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Early Stages of Host Invasion by Pseudomonas Aeruginosa and Effect of Cyclic Diguanylate Signaling

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Early Stages of Host Invasion by Pseudomonas Aeruginosa and Effect of Cyclic Diguanylate Signaling EARLY STAGES OF HOST INVASION BY PSEUDOMONAS AERUGINOSA AND EFFECT OF CYCLIC DIGUANYLATE SIGNALING AYSHWARYA RAVICHANDRAN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I express my heartfelt gratitude to my supervisor, A/P Sanjay Swarup for his constant guidance and supervision throughout the period of this project. I sincerely thank National University of Singapore for providing me with Research Scholarship to complete this project. I would also like to thank Research Centre for Excellence in Mechanobiology for funding part of this study and support. I am extremely thankful to Dr. Yasushi Ishihama , Keio University, Japan for performing phoshoproteome analysis on our samples without which my publication would not have been possible. Helium-ion imaging was conducted under Dr. Daniel Pickard and I am thankful for his guidance and facility. I extend my sincere gratitude to Dr. Gerard Michel , Centre National de la Recherche Scientifique , France for his kind gesture of sending antibodies and guidance in P. aeruginosa type II secretion system- related experiments. I would also like to thank Dr. Zhang Lian-Hui for providing workspace in his laboratory during the initial stages of this project and Dr. Ganesh Anand for his valuable scientific discussions time-to-time. I express my thanks to Malarmathy Ramachandran and Karen Lam who have been very instrumental in helping me with optimization of experimental methods used in this study. I express my gratitude to Protein and Proteomics centre for their mass spectrometry services, Electron microscopy and the confocal microscopy facilities at the Faculty of Medicine, and the Electron microscopy facility at Department of Biological Sciences. In this regard, I thank Ms.Michelle Mok, Ms. Wang Xianhui and Mdm. Loy Gek Luan . My thanks are due to our lab officers Ms. LiewChye Fong , Dennis Heng and Jiun Fu . I extend my gratitude to all my lab mates especially Chui Ching, Weiling and Tanujaa for their cooperation, help and constant support. I would also like to thank all theother undergraduates and attachment students who have in one way or other helped this project. I am lucky to have great friends at NUS especially Sheela, Gauri, Sravanthy, Karthik, and Prasanna for their criticism, discussions and moral support. I have been blessed with wonderful family that lives across the globe, a constant source of encouragement and love; especially my parents Dr. Ravichandran and Dr. Rajarajeswari , who inspired me to take up research. A special mention goes to Mrs. Chandrika and Mr. Nagarajan , my guardians in Singapore. Last but not least, my husband Mr. Vigneshwaran and parents-in-law have always been greatly supportive of my career endeavors. I have no words to thank these people, without whom I could not have endured this tough journey. CONTENTS ACKNOWLEDGEMENTS i SUMMARY vii ABBREVIATIONS ix LIST OF TABLES xii LIST OF FIGURES xiii PUBLICATIONS xv CHAPTER 1 INTRODUCTION 1.1 General Introduction 1 1.2 Objectives 3 CHAPTER 2 REVIEW OF LITERATURE 2.1 Bacterial invasion and infection mechanisms 5 2.2 Pseudomonas aeruginosa - an opportunistic pathogen 8 2.2.1 Chronic vs acute infection 9 2.3 Multifactorial nature of P. aeruginosa virulence mechanisms 10 2.4 Host surface-attachment, a key step in P. aeruginosa 12 invasion 2.5 Role of bacterial appendages in surface attachment 15 2.5.1. Flagellum- a primary adhesin 15 2.5.2. Type IV pili-mediated attachment 17 2.6 P. aeruginosa internalization by non-phagocytic cells 19 2.6.1. Host signaling pathways necessary for P. aeruginosa invasion 20 2.7 Role of secretion systems in bacterial invasion 23 ii 2.7.1. Type II secretion system (T2SS) in P. aeruginosa 25 2.8 Co-ordinated regulation of P. aeruginosa virulence mechanisms 29 2.9 C-di-GMP signaling 32 2.9.1. Role of c-di-GMP signaling in virulence regulation 34 2.9.2. MorA signaling 36 2.10 Bacterial Ser/Thr/Tyr phosphorylation system 38 CHAPTER 3 MATERIALS AND METHODS 3.1 Bacterial strains, plasmids and growth conditions 41 3.2 Gene expression studies 42 3.3 Cloning and genetic manipulation studies 43 3.4 Expression of recombinant proteins 45 3.5 Secretome analysis 46 3.5.1. Elastolytic activity assay 48 3.6 Intracellular protein extraction 49 3.7 Membrane protein preparation 49 3.8 Immunoblotting 50 3.9 Bacterial infection studies 51 3.9.1. Cell culture conditions 51 3.9.2. Infection assays 52 3.10 Extracellular matrix extraction 54 3.11 Sample preparation for Helium-ion microscopy 55 3.12 Phosphoproteome analysis 56 iii 3.12 .1. 2-Dimensional Electrophoresis (2-DE) of P. putida 56 protein samples 3.12 .2. Staining for phosphoproteins 61 3.12 .3. Sample preparation for phosphoproteome analysis by Nano-LC-MS-MS 62 3.12 .4. Analysis of LC-MS-MS data 64 CHAPTER 4 CYCLIC DIGUANYLATE SIGNALING AFFECTS P. AERUGINOSA ATTACHMENT AND ENTRY INTO LUNG FIBROBLASTS 4.1 BACKGROUND 66 4.2 RESULTS AND DISCUSSION 4.2.1. MorA affects bacterial attachment to host in P. aeruginosa 67 4.2.2 . Which appendage plays a major role in attachment changes due to MorA- flagellum or pili? 72 4.2.3. Investigation of entry mechanism 74 4.3 CONCLUSIONS AND FUTURE DIRECTIONS 75 CHAPTER 5 SECRETION OF EXTRACELLULAR PROTEASES IS AFFECTED BY CYCLIC DIGUANYLATE SENSOR REGULATOR MorAIN P. AERUGINOSA 5.1 BACKGROUND 79 5.2 RESULTS AND DISCUSSION 5.2.1. C-di-GMP signaling affects T2SS secretome in P. aeruginosa 81 5.2.2. Biological effects of increased extracellular protease levels 87 iv 5.2.3. MorA affects invasion efficiency of P. aeruginosa 89 5.2.4. Mechanism of c-di-GMP regulation of P. aeruginosa protease secretion 91 i) RNA levels of protease genes 91 ii) Protein levels of protease genes 93 iii) Levels of T2SS secreton assembly proteins 95 5.2.5. Does MorA affect invasion by degrading the extracellular matrix? 96 5.3 CONCLUSIONS AND FUTURE DIRECTIONS 99 CHAPTER 6 Ser/Thr/Tyr PHOSPHOPROTEOMES OF P. PUTIDA AND P. AERUGINOSA AND THEIR CROSSTALK WITH CYCLIC DIGUANYLATE SIGNALING 6.1 BACKGROUND 104 6.2 RESULTS AND DISCUSSION 6.2.1. Gel-based approach for identification of phosphoproteins 107 6.2.2. Phosphoproteome analysis of P. putida and P. aeruginosa by Nano-LC-MS/MS method 110 6.2.3. Crosstalk of MorA-c-di-GMP signaling and protein phosphorylation 124 6.3 CONCLUSIONS AND FUTURE DIRECTIONS 127 CHAPTER 7 CONCLUDING REMARKS 131 REFERENCES 132 v APPENDICES Supplementary information on gene cloning done in this I 152 study II Methods for Ser/Thr/Tyr Phosphoproteome analysis 157 III Supplementary information on host cell morphology 161 IV Gene regulatory network of promoters affected by MorA 162 MALDI-ToF-ToF spectra of secreted proteins affected by V MorA 163 VI MorA affects timing of flagellar biogenesis in P. aeruginosa 171 VII Crosstalk of MorA and acetyl phosphate (AcP) signaling 172 vi SUMMARY Bacterial invasion plays a critical role in the establishment of P. aeruginosa infection, which involves surface attachment of bacteria on the host cells followed by internalization/ tissue penetration. Major virulence factors aiding bacterial invasion are surface appendages and secreted proteases. The second messenger cyclic diguanylate (c- di-GMP) is well known to affect attachment of bacteria to surfaces, biofilm formation and related virulence phenomena. MorA, a global regulator containing a GGDEF-EAL domain has been previously shown to affect biofilm formation and timing of flagellar biogenesis in P. aeruginosa PAO1 strain, and fimbriae expression in other clinical strains. These domains are implicated in the turnover of c-di-GMP. This study provides evidence that the global regulator MorA affects P. aeruginosa attachment to host surface and levels of proteases secreted by the type II secretion system (T2SS) hence regulating the invasion capacity of the pathogen. This is the first report on control of c-di-GMP signaling on this secretion system. It was postulated that there may be a common post-transcriptional signal acting between the regulatorMorA and the effectors i.e. T2SS and pili/flagella since all the three are located at the bacterial poles. Results confirm that the effect of MorA signaling on T2SS is post-transcriptional. Data from this study suggest that the effect of MorA on host-surface attachment may be mediated by pili, a key surface appendage. Owing to growing importance of Ser/ Thr/ Tyr protein phosphorylation in bacteria, it was hypothesized to be the common phenomenon bridging the altered c-di-GMP levels and the observed effects on protease secretion and attachment to host surface. A comprehensive phosphoproteome analysis was conducted on P. aeruginosa and P. putida vii that revealed several interesting leads suggesting many virulence and survival mechanisms to be regulated by protein phosphorylation. This analysis uncovered a novel crosstalk between two bacterial signaling paradigms namely- c-di-GMP second messenger signaling and Ser/ Thr/ Tyr protein phosphorylation. Since not many Ser/Thr/Tyr kinases have been characterized in bacteria, a direct correlation of c-di-GMP levels and alteration in protein phosphorylation patterns need further investigation. viii ABBREVIATIONS 2-DE 2-Dimensional electrophoresis ABC ATP-binding cassette AckA acetate kinase AcP acetyl phosphate Acyl-HSL acyl homoserinelactone aGM asialoganglioside gangliotetrasylceramide Amp ampicillin AP alkaline phospahatase BSA bovine serum albumin cAMP cyclic adenosine monophosphate c-di-GMP cyclic di-guanylate monophosphate CF cystic fibrosis CFTR cystic
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