the proteins detected to be immunogenic were within the isoelectric point range of approximately 3 7, i.e. acidic to neutral. Moreover, the immunogenic proteins were well distributed within a wide range of molecular weight

115 | Page Chapter 5 Identification of immunogenic proteins

(around 22 kDa to 90 kDa). The low molecular weight and basic pI proteins did not seem to immunoreact, possibly because they were transferred on the membrane.

As mentioned in the previous chapter, 2D gels of the same sample may not behave in an identical manner; the differences can be noticed from the Western blot replicates A and B. Of particular interest was the region of the gels at molecular weights above 60 kDa, as no proteins could be detected in blot A whereas unresolved proteins of >60 kDa (which appear as bands rather than discrete spots) showed immunogenic reactions in blot B.

5.3.4.5 Pooled standard protein samples (DIGE)

Figure 5.8 shows an annotated Western blot of pooled protein samples from L. pneumophila and L. longbeachae as described for the pooled standard used for DIGE in the previous chapter.

Figure 5.8: Western blot of pooled total soluble proteins from L. pneumophila and L. longbeachae against serum sample C5D pI: Isoelectric point. kDa: kiloDalton. M: Marker - Precision Plus Protein Kaleidoscope protein ladder

Sample C5D, which was found to cross-react with all the Legionella species, was used to identify the proteins that might be responsible for the immunoreaction. This analysis was carried out to determine whether the antibodies from the serum sample could recognise the same proteins in both species and if any of the proteins were specific to

116 | Page Chapter 5 Identification of immunogenic proteins the Legionella genus. This was done in order to detect any novel genus-wide antigenic proteins and also to test the assumption that the serum sample may be from a patient infected with an etiological agent other than Legionella.

The protein identification data showed that 12 proteins were found from the annotated spots specifically from L. pneumophila and 18 from L. longbeachae. Eight proteins were found from both the species. Out of the 38 proteins identified, four were found to be potentially specific to Legionella but none were specific to either of the species.

5.3.4.6 Differential detection of immunoreactive proteins of the outer-membrane sub-proteome of L. longbeachae with serum samples B1G and C5D

Figures 5.9 shows the immunogenic reaction observed using Sample C5D. Only a few proteins were found immunoreactive with Sample C5D.

Figure 5.9: Western blot of outer-membrane sub-proteome of L. longbeachae against serum sample C5D pI: Isoelectric point. kDa: kiloDalton. M: Marker Fermentas PageRuler Plus Prestained protein ladder

Only six protein spots were successfully matched from the Western blot onto the preparative gel (Appendix 4) for protein identification. The region encircled in red shows immunoreactive protein spots, but no protein spots were identified in that region on the preparative gel. This could possibly be because the proteins were very low in abundance and were not stained with Coomassie blue, whereas the more sensitive immunoassay was able to show their presence.

Figure 5.10 shows a very strong Western blot reaction against outer-membrane proteins. Using smaller volumes of protein and lowering the volume (diluting) of the serum did not show any difference in the patterns of reaction, except for the developed colour

117 | Page Chapter 5 Identification of immunogenic proteins being slightly lighter. As it appears that the whole outer-membrane fraction was strongly immunoreactive, this suggests that most of the proteins which are surface- exposed are most likely to be immunogenic and also demonstrates that the method used for the isolation of the outer membrane proteins was successful. However, many proteins appear to be unresolved as protein spots, due to the fact that outer-membrane proteins are poorly soluble (mostly hydrophobic) and therefore not properly focussed on the immobilised pH gradient (IPG) strips during isoelectric focussing.

Figure 5.10: A: 2D gel image of outer membrane subproteome of L. longbeachae. B: Western blot of outer-membrane sub-proteome of L. longbeachae against serum sample B1G pI: Isoelectric point. kDa: kiloDalton. M: Marker Fermentas PageRuler Plus Prestained protein ladder

A greater number of proteins were found to be immunoreactive in Sample B1G compared to Sample C5D.

There are a few interesting points to note from these Western blot experiment. Firstly, the heat shock protein (Hsp60) which has constantly been identified as dominant immunoreactive protein of Legionella was localised at different spots on the gel, most probably because of post-translation modifications, and was still found immunoreactive irrespective of modifications. A few other proteins were also found to be resolved at different spots and were still immunoreactive. Secondly, several proteins were detected to show immunogenic reactions which have not been reported before, especially for L. longbeachae. Thirdly, the outer-membrane sub-proteome fraction showed immunoreaction throughout the whole blot, that is, across broader isoelectric points (3 10) and molecular weights (~20 100 kDa) as compared to the total soluble proteome fraction. As such, some proteins that were not detected from the total soluble proteins fraction were identified from the outer-memebrane sub-proteome. This was mainly because a different protein isolation protocol was used for the outer-membrane and a 118 | Page Chapter 5 Identification of immunogenic proteins chaotropic reagent was added to facilitate the solubilisation of membrane proteins. The use of the chaotropic reagent amidosulfobetaine (ABS-14) was found to be possibly problematic in the larger format 2D gels presented in the previous chapter and was therefore excluded from the sample buffer for the total soluble protein isolation.

5.3.5 Western blot data mining

Figure 5.11 illustrates the number of proteins identified as being either unique or common to a particular set using the data from the total soluble proteins and outer- membrane proteome against serum samples B1G and C5D.

A total of 187 proteins were identified to be potentially immunoreactive from the Western blot data of the total soluble proteins and outer-membrane subproteome of L. longbeachae against samples B1G and C5D. As can be noted, some of proteins were uniquely identified to groups A, B or C. Only one protein was identified to be common to all the four groups. Unsurprisingly, it was the 60 kDa heat shock protein, recognised as a major immunogenic protein in Legionella species, that cross-reacted with both serum samples and was found both as a cytosolic and surface-exposed protein.

Figure 5.11: Venn diagram of differentially identified proteins per proteome sub-type against different serum samples The letters A-D denote the proteome type and serum samples used. A: total soluble protein against Sample B1G, B: outer-membrane sub-proteome against Sample B1G, C: total soluble protein against Sample C5D and D: outer-membrane sub-proteome against Sample C5D.

Immunogenic reactions were found to occur with proteins potentially specific to the Legionella genus from set A, B and C, with only two proteins specific to C. No proteins specific to Legionella were detected to react to antibodies in set D, probably because not

119 | Page Chapter 5 Identification of immunogenic proteins all the proteins that showed immunogenic reactions were identified, as mentioned earlier in Section 5.3.3.5.

An interesting point to note though is the difference in the number of proteins that showed immunogenic reactions with the two different serum samples. It can be clearly seen that a higher number of proteins were immunoreactive with Sample B1G. About 80% of the proteins were specifically found to be potentially immunogenic to sets A and B combined. These data suggest that Sample C5D might have been collected from a patient who was infected with a Legionella species other than L. pneumophila or L. longbeachae that has homologous proteins to the ones identified as specific to Legionella. However, this cannot be confirmed with the lack of genome data.

The Venn diagram also demonstrates that the majority of proteins having differential immunoreaction are from the outer-membrane sub-proteome fraction. However, there were several proteins which were predicted to be of cytosolic cellular localisation by PSORTb. Nevertheless, they were all found to be potentially immunogenic and this may suggest that the proteins might have been translocated to the outer-membrane after synthesis.

5.3.6 Immunogenic proteins of interest

Table 5.2 summarises the proteins which could be of potential interest for use as biomarkers. Eighty-three proteins are tabulated based on the number of times they were identified as immunogenic proteins using the two serum samples (highlighted rows). The proteins were also selected based on their cellular localisation, specificity and function.

Several proteins are reported here for the first time to be immunogenic. A few of them, for example, Uroporphyrinogen III methylase HemX, Ankyrin repeat protein and Recombination associated protein, were found to be possibly specific to the Legionella genus. Moreover, there were a few other immunogenic proteins, such as the BNR/Asp- box repeat containing protein and Putative coiled-coil protein, which were also determined to be potentially specific to L. longbeachae. The Putative capsular polysaccharide biosynthesis protein was another such protein that was identified but its homolog does not seem to be present in the L. longbeachae D4968 isolate.

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Chapter 5 Identification of immunogenic proteins

Table 5.2: Immunogenic proteins of interest from total soluble proteins and outer-membrane sub- proteome of L. longbeachae

S. Accession No. Protein name Function Cellular Specificity No. localisation

1 D3HJ49_LEGLN Isocitrate dehydrogenase [NADP carbohydrate CY NS metabolism TCA cycle

2 D3HKF8_LEGLN S-adenosylmethionine synthase DNA methylation/gene CY NS expression regulation 3 D3HKN1_LEGLN Putative uncharacterized protein unknown UN SLG

4 D3HL63_LEGLN Cell division protein ftsZ cell division UN NS 5 D3HM81_LEGLN Putative uncharacterized protein unknown UN SLG

6 D3HN33_LEGLN ATP synthase subunit beta ATP synthesis CM NS 7 D3HN35_LEGLN ATP synthase subunit alpha ATP synthesis CM NS

8 D3HNC4_LEGLN Alkyl hydroperoxide reductase antioxidant activity CY NS 9 D3HND8_LEGLN Cysteine synthase transferase amino acid CY NS biosynthesis

10 D3HP07_LEGLN Transcription elongation protein transcription regulation CY NS nusA

11 D3HPB3_LEGLN Succinate dehydrogenase, iron carbohydrate CY NS sulfur protein metabolism TCA cycle 12 D3HPB6_LEGLN Succinyl-CoA ligase [ADP- ligase TCA cycle CY NS forming] subunit beta

13 D3HPI9_LEGLN Elongation factor Tu transcription regulation CY NS 14 D3HPM8_LEGLN DNA-directed RNA polymerase transcription regulation CY NS subunit alpha

15 D3HPX4_LEGLN Putative leucine dehydrogenase oxidoreductase CY NS

16 D3HPY7_LEGLN 60 kDa chaperonin chaperone - stress CY NS response/protein folding

17 D3HQP4_LEGLN Alanine dehydrogenase amino acid metabolism CY NS 18 D3HR81_LEGLN Putative outer membrane protein oxidoreductase UN NS

19 D3HRD0_LEGLN Putative long-chain fatty acid porin protein OM SLG transporter

20 D3HRR4_LEGLN Putative PPi dependent carbohydrate CY NS phosphofructokinase metabolism TCA cycle 21 D3HRW2_LEGLN Trigger factor cell division chaperone CY NS 22 D3HS36_LEGLN Putative carbonic anhydrase lyase CY NS

23 D3HSK4_LEGLN Putative uncharacterized protein unknown UN SLG 24 D3HSL9_LEGLN Similar to chloroperoxidase oxidoreductase CY NS 25 D3HT54_LEGLN Elongation factor Ts protein biosynthesis CY NS

26 D3HJ21_LEGLN UDP-N-acetylglucosamine 1- cell division cellwall CY NS carboxyvinyltransferase biogenesis 27 D3HJY7_LEGLN Putative 3-ketoacyl-CoA thiolase transferase CY NS (Thiolase I, acetyl-CoA transferase 28 D3HK05_LEGLN Periplasmic serine protease Do protease PP NS 29 D3HL55_LEGLN Putative adenosine deaminase hydrolase CY NS protein 30 D3HLF9_LEGLN Putative signal recognition particle GTPase activity CY NS protein Ffh 31 D3HMW3_LEGLN Putative aspartate aminotransferase transferase CY NS 32 D3HN80_LEGLN Putative uncharacterized protein unknown CY SLG 33 D3HPB7_LEGLN Succinyl-CoA ligase [ADP- ligase ATP binding CY NS forming] subunit alpha cofactor 34 D3HQG3_LEGLN Quinone oxidoreductase oxidoreductase CY NS (NADPH:quinone reductase 35 D3HQI2_LEGLN 2-amino-3-ketobutyrate coenzyme transferase CY NS A ligase 36 D3HR82_LEGLN Glycine--tRNA ligase alpha protein biosynthesis CY NS subunit 37 D3HRA3_LEGLN Acetyl-CoA acetyltransferase transferase CY NS 121 | Page

Chapter 5 Identification of immunogenic proteins

Table 5.2: Immunogenic proteins of interest from total soluble proteins and outer-membrane sub- proteome of L. longbeachae (continued)

S. Accession No. Protein name Function Cellular Specificity No. localisation 38 D3HSJ8_LEGLN Pyruvate dehydrogenase E1 alpha glycolysis CY NS subunit 39 D3HJR2_LEGLN Septum site-determining protein barrier septum selection ML NS (Cell division inhibitor) site 40 D3HMM6_LEGLN Glyceraldehyde 3-phosphate glucose metabolic CY NS dehydrogenase process 41 D3HPU5_LEGLN Protein-export protein SecB transport CY NS 42 D3HPZ0_LEGLN Putative 3-hydroxybutyrate hydroxybutyrate CY NS dehydrogenase dehydrogenase activity 43 D3HQE2_LEGLN Putative 4-methyl-5(B- vitamin biosynthesis UN NS hydroxyethyl)-thiazole monophosphate biosynthesis enzyme 44 D3HQL5_LEGLN 2,3,4,5-tetrahydropyridine-2,6- amino acid biosynthesis CY NS dicarboxylate N- succinyltransferase 45 D3HQM4_LEGLN Putative 3-hydroxyacyl-CoA oxidoreductase CY NS dehydrogenase type II 46 D3HT84_LEGLN Putative acetoacetate acetoacetate CY NS decarboxylase decarboxylase activity 47 D3HKX8_LEGLN Putative octaprenyl-diphosphate isoprene biosynthesis in CY NS synthase response to heat stress 48 D3HSJ6_LEGLN Branched-chain alpha-keto acid TCA cycle-transferase CY NS dehydrogenase subunit E2 49 D3HIZ7_LEGLN Putative aminotransferase class-V transferase CY NS 50 D3HP38_LEGLN Uroporphyrinogen III methylase transferase CM SLG HemX 51 D3HPZ7_LEGLN Putative uncharacterized protein phosphorylation CY SLB 52 D3HRJ7_LEGLN Ankyrin repeat protein unknown CY SLG 53 D3HTA1_LEGLN D-alanyl-D-alanine hydrolase CM NS carboxypeptidase (Penicillin- binding protein 5 54 D3HQI7_LEGLN IcmL-like protein potentially macrophage EX SLG killing

55 D3HRX4_LEGLN Recombination associated protein DNA recombination CY SLG

56 D3HJ70_LEGLN Peptidyl-prolyl cis-trans isomerase isomerase/rotamase OM NS 57 D3HKZ9_LEGLN Outer membrane protein assembly integral to outer OM NS factor membrane

58 D3HL98_LEGLN Component of the Dot/Icm Dot/Icm sceretion UN NS secretion system system

59 D3HNC7_LEGLN Major outer membrane protein integral to outer OM SLG homolog membrane

60 D3HNC8_LEGLN Major outer membrane protein integral to outer OM SLG membrane 61 D3HQ84_LEGLN Protein with a bacterial probable UN NS immunoglobulin-like domain intimin/invasin-like function

62 D3HQZ3_LEGLN BNR/Asp-box repeat containing bacterial neuraminidase EX SLB protein

63 D3HJ12_LEGLN Putative uncharacterized protein unknown UN SLB

64 D3HJ93_LEGLN Peptidoglycan-associated integral to membrane OM NS lipoprotein (19 kDa surface antigen) (PPL 65 D3HJV9_LEGLN Putative patatin-like phospholipase lipid metabolic process UN NS 66 D3HKW1_LEGLN Enhanced entry protein EnhC beta lactamase hydrolase EX SLG 67 D3HL27_LEGLN Putative ferredoxin--NADP oxidoreductase CY NS reductase

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Table 5.2: Immunogenic proteins of interest from total soluble proteins and outer-membrane sub- proteome of L. longbeachae (continued) S. Accession No. Protein name Function Cellular Specificity No. localisation 68 D3HLA3_LEGLN Putative uncharacterized protein unknown UN SLG lphA / IcmN (DotK 69 D3HLP1_LEGLN Putative oxidoreductase oxidoreductase activity CY NS FAD/NAD(P)-binding 70 D3HM82_LEGLN Putative coiled-coil protein unknown CY SLB 71 D3HMD2_LEGLN Putative capsular polysaccharide capsule biosynthesis CY SLB biosynthesis protein 72 D3HMI8_LEGLN Putative uncharacterized protein stress response UN SLG chaperone 73 D3HNA5_LEGLN Thaumatin domain-containing stress response mainly ML SLB protein due to infection in plants 74 D3HPC2_LEGLN Putative uncharacterized protein unknown UN SLG 75 D3HPC5_LEGLN Outer membrane lipoprotein LolB outer membrane protein OM SLG 76 D3HPX5_LEGLN Putative uncharacterized protein unknown UN NS 77 D3HQ11_LEGLN Putative uncharacterized protein / outer membrane OM NS Major outer membrane protein Momp 78 D3HQI0_LEGLN Putative outer membrane protein transporter activity OM NS TolC 79 D3HQK5_LEGLN Putative outer membrane protein outer membrane protein OM SLG 80 D3HQZ4_LEGLN Putative uncharacterized protein unknown UN SLG 81 D3HS13_LEGLN Putative uncharacterized protein unknown UN SLB 82 D3HSQ0_LEGLN Putative heat shock protein response to stress CY NS 83 D3HSX3_LEGLN Putative uncharacterized protein metallopeptidase activity UN SLB Specificity was determined by Basic Local Alignment Sequence Tool (NCBI-nr): < 35% sequence homology was considered as potentially specific. S. No.: Serial number. SLG: specific to Legionella genus, SLB: specific to L. longbeachae, NS: not specific. Cellular localisation was predicted by PSORTb. CY: Cytoplasmic, CM: Cytoplasmic membrane, EX: Extracellular, ML: Multiple localisation, PP: Periplasmic, OM: Outer membrane, UN: Unknown

5.4 DISCUSSION

5.4.1 Optimisation of isolation of outer-membrane sub-proteome

The isolation of the outer-membrane proteome was quite challenging. As outer membrane proteins are known to be hydrophobic and difficult to resolve with 2 D gel electrophoresis (Paul et al., 2013), a suitable protocol had to be devised. The protocol was improved by combining the incubation of the cell lysates in the presence of CHAPS and sodium carbonate to enrich the outer membrane proteins and ultracentrifugation for differential sedimentation of proteins. The addition of multiple chaotropic reagents to the sample solubilisation buffer was found to further improve the solubilisation of the outer membrane proteins. This resulted in improved resolution of the proteins during 2D gel electrophoresis.

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5.4.2 Differential immunoreaction of serum samples

This study was performed using blinded samples; one advantage of such an approach is that biases are avoided while performing the tests. As a result, any apparent limitations in the diagnostic tests currently in use could be identified. In this study, as serum samples were obtained from two different sources, the biases that may be introduced due to differences in sample collection, handling, processing and storage were overcome. A recent study carried out by Coura-Vital et al. (2014) identified limitations in the serology testing protocols used for the diagnosis of canine visceral leishmaniasis. By using their improved test, they concluded that the estimated incidence of the disease was far greater than what is reported by carrying out a study to eliminate any biases using a blinded approach. The authors also mentioned that using IFAT, which is a rather subjective method, can lead to erroneous interpretations and result in either false negative or positive test being reported.

The proper identification of an etiological agent is particularly important in surveillance programs involving notifiable diseases such as Legionellosis (which is notifiable in many countries). Furthermore, it is believed that cases of Legionnaires disease are under-reported due to a lack of proper diagnostic tests. Therefore, the inclusion of blinded samples in this study was found to show some rather different results than would be normally anticipated, as discussed below.

Serum sample C5D was found to cross-react with all the Legionella species in this study whereas Sample B1G was found to cross-react with eight species only. Very strong immunogenic reactions were observed from Sample B1G against proteins from L. longbeachae. Moreover, several potentially specific proteins immunogenic to L. longbeachae were identified with Sample B1G, suggesting that this sample had been collected from a patient who was probably infected with L. longbeachae. The fact that no or little immunoreaction was observed with 11 Legionella species indicates that immune responses may differ from species to species of Legionella.

Previous studies comparing two Burkholderia species (Shinoy et al., 2013) and different Legionella species (Sampson et al., 1986, Weber et al., 2012) have shown that there may be differences in immunogenic reactions across species of the same genus. A study by Casson et al. (2007) to determine immunoreaction with distantly related chlamydia- like organisms found that most of them were either non-cross-reactive or showed little 124 | Page

Chapter 5 Identification of immunogenic proteins cross-reaction. Therefore, it may be that different Legionella species decorate their cell surface with different antigens and induce dissimilar antibody production, which may not cross-react with other species. However, the little cross-reactivity observed by dot blot and ELISA could be due to the presence of polysaccharide from cell lysates which are immunoreactive. Polysaccharides have been described to be immunogenic in Legionella and they are used for the identification of different serogroups. These polysaccharides were not resolved on the SDS-PAGE gels and this could be the reason for no immunoreaction detected during Western blot.

Quite a few Legionella species have been classified as having several serogroups, which means that there are differing antigens in the serogroups of the same species. Therefore, dissimilar antibodies are produced for the different serogroups during infection. This suggests that con-generic species diversity is likely to be higher and thus different antigens may be required to be pooled together for genus-specific diagnosis.

However, other species such as L. parisiensis, L. tucsonensis and L. sainthelensii showed even stronger immunoreaction against Sample B1G, as compared to L. longbeachae. Although, several immunogenic proteins were identified to be potentially specific to L. longbeachae, the other three species do not have any genome sequence data available to determine whether homologs of those proteins are present. Therefore, it cannot be ascertained that the serum sample was from a patient infected with L. longbeachae. The significant cross-reactivity between the eight Legionella species with Sample B1G suggests that they may be closely related. However, as could be inferred from the phylogenetic analysis carried out by Rubin et al. (2005) L. longbeachae and L. sainthelensii (same sister clade) appeared to be closely related. L. parisiensis and L. tucsonensis were found to be distantly related but the latter two were from the same sister clade.

The 60 kDa heat shock protein (Hsp60) has been reported to be an immunodominant antigen of Legionella and cross-reactivity of this protein to Legionella species involved in this study has been reported in literature. Quite intriguingly, the antibody from Sample B1G that recognised the Hsp60 in L. longbeachae did not to react with heat shock protein from L. pneumophila and most probably to 10 other Legionella species, as evidenced from the Western blot analyses. One possible explanation is that the protein was not expressed in the other species. However, the data from Figure 5.8 showed that

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Chapter 5 Identification of immunogenic proteins

Hsp60 was expressed in L. pneumophila but did react with antibodies found in Sample C5D. Therefore, another possible explanation is that the epitope recognised by the antibody may not be present in the other species.

Referring to the previous chapter on the DIGE experiments and the identification of 135 proteins that were commonly expressed in both L .pneumophila and L. longbeachae, 50 of them were detected to be potentially immunogenic. It is interesting to note that none of the proteins from L. pneumophila were found to have immunogenic reactions against Sample B1G. One plausible explanation for such an observation could be that no cross- reactive epitopes were found in the non-reacting species, probably due to acquired mutations through evolution or host-factors which may influence the type of immune response.

5.4.3 Potential limitations of current diagnostic tests

Serum sample C5D was obtained from Source B which uses a commercial kit for identification of Legionella infection. This kit comprises heat-inactivated bacterial cells coated in the wells of a slide. The slide contains sets of wells with pooled L. pneumophila serogroups 1 6, L. pneumophila serogroup 7 14 and 6 pooled species (L. bozemanii serogroups 1 and 2, L. longbeachae serogroups 1 and 2, L. dumoffi, L. gormanii, L. micdadei and L. jordanis). Serum sample B1G did not show any immunogenic reaction with some of the Legionella species. This serum sample was obtained from Source A which uses an in-house developed IFA test (IFAT) based on the protocol described by Wilkinson et al. (1979). Serum samples are tested against only pooled L. pneumophila serogroups 1 6 and L. longbeachae by Source A (personal communication). As these diagnostic tests do not include all the Legionella species in the testing panel and the fact that cross-reactivity from other genera may occur, these tests appear to have some limitations.

As can be inferred from the results obtained in this study, it is possible that infection with other Legionella species may result in a different type of immune response. This in turn may not cross-react with the Legionella species in the list from the diagnostic kit as was observed with Sample B1G which did not cross-react with 11 Legionella species. If L. longbeachae was not included by Source A in their diagnostic assay, this serum sample might not have shown significant immunogenic reaction and a false negative test would have been reported. However, this may depend on the acceptable titre by the 126 | Page

Chapter 5 Identification of immunogenic proteins source as a very weak reaction was observed with L. pneumophila. This may be the reason why several Legionella species are under-reported.

It was also observed during the study that a higher number (over 18-fold) of specific immunoreactive proteins were detected with Sample B1G compared to Sample C5D. This suggests that Sample C5D might have been taken from a patient infected with a Legionella species other than L. pneumophila or L. longbeachae. Although a few proteins which are potentially specific to L. longbeachae were identified to cross-react during the immunoassays with Sample C5D, it is difficult to say that homologs of those proteins are not present in other Legionella species whose genome sequence is not available. It may be possible that homologs are present as was observed with the immunoreaction with Sample B1G, whereby seven other species showed similar patterns of reaction. Therefore, the tests being used by sources A and B may be appropriate for limited genus-level diagnosis but not species-specific detection.

5.4.4 Surface-exposed proteins as potential biomarker

Surface-exposed proteins are known to be mostly immunogenic and are the first point of contact with the host immune system. Also, most of the outer membrane proteins are known to be constitutive as they form part of the structural component of the cell. Therefore, identification of such antigenic proteins may serve as better targets for further analysis in terms of biomarker research.

In this study, it has been shown that the majority of the immunoreactive proteins were from the outer-membrane sub-proteome, although, they were predicted to be from different cellular locations. Furthermore, a very strong reaction was seen for the outer- membrane fraction by Western blot. Many cytoplasmic proteins might be exported to the surface of the cell and thus may have different functions. One such example is the protein Elongation factor Tu which is a cytosolic protein but has been demonstrated to be surface exposed and immunoreactive in many different bacterial species (Nieves et al., 2010, Lee et al., 2011, Granato et al., 2004). This protein has been demonstrated to be involved in pathogenesis (attachment to host cell) and is also a potential vaccine candidate.

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5.4.5 Immunogenic proteins of L. longbeachae

A few proteins which were determined to be potentially immunogenic for L. longbeachae in the previous chapter were found to immunoreact in this study suggesting that they are indeed immunogenic. The coiled-coil protein and the BNR/Asp-repeat box repeat-containing proteins are such examples and have not been reported before. They seem to be potentially specific to L. longbeachae. These proteins may possibly be involved in pathogenesis and can also be targeted as potential biomarkers.

The bacterial immunoglobulin domain-containing protein, which was reported in the previous chapter and identified to have homology to proteins of type V secretion system in Escherichia coli and Yersinia, was found to be potentially immunogenic. Although the reaction was not confirmed from the 2D gel electrophoresis of the total soluble proteins, one of the blots (Figure 5.10 B) showed an immunogenic reaction within the expected molecular weight range of the protein (~87 kDa). One reason for it not being identified could be because of its low abundance. However, this protein was identified from the outer-membrane sub-proteome Western blot to be immunoreactive but with a lower molecular weight (~60 kDa), most probably due to post-translational modification. This result suggests that the protein may indeed be involved in pathogenesis.

Another protein of interest is the Putative capsular polysaccharide biosynthesis protein which does not appear to be present in the L. longbeachae D4968 isolate, as inferred from the peptide mass fingerprinting and BLAST data. This may be because the gene has not been annotated or may be absent from the genome and therefore warrants further investigation. As the name suggests, this protein may be involved in the synthesis of the capsule and L. longbeachae NSW150 has been reported to contain a capsule (Cazalet et al., 2010). A search revealed that L. longbeachae D4968 appears to possess a gene that encodes the protein Putative capsule biosynthesis protein CapA which is potentially specific to L. longbeachae. However, an identical homolog is found in L. longbeachae NSW150. From the findings of this study that only the capsule biosynthesis protein belonging to L. longbeachae NSW150 was identified, it is speculated that only L. longbeachae NSW150, which is an Australian isolate, forms a capsular outer surrounding. Bacterial capsules are known to confer protection to the

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Chapter 5 Identification of immunogenic proteins organism and be involved in pathogenicity (Wilson et al., 2002). In case only L. longbeachae NSW150 produces a capsule, then this may be a possible explanation why the Australian isolates of L. longbeachae were found to be more virulent by Doyle et al. (2001).

5.5 CONCLUSION

In conclusion, this study demonstrated that immune response following Legionella infection may possibly be host and/or species dependent, and the current serological (immunoassays) diagnostic tests may have some shortcomings. It was also found that most of the immunogenic proteins were most likely surface exposed. As this study was more focussed on L. longbeachae, several immunogenic and species-specific proteins were identified which were most likely involved in virulence and pathogenesis. Several other immunogenic proteins which are potentially genus-specific were also identified. These data will help in selection of candidate proteins to be targeted as potential biomarkers and carefully design an improved diagnostic test.

The next chapter will describe the identification of surface exposed proteins, an attempt to affinity purify antigenic proteins and determination of antigenic epitopes.

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IDENTIFICATION OF ANTIGENIC EPITOPES

6.1 INTRODUCTION

Immunogenic molecules, such as proteins, trigger the bodys immune response and contain antigenic determinants known as epitopes. These epitopes are either continuous or discontinuous. Continuous epitopes, also called linear epitopes, are varying lengths of an uninterrupted stretch of amino acids. Conversely, discontinuous epitopes, also known as conformational epitopes, are distantly spread amino acid residues that are brought in close proximity to spatially organise and form an antigenic determinant due to the three-dimensional structure of the protein (Dhungana et al., 2009). With respect to bacterial antigens, these epitopes may be species-specific, genus-specific or even cross-react with other genera.

While congeneric cross-reactivity may be desirable, antibodies produced during infection from organisms of a particular genus can possibly show unwanted cross- reactivity with other genera due to the occurrence of homologous proteins. Several studies have demonstrated the ability of serum samples from patients with confirmed Legionella infection to cross-react with organisms of other genera. Similarly, serum samples from patients with non-Legionella infections have also been shown to cross- react with Legionella antigens (Gray et al., 1991, Boswell, 1996, Musso and Raoult, 1997). Therefore, the identification of Legionella genus and species-specific epitopes may help in the selection of antigenic determinants of interest for the development of specific diagnostic tests. In some cases, the epitopes can also be used as vaccine candidates.

Epitope mapping can be carried out either by in silico predictions or experimentally determined. Several software applications are available for the prediction of epitopes

130 | Page Chapter 6 Identification of antigenic epitopes from protein sequences, and one such example is the IMMUNE EPITOPE DATABASE (IEDB) AND ANALYSIS RESOURCE. Otherwise, epitope extraction can be performed in the laboratory using techniques such as the limited proteolysis method described by Dhungana et al. (2009). Immobilised antibodies can be used for affinity- binding of the antigenic proteins. As antibodies are highly resistant to proteolysis, the antigen-antibody complex can then be subjected to a proteolytic digestion to cleave regions of the antigen that are not bound to the antibody and then washed off. The bound peptides can then be eluted and subjected to further analysis, such as mass spectrometry, to determine the amino acid sequence.

6.2 EXPERIMENTAL DESIGN

6.2.1 Aims and objectives

In the previous chapter, it was pointed out that there might be differences in immune responses due to different Legionella species. As some of the species did not show cross-reaction in Western blot experiments, the objective was to select a panel of proteins for screening in order to determine potential genus-specific as well as species- specific antigenic epitopes. Such epitopes can be pooled or used individually and applied in the development of an improved diagnostic test for genus and species identification.

6.2.2 Experimental procedures

Based on earlier results, a set of immunogenic proteins was identified as containing potential genus-specific and species-specific (L. longbeachae) antigenic determinants. A trypsin shaving of L. longbeachae was also carried out to determine which proteins were surface exposed. Affinity purification of antigenic proteins was also attempted in order to identify epitopes experimentally.

It was hypothesised that during the tryptic digestion of proteins picked from gels, which was intended to liberate the peptides and allow their identification using mass spectrometry, the trypsin activity sites on the outer-membrane proteins would have been more accessible. Those peptides were also likely to be more hydrophilic as determined from the amino acid sequence, and also due to the fact that they were soluble in the aqueous extraction solution and were eluted from the hydrophobic binding of the C18

131 | Page Chapter 6 Identification of antigenic epitopes column during mass spectrometric analysis. It was concluded that these peptides were most likely surface exposed and could be targeted as prospective antigenic sites. With this as a starting point, proteins found in all Legionella species or L. longbeachae- specific by bioinformatics analysis were subjected to further investigations. A linear B- cell epitopes prediction was then carried out using the IEDB software. Selected peptides were then synthesised based on their specificity and hydrophilicity, and then subjected to immunoassays. Previously identified immunogenic proteins were also screened as potential candidates for use as biomarkers. As mentioned in the earlier chapters, L. pneumophila has been well studied so this chapter was focussed on obtaining data from L. longbeachae.

6.3 RESULTS

6.3.1 Trypsin shaving

Table 6.1 provides a list of proteins identified from the trypsin shaving experiment that are most likely surface exposed. As described in the experimental procedure, the peptides that were identified by mass spectrometry as a result of trypsin shaving would have been surface exposed and easily accessible.

As surface exposed proteins are the first point of protein-protein interaction between the pathogen and the host, and, as discussed earlier, these proteins being more likely constitutive, they can be targeted as potential biomarkers. These data also indicate that there was no contamination from cytosolic proteins, as most of the proteins have been identified to originate from the outer membrane and secretome fractions (Chapters 4 and 5). Moreover, whilst several of the identified proteins have homologs in other organisms, some are potentially specific to L. longbeachae and a few to the Legionella genus as determined by BLAST analysis.

Proteins which normally belong to the cytoplasmic fraction, such as ribosomal proteins have been reported to be found on cell surface and may therefore have multiple localisations. Similar proteins were identified during the trypsin shaving experiment. However, little is known regarding their involvement in pathogenesis and triggering immune response. The peptidyl-prolyl cis-trans isomerase (MIP) and the enhanced entry (EnhC) proteins were of particular interest as they have been reported to be pathogenesis-related and were found to be surface-exposed. 132 | Page Chapter 6 Identification of antigenic epitopes

Table 6.1: Proteins identified from trypsin shaving

S. UniProt Accession Protein pI MW No. No. 1 D3HR81_LEGLN Putative outer membrane protein 8.37 28558 2 D3HL98_LEGLN Component of the Dot/Icm secretion system 5.45 30495 3 D3HKG1_LEGLN Chaperone protein DnaK 4.91 70275 4 D3HPK9_LEGLN 30S ribosomal protein S3 10.08 24047 5 D3HP07_LEGLN Transcription elongation protein nusA 4.55 54931 6 D3HJ85_LEGLN Putative phospholipid binding protein 9.09 11988 7 D3HPJ3_LEGLN 50S ribosomal protein L1 9.59 24488 8 D3HMQ0_LEGLN Thiol:disulfide interchange protein DsbA 8.7 23274 9 D3HSL3_LEGLN PQQ (Pyrrolo-quinoline quinone) enzyme 9.14 41769 10 D3HPB6_LEGLN Succinyl-CoA ligase [ADP-forming] subunit 5.16 41777 11 D3HL13_LEGLN Protease subunit HflK 5.74 42043 12 D3HJ70_LEGLN Peptidyl-prolyl cis-trans isomerase 8.57 24647 13 D3HT32_LEGLN Aconitate hydratase 6.16 98803 14 D3HN80_LEGLN Putative uncharacterized protein 5.98 35268 15 D3HPK0_LEGLN Translation elongation factor G 5.09 77131 16 D3HTD3_LEGLN 30S ribosomal protein S1 4.95 61632 17 D3HP47_LEGLN Peptidyl-prolyl cis-trans isomerase 5.75 18008 18 D3HQJ5_LEGLN RND efflux membrane fusion protein, acriflavin 9.28 46292 19 D3HPY7_LEGLN 60 kDa chaperonin 5.16 58386 20 D3HKZ8_LEGLN Putative outer membrane proteins 9.28 18745 21 D3HPJ5_LEGLN 50S ribosomal protein L7/L12 4.58 12784 22 D3HPM2_LEGLN 50S ribosomal protein L15 10.44 15421 23 D3HPY6_LEGLN 10 kDa chaperonin 5.84 10483 24 D3HJ49_LEGLN Isocitrate dehydrogenase [NADP 5.91 45960 25 D3HRH0_LEGLN Putative Acid sphingomyelinase-like 8.42 43992 26 D3HNE4_LEGLN Thioredoxin 4.85 12018 27 D3HKW1_LEGLN Enhanced entry protein EnhC 5.28 134011 28 D3HRR4_LEGLN Putative PPi dependent phosphofructokinase 5.95 44976 29 D3HM66_LEGLN Putative uncharacterized protein 8.96 40615 pI: Isoelectric point, MW: Molecular weight (kDa). S. No.: serial number

6.3.2 Affinity purification of antibodies and antigens

6.3.2.1 Affinity purification of IgG from serum sample

From the data presented in Chapter 5, it was evident that serum samples C5D and U1S showed immunoreactivity to L. pneumophila and L. longbeachae proteins. Thus, these samples were selected for affinity purification of immunoglobulin G (IgG).

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Figure 6.1 shows SDS-PAGE gels samples C5D and U1S for the different fractions of eluted IgG after affinity purification with anti-human IgG.

Figure 6.1: Affinity purification of IgG from serum sample C5D and U1S A: Sample C5D, B: Sample U1S. Lanes 1-10 represent: 1: crude serum sample (before binding), 2: unbound proteins after incubation with immobilised anti-human IgG, 3: First fraction of elution after washing, 4 8: fractions 2 6 of elution respectively, 9: pooled eluted fractions 5-8, M: Marker - Precision Plus Protein Kaleidoscope protein ladder. kDa: Kilodalton. B1-B4: bands 1-4.

N-hydroxysuccinimide (NHS) activated agarose columns were used to first immobilise anti-human IgG. After blocking the unoccupied ligands, the serum samples were added to the columns and incubated for affinity purification of the IgG. After the washing steps to remove any unbound materials, the IgG was eluted. The purified IgG was then immobilised on a fresh column to capture the antigens from cell lysates. The antibodies were affinity purified from the serum samples before affinity purification of the antigens as human serum contains many protein species of which albumin is one of the major constituents. If serum samples were to be immobilised using NHS activated agarose, other proteins would have competed with the antibodies, including those from different classes, IgA and IgM, and occupied most of the binding spaces. Therefore, pure anti- human IgG was immobilised on NHS activated agarose and the unoccupied space was then blocked with Tris buffer. This was then used to selectively purify IgG from the human serum samples. The labelled bands were excised and subjected to tryptic digestion. Peptide mass fingerprinting was then carried out to determine the identity of the proteins.

Table 6.2 shows the proteins identified from the peptide mass fingerprinting of the four protein bands from Figure 6.1A. The heavy and light chains of the IgG are known to have a molecular weight of approximately 50 and 25 kilodaltons, respectively, under

134 | Page Chapter 6 Identification of antigenic epitopes denaturing conditions. The bands B3 and B4 were identified to be of similar molecular weights; peptide mass fingerprinting also confirmed the identity of the proteins. These results suggest that the affinity purification of IgG was successful. However, the eluted fractions were contaminated with serum proteins, such as albumin and serum transferrin, that form the major proteinaceous components of the serum.

Table 6.2: Protein identification by peptide mass fingerprinting

Protein NCBInr Molecular Protein band Accession weight No. (kDa) B1 gi|110590597 76810 Chain A, Apo-Human Serum Transferrin (Non-Glycosylated) B1 gi|51476390 71353 hypothetical protein [Homo sapiens] B1 gi|41388180 65126 monoclonal IgM antibody heavy chain [Homo sapiens] B1 gi|222978 30842 Ig M Fc B1 gi|229601 49801 Ig G1 H Nie B1 gi|519674478 13474 immunoglobulin A heavy chain variable region, partial [Homo sapiens] B2 gi|582045552 68239 Chain D, Ternary Complex Between Neonatal Fc Receptor, Serum Albumin And Fc B2 gi|177933 45567 alpha-1-antichymotrypsin precursor, partial [Homo sapiens] B3 gi|28590 71246 unnamed protein product [Homo sapiens] B3 gi|177827 46787 alpha-1-antitrypsin [Homo sapiens] B3 gi|304563032 14630 immunoglobulin gamma 1 heavy chain variable region [Homo sapiens] B3 gi|229601 49801 Ig G1 H Nie B3 gi|323432985 16574 immunoglobulin variable region [Homo sapiens] B3 gi|119601993 23448 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3, isoform CRA_a [Homo sapiens] B3 gi|10334541 42301 immunoglobulin heavy chain [Homo sapiens] B3 gi|41388180 65126 monoclonal IgM antibody heavy chain [Homo sapiens] B4 gi|21669315 28556 immunoglobulin kappa light chain VLJ region [Homo sapiens] B4 gi|4176418 23690 IgG kappa chain [Homo sapiens] B4 gi|11275306 23651 anti TNF-alpha antibody light-chain Fab fragment [Homo sapiens] B4 gi|229526 23779 protein Rei,Bence-Jones B4 gi|76252621 10791 immunoglobulin kappa light chain variable region [Homo sapiens] B4 gi|507342 12914 immunoglobulin kappa light chain V-Jk4 [Homo sapiens] B4 gi|337758 25495 pre-serum amyloid P component [Homo sapiens] B4 gi|229601 49801 Ig G1 H Nie B4 gi|125795 11742 RecName: Full=Ig kappa chain V-III region B6 B4 gi|170684534 23371 immunoglobulin lambda 2 light chain [Homo sapiens]

Figure 6.2 shows the results of ELISA performed against the affinity purified IgG to verify its activity. The purified antibodies were found to immunoreact with the total soluble proteins of L. pneumophila and L. longbeachae. In this experiment, Sample

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D3W was used a negative control to calculate the cut-off filter. It can be seen that the OD value for the affinity purified IgG was significantly lower than that of Sample B1G, which was used for the affinity purification. Nevertheless, the purified IgG showed immunoreaction, which suggests that the antibodies retained their functional activity even after the immunoaffinity purification process.

Indirect ELISA for verifying activity of immunopurified IgG 3

2.5 2 1.5 1 OD at 450nm 0.5 0 A2X B1G R2B H5J U1S C5D AP Antibody source

Figure 6.2: Activity of affinity purified IgG AP: Affinity purified IgG from serum sample C5D. OD: Optical density.

6.3.2.2 Affinity purification of antigens

The purified IgG was immobilised to affinity purify the antigens but no proteins were visible on SDS-PAGE in the eluted fractions. It was hypothesised that the proteins were low in abundance and, therefore, the eluted fractions were pooled and then concentrated using Amicon centrifugal filter units. The low pH (2) buffer used for elution was also exchanged immediately to retain protein integrity, but there were no significant improvements. As a result, the in silico prediction of epitopes method was preferred and adopted.

6.3.3 In silico prediction of antigenic epitopes

Table 6.3 provides a list of proteins that were selected as potential biomarkers for in silico prediction of epitopes. These proteins were chosen based on the immunoreaction results shown in Chapter 5. The proteins were also selected based on their genus- specificity, species-specificity, function and cellular localisation. All the proteins tabulated were found to be most likely surface-exposed, as they were identified from the outer-membrane fraction or by trypsin shaving of polypeptides found on the surface of the bacteria. 136 | Page

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Table 6.3: Proteins selected for further analysis as potential biomarker

S. UniProt Accession Protein Specificity No. No. 1 D3HQ84_LEGLN Protein with a bacterial immunoglobulin-like domain SLB* 2 D3HJ70_LEGLN/ Peptidyl-prolyl cis-trans isomerase NS 3 D3HJ93_LEGLN Peptidoglycan associated lipoprotein (19kDa) NS 4 D3HRD0_LEGLN Putative long-chain fatty acid chain transporter SLG 5 D3HP38_LEGLN Uroporphyrinogen III methylase HemX SLG 6 D3HRJ7_LEGLN Ankyrin repeat protein SLG 7 D3HQ17_LEGLN IcmL-like protein SLG 8 D3HRX4_LEGLN Recombination associated protein SLG 9 D3HNC7_LEGLN/ Major outer membrane protein homolog SLG 10 D3HKW1_LEGLN Enhanced entry protein SLG 11 D3HLA3_LEGLN Putative uncharacterised protein lphA/IcmN (Dot K) SLG 12 D3HPC5_LEGLN Outer-membrane lipoprotein Lolb SLG 13 D3HQK5_LEGLN Putative outer membrane protein SLG 14 D3HQZ3_LEGLN BNR/Asp-box repeat containing protein SLB 15 D3HM82_LEGLN Putative coil-coil protein SLB 16 D3HMD2_LEGLN Putative capsular polysaccharide biosynthesis protein SLB 17 D3HNA5_LEGLN Thaumatin-domain containing protein SLB

Specificity: >35% sequence homology was determined to be non-specific. SLB*: found only in L. longbeachae for Legionella genus but homolog present in other genera, NS: non-specific, SLG: specific to Legionella genus, SLB: specific to L. longbeachae.

Firstly, the proteins of interest found to be Legionella genus-specific were investigated for the presence of their homologs in as many Legionella congeneric species as possible using the UniProt database. The proteins identified as having sequence data available for homologs in several congeneric species were aligned for the genus-specific proteins to determine conserved regions and their specificity concurrently with epitope prediction.

All regions that were found conserved in Legionella were then assessed for their hydrophilicity using bioinformatics tools. The peptides identified by mass spectrometry of the proteins of interest were further analysed to verify whether they matched the regions of interest; conserved, specific and hydrophilic linear amino acid sequences. Lists of peptides that may be antigenic (epitope) were generated using the IEDB software for epitope prediction from selected proteins. The overall screening process resulted in the final selection of five different peptides.

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Chapter 6 Identification of antigenic epitopes

Two of the peptides were derived from the genus-specific proteins Peptidyl-prolyl cis- trans isomerase (MIP) and Peptidoglycan associated lipoprotein (PAL). Another peptide, derived from Enhanced entry protein (EnhC), was L. longbeachae specific and has a homolog in other Legionella species. The other two peptides were derived from the L. longbeachae-specific proteins Protein with a bacterial immunoglobulin-like domain (BIDC) and Thaumatin-domain containing protein (ThDC). Peptides were not designed from the other proteins as they were either not found to contain significant conserved sequences across the different Legionella species or data were not available for other congeneric species. Moreover, certain conserved regions were found to have many hydrophobic amino acid residues and were therefore not considered for peptide synthesis, as there was a significant likelihood that they were insoluble and would therefore be inappropriate for the assays used.

The prediction of hydrophylicity was based of the grand average hydropathy (GRAVY) value, whereby a high positive value indicates high hydrophobicity. The peptides were also cross-checked for their solubility from https://www.genscript.com/ssl- bin/site2/peptide_calculation.cgi. Some of the peptides, although found to be conserved in Legionella species, showed some similarity to proteins from other genera and were therefore excluded.

Table 6.4 lists the peptides that were designed as a result of epitope prediction. There were twenty-one species that had their amino acid sequence data available on the UniProt database for MIP protein, whereas only 3 species (L. longbeachae, L. pneumophila and L. drancourtii) had sequence data available for PAL protein.

Table 6.4: Peptides designed on the basis of in silico epitope predictions

Protein Peptide Specificity GRAVY value Peptidyl-prolyl cis-trans isomerase (MIP) KDKLSYSIGADL Genus level -0.35

Peptidoglycan associated lipoprotein QEPGESYTTQ Genus Level -1.95

Enhanced entry protein DKGNEKAMLA L. longbeachae -0.94

Protein with a bacterial immunoglobulin-like domain DSPGAAVGKG L. longbeachae -0.32

Thaumatin-domain containing protein RFTATDATPS L. longbeachae -0.61

GRAVY value was predicted from http://www.gravy-calculator.de/index.php

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Chapter 6 Identification of antigenic epitopes

Figure 6.3 illustrates an example of an alignment of the amino acid sequence for the homologs of the Peptidyl-prolyl cis-trans isomerase (MIP) protein found in all the Legionella species. However, only twenty-one species were found to have the sequence data available for this protein. Nevertheless, the alignment showed a few stretches of amino acid sequences that are conserved in all species.

The amino acid sequences have been highlighted according to the peptides that were identified to the MIP protein by peptide mass fingerprinting. The region highlighted in grey represents the peptides that were identified from the total cell soluble proteome and outer-membrane sub-proteome. The amino acid sequences highlighted in aqua blue were identified from the total soluble proteome only. The region highlighted in pink indicates that the peptide was identified from the outer-membrane sub-proteome only. The peptide sequence highlighted in orange was identified from both the trypsin shaving and total soluble proteome analyses. The amino acid residues highlighted in yellow represent overlapping peptide sequences.

Only the peptide sequences KLSYSIGADLGK and SVGGPIGPNETLIFK were found to belong to a conserved region of a reasonable stretch of amino acid residues. In general, linear epitopes normally consist of 5-10 amino acids (Zhao and Chait, 1994). However, it was anticipated that the larger the stretch of the amino acids chosen, the more likely the sequence was to be specific to Legionella. BLAST (UniProt and NCBInr) analyses revealed that although the peptide KLSYSIGADLGK shared some similarity with other organisms, mainly marine proteobacteria, they are not known to cause pneumonia. On the other hand, the peptide SVGGPIGPNETLIFK was found to share significant homology with organisms such as Coxiella burnetti, which is closely related to Legionella and is also known to cause pneumonia. Moreover, the GRAVY score was found to be +0.2, which means it is more hydrophobic than the other peptide.

The peptide sequences in bold red are the peptides which were predicted as epitopes via IEDB. Based on the conserved region, hydrophilicity, specificity and predicted antigenic epitopes, the peptide KDKLSYSIGADL highlighted in green was selected for synthesis to determine its immunogenicity.

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1 20 40 60 80 100 120 Chapter 6 longbeachae MKMKLVTAAIMGLAMSTAMAATDATSLTTDKDKLSYSIGADLGKNFKNQGIDINPDVLAKGMQDGMSGAQLILTEEQMKDVLSKFQKDLMAKRSAEFNKKAEENKAKGDAFLSANKSKPGIVV L. sainthelensi MKMKLVTAAIMGLAMSTAMAATDATSLTTDKDKLSYSIGADLGKNFKNQGIDINPDVLAKGMQDGMSGAQLILTEEQMKDVLSKFQKDLMAKRSAEFNKKAEENKAKGEAFLSANKSKPGIVV

L. cincinatiensis MKMKLVTAAIMGLAMSTAMATTDATSLTTDKDKLSYSIGADLGKNFKNQGIDINPEALAKGMQDGMSGAQLILTEEQMKDVLSKFQKDLMAKRSAEFNKKAEENKAKGEAFLSANKSKPGIVV L. santicrusis MKMKLVTAAIMGLAMSTAMAATDATSLTTDKDKLSYSIGADLGKNFKNQGIDINPEALAKGMQDGMSGAQLILTEEQMKDVLSKFQKDLMAKRSAEFNKKAEENKAKGDAFLSANKSKPGIVV L. cherrii MKMKLVTAAIMGLAMSTAMAATDATSLVTDKDKLSYSIGADLGKNFKNQGIDINPDALAKGMQDGMSGAQLILTEQQMKDVLNKFQKDLMAKRSAEFNKKAEENKAKGEAFLSTNKSKSGVVV L. gratiana MKMKLVTAAIMGLAMSTAMAATDATSLATDKDKLSYSIGADLGKNFKNQGIEVNPEVLAKGMQDGMSGAQLILTEEQMKDVLSKFQKDLMAKRSAEFNKKAEENKAKGDAFLSANKAKPGVVA

L. steigerwaltii MKMKLVTAAIMGLAMSTAMAATDATSLVTDKDKLSYSIGADLGKNFKNQGIDINPDALAKGMQDGMSGAQLILTEQQMKDVLNKFQKDLMAKRSAEFNKKAEENKAKGEAFLSSNKSKSGVVV L. dumoffii MKMKLVTAAIMGLAMSTAMAATDATSLVTDKDKLSYSIGADLGKNFKNQGIDINPEALAKGMQDGMSGAQLILTEQQMKDVLNKFQKDLMAKRSAEFNKKAEENKAKGEAFLSSNKSKSGVVV L. gormanii MKMKLVTAAIMGLAMSTAMAATDATSLVTDKDKLSYSIGADLGKNFKNQGIDINPEALAKGMQDGMSGAQLILTEQQMKDVLNKFQKDLMAKRSAEFNKKAEENKSKGEAFLSTNKSKSGVVV L. parisiensis MKMKLVTAAVMGLAMSTVMAATDATSLVTDKDKLSYSIGADLGKNFKNQGIDINPDALAKGMQDGMSGAQLILTEQQMKDVLNKFQKELMAKRSAEFNKKAEENKSKGDAFLSTNKSKSGVTV

L. tucsonensis MKMKLVTAAVMGLAMSTVIAATDATSLVTDKDKLSYSIGADLGKNFKNQGIDINPDALAKGMQDGMSGAQLILTEQQMKDVLNKFQKELMAKRSAEFNKKAEENKSKGETFLSTNKSKSGVVV L. bozemanii MKMKLVTAAVMGLAMSTVIAATDATSLVTDKDKLSYSIGADLGKNFKNQGIDINPDALAKGMQDGMSGAQLILTEQQMKDVLNKFQKELMAKRSAEFNKKAEENKSKGEAFLSTNKSKSGVVV L. wadsworthii MKMKLVNAAILGLAMSTAMAATDATSLVTDKDKLSYSIGADLGKNFKTQGIDINPDALAKGMQDGMSGAQLILTEQQMKDVLNKFQKDLMAKRSAEFNKKAEENKSKGDSFLSSNKSKSGVVV L. anisa MKMKLVTAAVMGLAMSTVMA-ADATSLVTDKDKLSYSIGADLGKNFKNQGIDINPDALAKGMQDGMSGAQLILTEQQMKDVLNKFQKELMAKRSAEFNKKAEENKSKGDAFLSTNKSKSGVTV

L. quateirensis MKMKLVTAAVLGLAMSTAMA-TDATSLPTDKDKLSYSIGADLGKNFKNQGIDVNPEALAKGMQDGMSGAQLILTEQQMKDVLNKFQKDLMAKRTSEFNKKAEENKSKGEAFLTENKSKTGVVV L. moravica MKMKLVTAAVLGLAMSSAMA-TDATSLPTDKDKLSYSIGADLGKNFKNQGIDVNPEALAKGMQDGMSGAQLILTEQQMKDVLNKFQKDLMAKRTSEFNKKAEENKSKGEAFLTENKSKTGVVV L. micdadei MKMKLVTAAVMGLAMSTAMAATDATSLATDKDKLSYSIGADLGKNFKNQGIDVNPEAMAKGMQDAMSGAQLALTEQQMKDVLNKFQKDLMAKRTAEFNKKADENKVKGEAFLTENKNKPGVVV L. pneumophila MKMKLVTAAVMGLAMSTAMAATDATSLATDKDKLSYSIGADLGKNFKNQGIDVNPEAMAKGMQDAMSGAQLALTEQQMKDVLNKFQKDLMAKRTAEFNKKADENKVKGEAFLTENKNKPGVVV

L. shakespearei MKMKLVTAAVMGLALSTAMAAPDATSLPTDKDKLSYSIGADLGKNFKTQGIDINPEALAKGMQDGMSGTQLILTEQQMKDVLNKFQKDLMAKRTSEFNKKADENKSKGEAFLVENKGKTGVVV L. drancourtii MKMRLVTAAVLGLALSTAMAA---DALVTDKDKLSYSIGADLGKNFKTQGIDINPEALAKGMQDGMSGGQLILTEQQMKDVLNKFQKDLMAKRSADFNKKADENKVKGEAFLTSNKAKTGVVV L. worsleiensis MKMKLVAAAVLGLAMSGAMA-ADATSLTTDKDKLSYSIGADLGKNFKNQGIDVNPEALAKGMQDGMSGTQLILTEQQMKDVLNKFQKDLMAKRTSEFNKKADENKLKGEAFLTANKTKAGVVV ***:** **::***:* .:* :* *******************.***::**:.:******.*** ** ***:******.****:*****:::*****:*** **::** ** * *:..

L. longbeachae LPSGLQYKIIDAGTGAKPGKSDTVTVEYTGTLIDGTVFDSTEKAGKPATFQVSQVIPGWTEALQLMPAGSTWEVFVPADLAYGPRSVGGPIGPNETLIFKIHLISVKKAA- 233 L. sainthelensi LPSGLQYKIIDAGTGAKPGKSDTVTVEYTGTLIDGTVFDSTEKAGKPATFQVSQVIPGWTEALQLMPSGSTWEVYVPADLAYGPRSVGGPIGPNETLIFKIHLISVKKAA- 233 L. cincinatiensis LPSGLQYKIIDAGTGAKPGKSDTVTVEYTGTLIDGTVFDSTEKAGKPATFQVSQVIPGWTEALQLMPSGSTWEVYVPADLAYGPRSVGGPIGPNETLIFKIHLISVKKAA- 233

L. santicrusis LPSGLQYKIIDAGTGSKPGKSDTVTVEYTGTLIDGTVFDSTEKAGKPATFQVSQVIPGWTEALQLMPSGSTWEVYVPADLAYGSRSVGGPIGPNETLIFKIHLISVKKAA- 233 L. cherrii LPSGLQYKIIEAGTGAKPGKSDTVTVEYTGTLIDGTVFDSTEKAGKPATFQVSQVIPGWTEALQLMPAGSTWEIFVPSDLAYGPRSVGGPIGPNETLIFKIHLISVKKAA- 233 L. gratiana LPSGLQYKIIDAGNGTKPGKSDTVTVEYTGTLIDGTVFDSTEKAGKPATFQVSQVIPGWTEALQLMPAGSTWEVYVPSDLAYGPRSVGGPIGPNETLIFKIHLISVKKEA- 233 L. steigerwaltii LPSGLQYKIIEAGTGNKPGKSDTVTVEYTGTLIDGTVFDSTEKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIFVPSDLAYGPRSVGGPIGPNETLIFKIHLISVKKA-- 232

L. dumoffii LPSGLQYKVIEAGSGAKPGKSDTVTVEYTGTLIDGTVFDSTEKAGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPSNLAYGPRSVGGPIGPNETLIFKIHLISVKKASA 234 Identification of antigenic epitopes L. gormanii LPSGLQYKVIEAGTGSKPGKSDTVTVEYTGTLIDGTVFDSTEKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPSDLAYGPRSVGGPIGPNETLIFKIHLISVKKA-- 232 L. parisiensis LPSGLQYKVIEAGTGNKPGKSDTVTVEYTGTLIDGTVFDSTEKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPSDLAYGPRSVGGPIGPNETLIFKIHLISVKKSA- 233 L. tucsonensis LPSGLQYKIIEAGTGNKPGKSDTVTVEYTGTLIDGTVFDSTEKAGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPSDLAYGPRSVGGPIGPNETLIFKIHLISVKKSA- 233 L. bozemanii LPSGLQYKVIEAGTGNKPGKSDTVTVEYTGTLIDGTVFDSTEKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPSDLAYGPRSVGGPIGPNETLIFKIHLISVKKSA- 233 L. wadsworthii LPSGLQYKVIEAGTGNKPSKTDTVTVEYTGTLIDGTVFDSTEKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPSDLAYGPRSVGGPIGPNETLIFKIHLISVKKASA 234 L. anisa LPSGLQYKVIEAGTGNKPGKADTVTVEYTGTLIDGTVFDSTEKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPSDLAYGPRSVGGPIGPNETLIFKIHLISVKKSA- 232 L. quateirensis LPSGLQYKIIDAGTGAKPGKSDTVTVEYTGRLIDGTVFDSTEKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPASLAYGPRSVGGPIGPNETLIFKIHLISVKKSAS 233 L. moravica LPSGLQYKIIDAGTGAKPGKTDTVTVEYTGRLIDGTVFDSTEKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPASLAYGPRSVGGPIGPNETLIFKIHLISVKKSAS 233 L. micdadei LPSGLQYKVINAGNGVKPGKSDTVTVEYTGRLIDGTVFDSTEKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPSGLAYGPRSVGGPIGPNETLIFKIHLISVKKSS- 233 L. pneumophila LPSGLQYKVINSGNGVKPGKSDTVTVEYTGRLIDGTVFDSTEKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPSGLAYGPRSVGGPIGPNETLIFKIHLISVKKSS- 233 140 L. shakespearei LPSGLQYKIIDAGTGAKPGKSDTVTVEYTGRLIDGTVFDSTDKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPSALAYGSRSVGGPIGPNETLIFKIHLISVKKTEA 234 L. drancourtii LPSGLQYKILEAGTGAKPSKTDTVTVDYTGTLIDGTVFDSTQKTGKPATFQVSQVIPGWTEALQLMPAGSTWEVYVPADLAYGPRSVGGPIGPNETLIFKIHLISVKKAA- 230

I Page L. worsleiensis LPSGLQYKILDAGTGTKPGKSDTVTVEYTGRLIDGTIFDSSDKTGKPATFQVSQVIPGWTEALQLMPAGSTWEIYVPAALAYGSRSIGGPIGPNETLIFKIHLISVKKSDA 233

********::::*.* **.*:*****:*** *****:***::*:***********************:*****::**: **** **:********************* Figure 6.3: Peptidyl-prolyl cis-trans isomerase (MIP) protein sequence alignment for identification of conserved regions

Chapter 6 Identification of antigenic epitopes

6.3.4 Determination of antigenicity of peptides

Enzyme linked immunosorbent assay (ELISA) and Dot blot were carried out with the designed peptides to determine their immunogenicity. However, none of the peptides appeared to cross-react, and one possible explanation would be that the peptides were not bound. Therefore, the Maxisorp Nunc ELISA plate was used, as it has a higher binding affinity compared to normal 96 well plates, but there were no significant differences. The absence of a reaction could be attributed to the peptides either not being immunogenic or being washed off owing to its small size.

6.4 DISCUSSION

6.4.1 Challenges in affinity purification of proteins

As evidenced in the data presented, the affinity purification of the IgG was successful although contamination with other proteins was apparent. However, in future developments, this issue could be addressed by using the depletion method, whereby unwanted proteins are selectively eliminated from a mixture, especially in serum samples.

There were no proteins found to be affinity purified by the method used. One explanation could be that, even after enrichment was attempted, the immunogenic proteins were still below the detection limit. As observed from the mass fingerprinting data of the affinity purified IgG, there were contaminants still present and these could have interfered with the purification process. Alternatively, the amount of the immobilised IgG could have been so small that there were not enough antibodies to bind to the antigens.

One of the suspected reasons for the non-binding of the antigens to the immobilised antibodies was probably related to the very low pH (2) of the buffer used in the elution of the affinity purified IgG, which could have affected the integrity of the protein. To verify this assumption a further ELISA test was carried out to determine the presence of immunoreactive antibodies and a reaction was still observed with the antigens. Therefore, the results suggest that the concentration of IgG and antigens should have been higher to achieve a better yield, above the level of detection. However, this is particularly difficult to achieve. As mentioned earlier, in such studies, human serum

141 | Page Chapter 6 Identification of antigenic epitopes samples can be one of the limiting factors as only small quantities are available. Therefore, to conduct such studies, we may have to rely on in silico predictions in order to minimise wastage of the precious samples.

6.4.2 Limitations of in silico prediction of epitopes and peptide design

The in silico prediction of epitopes has been successfully applied in the detection of potential antigenic determinants for their use in the development of improved diagnostic tests. One such example is the study carried out by Maksimov et al. (2012) whereby the authors identified eight novel antigenic peptides that could be used in toxoplasmosis diagnosis. In this study, none of the predicted epitopes were found to be immunoreactive, probably because they were not antigenic. As such, epitope prediction can be carried out using several prediction software applications and the use of consensus peptides as the algorithms may differ.

The fact that this method was used to predict linear epitopes based on conserved regions meant that the number of peptides that could be synthesised was limited and hence a larger panel of epitopes was not included. This approach may thus have some limitations. Furthermore, in general most of the epitopes are known to be discontinuous; up to 90% (Sweredoski and Baldi, 2008), which are protein structure dependent rather than the linear amino acid sequence. As a result, the probability of identifying such epitopes appears to be comparatively lower. However, identifying discontinuous genus- specific epitopes can be even more challenging.

6.4.3 Lack of genomic data and its implications

There are very little genomic data available for other Legionella species. This rendered the identification of genus-specific epitopes very difficult and was limited to only a few proteins for which sequence data was available. This resulted in only two peptides that were targeted to recognise antigenic determinants from conserved amino acid sequences within the Legionella genus. The results based on two peptides are therefore insufficient to determine whether conserved linear epitopes exist for the proteins studied. As identified in the comparative analyses of the proteomes of L. longbeachae and L. pneumophila, the differential expression of proteins and the degree of variation in the amino acid sequence of homologous proteins may prove identification of conserved

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Chapter 6 Identification of antigenic epitopes linear epitopes difficult. This study suggests that there is a need for more genome information for other Legionella species.

6.5 CONCLUSION

This study demonstrated that limitations were encountered using the approach of identifying epitopes experimentally in the laboratory. The volume of serum samples available was a shortcoming for the experiments as they had to be used sparingly to perform all the required assays. The approach used for in silico prediction of the epitopes requires improvement, firstly because limited genomic data is available for the majority of Legionella species implicated in causing infection in human and secondly because most of the epitopes are discontinuous. Further works addressing these issues may have better outcomes.

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GENERAL DISCUSSION AND CONCLUSIONS

7.1 OVERVIEW

An empiric method of antibiotic prescription is commonly used in the treatment of community-acquired pneumonia. However, due to the inefficacy of such a treatment method amongst Legionnaires disease patients, specific antibiotic therapy is generally required. Therefore, for the administration of efficient antibiotics, a suitable diagnostic test is essential to identify the etiological agent. There are currently several serological tests commercially available for the diagnosis of legionellosis. Nonetheless, a review of the literature showed that these tests have several limitations, such as the inability to diagnose patients infected with all the pathogenic Legionella species, a long turnaround time for test results and a lack in sensitivity and specificity.

Legionellosis is a notifiable disease in many countries. However, owing to the absence of a genus-wide diagnostic test, many cases are most likely unreported. Moreover, improper diagnosis may have serious consequences such as increased risks to the patients health, emergence of drug-resistant pathogens, greater economic burden and inefficient surveillance programs, which are particularly important in case of an outbreak. Thus, there is a real need for an improved test for the diagnosis of Legionnaires disease, mainly due to non-pneumophila Legionella species.

Moreover, there are other Legionella species such as L. longbeachae which are being increasingly reported to cause disease in human. L. longbeachae has been found to be more prevalent in Australasian region. Also, the Australian clinical isolates have been reported to be more virulent to strains from other geographical regions. Therefore, correct diagnosis may prove helpful in epidemiological studies also

144 | Page Chapter 7 General discussion and conclusions

The aim of this study was to identify genus-specific and species-specific epitopes that can be targeted as potential biomarkers. To achieve this aim, several objectives were set to: • optimise sample preparation for the analysis of proteomes by 2-Dimensional gel electrophoresis for subsequent analyses • perform comparative analyses of the different sub-proteomes of L. pneumophila and L. longbeachae to determine differentially expressed proteins under similar growth conditions • revise an ELISA-based protocol for blind-testing of human serum samples to identify those containing antibodies against Legionella antigens (some of which had previously been identified to contain antibodies against Legionella from two different sources where Legionnaires disease diagnosis is routinely performed) • identify genus-specific and L. longbeachae-specific immunogenic proteins as potential biomarkers • carry out epitope mapping experimentally by affinity purification methods, in silico prediction of antigenic determinants, and test for antigenicity.

The main findings of this study demonstrate that most of the objectives were successfully addressed and have brought significant contributions to the fields of proteomics and immunology, as further discussed below.

The key for successful 2-Dimensional gel-based proteomics analyses is appropriate sample preparation. As such, protocols for performing 2-D gel electrophoresis available in literature have to be optimised according to the type of organism and proteome. Satisfactory protein separation and reproducibility can be evidenced from the DIGE gels (Chapter 4) as a result of appropriate optimised protocols used for sample preparation and performing 2-D gel electrophoresis. As there have been, to the best of our knowledge, no reports on the proteome of L. longbeachae, the protocols optimised in this work will prove helpful for future studies.

To date, a comparative proteomic analysis of two congeneric species using the DIGE method has only been reported recently during the time of the current study (Shinoy et al. (2013). However, the comparative proteomics analysis of congeneric Burkholderia species carried out was aimed at identifying immunogenic proteins only. The present work demonstrated that, except for the investigation of differential quantitative

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Chapter 7 General discussion and conclusions expression of proteins under different states, for example, diseased vs. normal or study of the effects of different treatment conditions in the same species, proteome analysis of congeneric species may also reveal other interesting physiological features.

As discussed in Chapter 4, both L. pneumophila and L. longbeachae were found to differentially express proteins under similar growth conditions. Whilst some proteins were specific to L. longbeachae, others were specific to L. pneumophila. This strongly suggests that there exist inherent physiological differences between the two Legionella species, which may reflect in their differential ability to cause infection using different pathogenesis strategies, and be due to their adaptation to dissimilar ecological niches. DIGE has the ability to show biological variations with the inclusion of internal standards in every gel and multiplexing with several replicates. The difference in the quantitative expression of several homologous proteins, as observed from the DIGE experiment, is therefore also suggestive of the different lifestyles of the Legionella species. These physiological differences may be suggestive of variations in the biochemical pathways of the bacteria. Therefore, further investigations may reveal more information regarding metabolic pathways that can assist in the formulation of better growth media, as many Legionella species are still difficult to grow under in vitro conditions.

An investigation of the immunogenicity of the proteins isolated from L. pneumophila and L. longbeachae in Chapter 5 generated rather distinctive results that, to the best of our knowledge, have never been reported in literature before. Human serum samples identified to contain antibodies against Legionella antigens were used to identify genus- specific and L. longbeachae-specific immunogenic proteins to be targeted as potential biomarkers. This experiment revealed that little to no cross-reactivity exists across the Legionella species proteins. The immunodominant Heat shock protein (Hsp60) from L. pneumophila was found most likely not to cross-react with antibodies present in two different serum samples while it reacted with Hsp60 in L. longbeachae.

Moreover, out of the fifty commonly expressed homologous proteins determined from the DIGE experiment which were immunoreactive in L. longbeachae, none were immunoreactive in L. pneumophila. This observation suggests that the proteins responsible for the differential immune response may be Legionella species dependent or the epitopes on the homologous immunogenic proteins may vary from species to

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Chapter 7 General discussion and conclusions species. Thus, it appears that the Legionella genus-specific proteins, described in literature as potential biomarkers for the diagnosis of Legionnaires disease, may give misleading results, unless genus-specific epitopes are identified. Moreover, this study revealed that there may be limitations in the serological tests currently in use, and a plausible reason for the generally recognised assumption that several Legionella species are under-reported.

The attempts to identify genus-specific and L. longbeachae-specific epitopes using two methods, experimental epitope mapping and in silico prediction of antigenic determinants, were rather unsuccessful. Although affinity purification of IgG was achieved and the purified antibody was found to retain activity, an attempt to affinity purify antigenic proteins was ineffective. This could possibly be because of the low volume of serum samples available for affinity purification of the antibodies and, therefore, the final amount of antibodies available for affinity purification of antigens could have been inadequate. A larger volume of serum sample may therefore alleviate this problem. This approach of epitope mapping may be particularly useful where several proteins are being screened for ultimately selecting a few as potential biomarkers. Accordingly, protein expression and antibody production may not be recommended, mainly from an economical viewpoint.

The in silico prediction of epitopes was also proven to be ineffectual. This approach of finding antigenic determinants was mainly affected due to the limited genome sequence data available for the majority of the Legionella species. There could be a few reasons why the experiments were unsuccessful. Firstly, the designed peptides could have been too small and might not have bound to the ELISA plates, and shown false negative result if they were washed off. Secondly, the peptides may not be antigenic as they were from linear stretches of amino acid sequences, and most of the epitopes are known to be discontinuous in nature. Thirdly, only one peptide per protein was designed based on this approach, and may consequently be too stringent. While this method can be appropriate for the identification of epitopes from individual species using a larger peptide library, as has been demonstrated in literature with several successful studies, identification of genus-specific epitopes via in silico prediction can be challenging. Again, the approach used in this study, i.e. finding conserved, linear, specific and hydrophilic peptides, to the best of our knowledge has not been reported in literature.

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Chapter 7 General discussion and conclusions

Overall, this study showed promising results in terms of how diagnostic tests could be improved. As it was found that there exists a large degree of variation in the proteomes of L. longbeachae and L. pneumophila under similar growth conditions, a thorough study of the metabolic pathways in different Legionella species may assist in the formulation of better culture media for those species that are difficult to grow in vitro. We propose the use of pooled genus-specific antigens for genus-level diagnosis and the inclusion of a panel of species-specific antigens in parallel for species identification.

As suggested by Rifai et al. (2006) the journey of protein biomarker discovery to its validation can be a long and uncertain path.

7.2 RECOMMENDATIONS FOR FUTURE WORK

During this study, a few limitations have been identified which can be addressed based on the following recommendations:

• performing further immunoassay tests with larger number of serum samples (animal studies or legionellosis patients with culture proven diagnosis) will provide statistically significant data and hence validate the observation of differential immune response due to different Legionella species • collection of larger volumes of serum samples for affinity purification of antibodies and antigens and epitope mapping • conjugating the peptides with a carrier protein to help coat the ELISA plate and also to be able to determine antigenicity via different immunoassays such as Western blot and dot-blot.

Also, as genome sequencing is becoming increasingly more affordable, the genome sequence of a few other species of interest may help in the:

. selection of genus-specific and species-specific epitopes for development of diagnostic test . exploration of the metabolic pathways for identification of differential substrate requirements of different Legionella species and hence assist in formulation of improved culture media . identification of novel virulence and pathogenesis related factors.

148 | Page List of references

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Appendices

APPENDICES

Appendix 1

L. longbeachae secretome map

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Appendix 2

L. pneumophila secretome 2D map

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Appendix 3

L. longbeachae total soluble proteome map

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Appendix 4

L. longbeachae outer-membrane sub-proteome map

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Appendix 5

DIGE analysis L. longbeachae NSW150 vs. D4968

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Appendix 6

DIGE analysis L. pneumophila vs. L. longbeachae

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Appendix 7

DIGE analysis L. longbeachae vs. L. pneumophila

181 | Page Appendices

Appendix 8

DIGE gels

Gel 4 Gel1

Gel 2 Gel 5

Gel 3 Gel 6

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Appendix 9

DIGE statistics

Spot on preparative Av. 1- Pos. Master No. gel T-test Ratio ANOVA Species 1 1778 99 0.00028 -2.57 1.10E-05 L. longbeachae 2 8 - 0.00011 2.14 0.0042 L. longbeachae 3 68 - 5.80E-05 26.21 0.00014 L. longbeachae 4 101 - 0.00036 7.8 4.50E-06 L. longbeachae 5 185 - 0.00057 8.93 6.70E-06 L. longbeachae 6 218 17 9.80E-06 6.07 2.60E-07 L. longbeachae 7 221 4 0.00029 1.65 0.00017 L. longbeachae 8 230 - 5.20E-05 20.51 6.20E-06 L. longbeachae 9 315 - 0.00035 5.68 1.80E-05 L. longbeachae 10 319 - 3.50E-05 4.18 1.60E-06 L. longbeachae 11 404 - 1.80E-06 8.96 1.10E-08 L. longbeachae 12 423 - 2.00E-05 10.22 5.70E-06 L. longbeachae 13 431 - 0.0001 3.46 7.40E-06 L. longbeachae 14 455 - 6.50E-05 10 4.00E-06 L. longbeachae 15 472 - 0.00035 16.01 1.30E-05 L. longbeachae 16 494 - 0.0001 13.11 6.90E-06 L. longbeachae 17 520 - 0.00032 4.37 4.00E-05 L. longbeachae 18 535 - 0.00054 2.28 3.10E-05 L. longbeachae 19 551 - 9.70E-05 7.19 8.30E-06 L. longbeachae 20 561 - 0.00099 2.47 0.0012 L. longbeachae 21 603 70 0.00046 16.2 1.90E-05 L. longbeachae 22 621 158 0.00016 3.47 1.90E-05 L. longbeachae 23 625 - 0.00057 12.19 0.0043 L. longbeachae 24 626 19 0.0003 3.4 0.00012 L. longbeachae 25 636 71 0.00017 19.41 3.60E-06 L. longbeachae 26 640 - 3.20E-05 3.83 2.20E-06 L. longbeachae 27 660 4 0.00016 5.79 7.80E-05 L. longbeachae 28 665 7 8.40E-05 2.78 9.30E-06 L. longbeachae 29 667 - 6.10E-05 14.5 1.80E-06 L. longbeachae 30 681 195 0.00021 3.52 3.80E-05 L. longbeachae 31 720 - 0.00068 5.04 7.90E-05 L. longbeachae 32 740 72 1.80E-05 18.74 2.20E-07 L. longbeachae 33 743 198 1.60E-08 16.45 5.50E-10 L. longbeachae 34 835 - 0.00022 6.84 6.20E-06 L. longbeachae 35 858 196 0.00031 4.89 3.80E-06 L. longbeachae 36 894 - 0.00018 3.47 8.60E-06 L. longbeachae 37 935 not present 0.0003 6.97 7.70E-05 L. longbeachae 38 999 not present 0.00027 3.79 0.00089 L. longbeachae 39 1015 134 0.00035 3.36 2.30E-05 L. longbeachae 40 1016 - 6.60E-05 23.19 6.20E-06 L. longbeachae 41 1019 65 0.00067 10.76 0.00078 L. longbeachae 42 1022 77 0.00039 3.28 1.90E-05 L. longbeachae 43 1050 - 0.00076 8.19 5.00E-05 L. longbeachae

183 | Page

Appendices

Spot on preparative Av. 1- Pos. Master No. gel T-test Ratio ANOVA Species 44 1065 - 7.80E-05 14.11 2.40E-05 L. longbeachae 45 1066 - 1.50E-06 29.22 4.10E-08 L. longbeachae 46 1081 167 1.90E-06 15.76 8.30E-07 L. longbeachae 47 1082 168 0.00021 11.76 0.00036 L. longbeachae 48 1085 - 1.20E-05 32.51 7.20E-08 L. longbeachae 49 1097 - 0.00033 3.62 8.60E-05 L. longbeachae 50 1104 - 4.40E-06 6.61 2.10E-08 L. longbeachae 51 1111 - 5.30E-05 3.15 2.00E-06 L. longbeachae 52 1113 - 5.10E-05 3.94 7.00E-06 L. longbeachae 53 1131 - 8.40E-07 44.98 2.90E-09 L. longbeachae 54 1133 - 0.00011 16.1 2.50E-05 L. longbeachae 55 1134 - 3.10E-06 34.5 1.90E-08 L. longbeachae 56 1141 169 6.20E-06 22.65 4.40E-08 L. longbeachae 57 1143 107 1.50E-06 10.54 1.60E-08 L. longbeachae 58 1144 136 0.00019 14.64 4.50E-05 L. longbeachae 59 1145 169 7.60E-08 13.33 2.70E-10 L. longbeachae 60 1147 107 2.80E-05 6.81 8.70E-07 L. longbeachae 61 1160 170 1.80E-06 5.07 7.50E-09 L. longbeachae 62 1166 137 0.00012 2.43 2.10E-05 L. longbeachae 63 1168 197 0.00032 2.55 2.00E-05 L. longbeachae 64 1169 181 0.00029 3.12 0.00066 L. longbeachae 65 1172 183 0.00013 2.55 0.00033 L. longbeachae 66 1178 - 6.70E-05 7.68 4.40E-06 L. longbeachae 67 1180 - 1.80E-07 17 8.30E-10 L. longbeachae 68 1198 106 0.00091 8.31 8.70E-05 L. longbeachae 69 1245 138 1.50E-06 6.3 8.20E-09 L. longbeachae 70 1281 111 0.00051 3.16 3.50E-05 L. longbeachae 71 1293 not present 3.80E-06 20.49 2.90E-06 L. longbeachae 72 1300 60 6.20E-06 10.79 5.30E-05 L. longbeachae 73 1314 150 0.00071 2.71 0.00011 L. longbeachae 74 1317 139 0.00024 3.87 2.70E-05 L. longbeachae 75 1349 140 6.00E-06 10.67 6.90E-08 L. longbeachae 76 1357 108 8.60E-06 5.34 8.50E-08 L. longbeachae 77 1358 109 0.00023 4.37 1.20E-05 L. longbeachae 78 1377 50 0.00063 3.09 0.00016 L. longbeachae 79 1385 - 0.00018 7.4 4.90E-06 L. longbeachae 80 1391 - 0.00011 2.62 0.00011 L. longbeachae 81 1393 95 0.0001 3.35 1.40E-05 L. longbeachae 82 1398 114 0.00092 6.62 6.50E-05 L. longbeachae 83 1412 - 4.10E-07 5.55 1.10E-09 L. longbeachae 84 1426 not present 0.00012 2.16 0.017 L. longbeachae 85 1431 55 5.70E-05 4.07 1.10E-06 L. longbeachae 86 1452 117 4.80E-06 6.08 1.10E-07 L. longbeachae 87 1460 not present 2.30E-05 9.27 1.60E-06 L. longbeachae 88 1470 34 6.30E-05 4.71 1.40E-06 L. longbeachae 89 1472 - 1.60E-05 9.05 6.80E-07 L. longbeachae 90 1478 186 2.80E-06 12.87 2.50E-08 L. longbeachae 91 1479 186 8.30E-07 18.75 3.90E-09 L. longbeachae 184 | Page

Appendices

Spot on preparative Av. 1- Pos. Master No. gel T-test Ratio ANOVA Species 92 1484 115 1.20E-05 11.12 5.20E-07 L. longbeachae 93 1501 144 0.00077 3.68 0.00018 L. longbeachae 94 1509 29 0.00041 2.31 3.70E-05 L. longbeachae 95 1518 - 1.50E-06 17.94 8.30E-08 L. longbeachae 96 1521 - 0.00017 3.04 4.30E-05 L. longbeachae 97 1524 - 4.10E-05 4.27 4.00E-05 L. longbeachae 98 1532 175 3.80E-05 7.7 6.10E-07 L. longbeachae 99 1534 175 4.70E-06 15.16 3.00E-08 L. longbeachae 100 1545 - 2.20E-08 58.93 4.00E-11 L. longbeachae 101 1550 123 0.00063 2.04 8.00E-05 L. longbeachae 102 1555 123 7.90E-06 2.73 1.10E-05 L. longbeachae 103 1590 93 7.00E-06 9.1 1.50E-07 L. longbeachae 104 1591 209 0.00037 4.01 3.00E-05 L. longbeachae 105 1593 99 2.60E-06 7.22 1.10E-06 L. longbeachae 106 1595 - 0.00056 3.63 9.10E-05 L. longbeachae 107 1598 99 4.50E-06 12.71 9.00E-08 L. longbeachae 108 1599 118 8.40E-05 3.06 0.00089 L. longbeachae 109 1614 208 1.50E-08 10.05 1.90E-11 L. longbeachae 110 1615 206 4.50E-05 6.15 7.00E-07 L. longbeachae 111 1628 174 3.20E-06 4.89 9.90E-08 L. longbeachae 112 1647 207 0.00011 16.56 1.90E-05 L. longbeachae 113 1655 - 3.50E-07 216.26 2.00E-09 L. longbeachae 114 1656 - 1.50E-05 48.76 7.30E-07 L. longbeachae 115 1668 112 1.00E-05 14.66 1.00E-07 L. longbeachae 116 1679 153 3.60E-07 1.94 0.00018 L. longbeachae 117 1684 113 0.00011 4.49 3.20E-05 L. longbeachae 118 1691 - 8.70E-05 4.79 1.60E-05 L. longbeachae 119 1693 176 3.70E-06 4.18 5.00E-07 L. longbeachae 120 1695 - 3.90E-05 6.1 8.50E-07 L. longbeachae 121 1705 - 8.40E-05 4.03 8.50E-06 L. longbeachae 122 1717 - 1.40E-05 22.6 1.10E-07 L. longbeachae 123 1718 94 2.70E-06 9.96 1.80E-07 L. longbeachae 124 1719 - 0.00012 3.71 6.70E-06 L. longbeachae 125 1728 - 0.00069 2.58 0.008 L. longbeachae 126 1729 - 1.50E-06 13.16 4.30E-08 L. longbeachae 127 1734 - 1.60E-06 28.88 9.70E-09 L. longbeachae 128 1735 - 1.50E-06 60.26 1.70E-08 L. longbeachae 129 1738 - 1.70E-06 62.23 9.60E-07 L. longbeachae 130 1739 - 5.90E-05 67.34 1.80E-06 L. longbeachae 131 1752 - 6.20E-05 5.62 2.50E-05 L. longbeachae 132 1775 - 3.40E-07 13.63 9.70E-09 L. longbeachae 133 1791 - 3.50E-05 14.68 0.00069 L. longbeachae 134 1801 - 2.40E-07 53.87 1.90E-08 L. longbeachae 135 1811 - 0.00063 2.77 0.0093 L. longbeachae 136 1826 - 5.50E-06 9.91 1.10E-07 L. longbeachae 137 1828 - 4.90E-06 18.34 5.50E-08 L. longbeachae 138 1837 - 6.60E-07 54.5 9.20E-10 L. longbeachae 139 1838 - 0.00077 13.86 3.90E-05 L. longbeachae 185 | Page

Appendices

Spot on preparative Av. 1- Pos. Master No. gel T-test Ratio ANOVA Species 140 1862 - 3.90E-05 9.12 7.80E-06 L. longbeachae 141 1863 - 5.10E-08 16.54 3.50E-09 L. longbeachae 142 1884 - 1.30E-05 9.73 2.10E-05 L. longbeachae 143 1934 - 0.00041 4.43 4.00E-05 L. longbeachae 144 1938 - 0.00041 8.79 0.00023 L. longbeachae 145 1955 - 4.40E-05 9.96 0.0052 L. longbeachae 146 1982 - 6.70E-05 2.82 1.60E-05 L. longbeachae 146 60 - 0.00023 -2.89 2.60E-05 L. longbeachae 147 144 - 0.00082 -2.56 0.0067 L. pneumophila 148 193 218 1.30E-05 -8.26 1.70E-06 L. pneumophila 149 202 218 5.10E-05 -12.37 2.30E-05 L. pneumophila 150 269 - 1.10E-05 -19.87 1.40E-07 L. pneumophila 151 273 - 0.00038 -9.59 0.00019 L. pneumophila 152 282 37 4.00E-06 -8.38 7.50E-06 L. pneumophila 153 605 25 4.90E-06 -12.81 2.80E-08 L. pneumophila 154 609 174 0.00012 -10.06 2.00E-05 L. pneumophila 155 689 165 2.10E-07 -21.4 8.40E-10 L. pneumophila 156 714 22 0.00039 -14.08 4.30E-05 L. pneumophila 157 717 210 8.00E-07 -11.43 3.90E-05 L. pneumophila 158 724 165 5.10E-05 -4.86 0.00054 L. pneumophila 159 739 22 0.0003 -17.07 0.0004 L. pneumophila 160 765 - 0.00056 -3.58 0.00033 L. pneumophila 161 776 6 0.00048 -10.94 0.00015 L. pneumophila 162 799 39 0.00094 -6.85 0.00064 L. pneumophila 163 814 225 0.00062 -22.06 2.70E-05 L. pneumophila 164 822 6 9.30E-05 -10.23 0.00013 L. pneumophila 165 830 118 4.90E-06 -5.14 0.00012 L. pneumophila 166 837 168 3.90E-06 -2.69 0.00065 L. pneumophila 167 838 117 3.10E-05 -4.09 0.00039 L. pneumophila 168 854 27 0.00051 -2.79 0.0077 L. pneumophila 169 861 26 0.00013 -7.13 1.10E-05 L. pneumophila 170 868 171 0.00013 -12.04 5.40E-05 L. pneumophila 171 892 116 2.30E-06 -6.42 8.70E-07 L. pneumophila 172 909 40 0.00057 -10.24 0.00024 L. pneumophila 173 921 23 0.00037 -3.94 0.0011 L. pneumophila 174 972 191 0.0006 -11.95 6.10E-06 L. pneumophila 175 981 119 2.80E-05 -6.66 1.70E-05 L. pneumophila 176 987 120 0.00023 -7.78 6.30E-05 L. pneumophila 177 989 180 0.00054 -3.03 0.00015 L. pneumophila 178 996 193 3.80E-05 -23.77 1.40E-06 L. pneumophila 179 1005 189 4.10E-07 -13.96 5.80E-08 L. pneumophila 180 1017 42 3.60E-06 -5.53 7.00E-07 L. pneumophila 181 1021 122 7.40E-06 -2.43 6.70E-06 L. pneumophila 182 1026 194 3.10E-06 -4.14 3.60E-06 L. pneumophila 183 1049 188 7.30E-05 -4.32 0.002 L. pneumophila 184 1058 35 4.10E-05 -3.66 3.10E-06 L. pneumophila 185 1078 18 2.20E-05 -12.37 5.40E-06 L. pneumophila 186 1079 124 0.00017 -4.23 2.70E-05 L. pneumophila 186 | Page

Appendices

Spot on preparative Av. 1- Pos. Master No. gel T-test Ratio ANOVA Species 187 1099 - 0.00039 -6.22 0.00043 L. pneumophila 188 1121 287 0.00018 -2.63 2.10E-05 L. pneumophila 189 1137 71 5.90E-05 -15.59 2.60E-06 L. pneumophila 190 1140 234 9.60E-05 -7.9 2.70E-05 L. pneumophila 191 1161 20 0.00012 -9.68 8.40E-05 L. pneumophila 192 1191 134 0.00024 -10.32 1.00E-05 L. pneumophila 193 1197 135 0.00014 -4.82 4.40E-05 L. pneumophila 194 1199 75 5.80E-06 -8.99 1.80E-07 L. pneumophila 195 1207 266 2.60E-06 -6.01 1.40E-05 L. pneumophila 196 1213 236 0.00066 -6.91 0.00013 L. pneumophila 197 1215 290 2.70E-06 -39 3.20E-07 L. pneumophila 198 1217 236 0.00052 -8.7 0.00016 L. pneumophila 199 1233 77 8.90E-05 -5.4 3.60E-05 L. pneumophila 200 1239 136 3.50E-05 -15.82 1.00E-06 L. pneumophila 201 1240 284 5.70E-07 -45.1 2.40E-08 L. pneumophila 202 1289 272 8.90E-05 -3.83 2.00E-05 L. pneumophila 203 1290 137 0.00049 -8.41 9.80E-05 L. pneumophila 204 1296 - 7.70E-05 -14.12 4.00E-05 L. pneumophila 205 1298 138 8.30E-07 -21.66 2.30E-07 L. pneumophila 206 1311 285 0.00014 -4.2 2.80E-05 L. pneumophila 207 1326 231 2.90E-06 -10.76 6.50E-07 L. pneumophila 208 1327 231 0.00093 -4.29 0.0011 L. pneumophila 209 1329 91 8.80E-05 -18.75 4.20E-05 L. pneumophila 210 1334 265 4.30E-07 -7.46 5.00E-07 L. pneumophila 211 1335 265 0.0005 -3.79 1.60E-05 L. pneumophila 212 1354 - 8.60E-05 -3.03 4.50E-06 L. pneumophila 213 1359 - 2.30E-05 -3.31 0.00036 L. pneumophila 214 1360 - 3.70E-06 -9.24 5.40E-07 L. pneumophila 215 1370 - 8.00E-07 -13.65 3.90E-08 L. pneumophila 216 1380 44 0.00034 -3.24 0.0043 L. pneumophila 217 1386 139 1.70E-06 -22.89 9.20E-07 L. pneumophila 218 1416 88 1.30E-06 -5.64 0.00029 L. pneumophila 219 1428 270 0.00012 -2.67 0.00022 L. pneumophila 220 1429 97 1.50E-05 -9.78 0.00013 L. pneumophila 221 1436 - 5.30E-08 -50.55 3.00E-10 L. pneumophila 222 1438 232 4.20E-06 -64.09 1.80E-07 L. pneumophila 223 1439 232 6.80E-06 -56.02 4.20E-07 L. pneumophila 224 1444 232 1.50E-06 -25.49 2.90E-08 L. pneumophila 225 1448 89 4.80E-07 -5.88 6.40E-07 L. pneumophila 226 1449 102 3.20E-05 -4.2 3.40E-07 L. pneumophila 227 1466 8 0.00067 -5.2 0.00027 L. pneumophila 228 1474 239 1.90E-06 -5.19 1.00E-06 L. pneumophila 229 1494 100 2.30E-05 -4.7 5.30E-07 L. pneumophila 230 1495 101 4.00E-05 -5.93 1.90E-05 L. pneumophila 231 1496 107 1.80E-05 -9.76 3.40E-07 L. pneumophila 232 1499 291 0.00025 -10.47 0.0014 L. pneumophila 233 1547 108 1.90E-05 -12.67 3.90E-06 L. pneumophila 234 1549 240 0.00014 -3.19 0.00024 L. pneumophila 187 | Page

Appendices

Spot on preparative Av. 1- Pos. Master No. gel T-test Ratio ANOVA Species 235 1557 279 6.20E-06 -23.63 3.40E-06 L. pneumophila 236 1561 269 3.10E-07 -8.59 7.80E-07 L. pneumophila 237 1576 244 0.00019 -2.26 1.20E-05 L. pneumophila 238 1582 109 0.00061 -3.25 0.00017 L. pneumophila 239 1583 246 1.30E-05 -5.67 6.40E-07 L. pneumophila 240 1584 246 1.20E-05 -6.36 7.30E-07 L. pneumophila 241 1601 49 0.00096 -6.47 0.00043 L. pneumophila 242 1618 141 1.60E-06 -11.08 1.10E-05 L. pneumophila 243 1625 110 9.80E-06 -29.9 7.60E-07 L. pneumophila 244 1630 278 0.00052 -7.1 0.00036 L. pneumophila 245 1632 278 4.80E-05 -6.99 6.90E-06 L. pneumophila 246 1635 55 0.00064 -18.92 9.20E-05 L. pneumophila 247 1636 111 0.00026 -15.76 0.00026 L. pneumophila 248 1637 50 0.00064 -6.05 0.00022 L. pneumophila 249 1640 281 0.00016 -3.1 3.90E-05 L. pneumophila 250 1648 142 8.30E-07 -25.8 2.10E-08 L. pneumophila 251 1650 245 0.00012 -5.08 2.20E-05 L. pneumophila 252 1657 - 1.70E-05 -19.21 1.20E-05 L. pneumophila 253 1662 57 3.80E-07 -7.57 2.10E-09 L. pneumophila 254 1690 148 0.00011 -5.8 5.40E-05 L. pneumophila 255 1698 146 0.0009 -4.94 0.00027 L. pneumophila 256 1704 52 4.60E-05 -10.89 1.20E-05 L. pneumophila 257 1756 104 2.10E-05 -3.66 7.00E-07 L. pneumophila 258 1770 160 1.10E-07 -15.67 9.60E-10 L. pneumophila 259 1774 61 6.50E-05 -19.11 2.30E-05 L. pneumophila 260 1785 299 4.70E-05 -14.82 2.50E-06 L. pneumophila 261 1802 64 0.00016 -6.53 3.90E-05 L. pneumophila 262 1803 63 3.20E-05 -12.79 2.50E-06 L. pneumophila 263 1843 248 3.00E-07 -18.5 2.10E-08 L. pneumophila 264 1856 260 0.00072 -1.84 0.002 L. pneumophila 265 1860 156 0.00018 -4.01 1.40E-05 L. pneumophila 266 1911 300 0.00017 -24.78 4.60E-05 L. pneumophila 267 1937 261 7.00E-05 -11.06 2.70E-06 L. pneumophila 268 2002 262 0.00012 -6.21 0.0012 L. pneumophila 269 2047 263 7.80E-07 -34.22 2.30E-06 L. pneumophila 270 1778 65 0.00028 -2.57 1.10E-05 L. pneumophila

188 | Page

Appendices

Appendix 10

Ethics approval

To: Associate Professor Francois Malherbe/Mr Kaylass Poorun, FLSS

Dear Francois and Kaylass

SUHREC Project 2012/040 Immunoproteomic identification of biomarkers for diagnosis of Legionellosis [1] Associate Prof Francois Malherbe, FLSS; Mr Kaylass Poorun, Associate Professor Enzo Palombo, Professor Linda Blackall Approved Duration: 05/09/2012 To 31/08/2013 [Adjusted]

I refer to your request to reactivate the above project given a new New South Wales context for the project and in light of similar research covered in approved SUHREC Project 2012/156. The request, as per the cover letter and protocol emailed on 27 August 2012, was put to the Acting Chair of SUHREC for expedited review. The Acting Chair took particular note of the case put forward concerning the use of already de-identified samples and that the methods of analysis and intended outputs as given will not lead to identification of individuals. I am pleased to advise that, as submitted to date, the project has approval to proceed in line with standard on-going ethics clearance conditions here outlined (as applicable). - All human research activity undertaken under Swinburne auspices must conform to Swinburne and external regulatory standards, including the National Statement on Ethical Conduct in Human Research and with respect to secure data use, retention and disposal. - The named Swinburne Chief Investigator/Supervisor remains responsible for any personnel appointed to or associated with the project being made aware of ethics clearance conditions, including research and consent procedures or instruments approved. Any change in chief investigator/supervisor requires timely notification and SUHREC endorsement. - The above project has been approved as submitted for ethical review by or on behalf of SUHREC. Amendments to approved procedures or instruments ordinarily require prior ethical appraisal/ clearance. SUHREC must be notified immediately or as soon as possible thereafter of (a) any serious or unexpected adverse effects on participants and any redress measures; (b) proposed changes in protocols; and (c) unforeseen events which might affect continued ethical acceptability of the project. - At a minimum, an annual report on the progress of the project is required as well as at the conclusion (or abandonment) of the project. - A duly authorised external or internal audit of the project may be undertaken at any time. Please contact me the Research Office you have any queries about the ethical review process, citing the SUHREC project number. Copies of clearance emails should be retained as part of project record- keeping.

Best wishes for the project.

Yours sincerely

Keith ------Keith Wilkins Secretary, SUHREC & Research Ethics Officer Swinburne Research (H68) Swinburne University of Technology P O Box 218 HAWTHORN VIC 3122 Tel +61 3 9214 5218 Fax +61 3 9214 5267 189 | Page

Conferences

CONFERENCES

Poster Presentations

A proteomic approach for identification of biomarkers for legionellosis, The Australian Society for Microbiology Annual Scientific Meeting (ASM) 2012, Brisbane Convention and Exhibition Centre, Brisbane, Australia (1 4July 2012)

Immunoproteomic identification of biomarkers for diagnosis of legionellosis, The 8th International conference on LEGIONELLA 2013, Melbourne Convention and Exhibition Centre, Melbourne, Australia (29 October 1 November), 2013

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