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Analysis of Genes Encoding Outer Membrane Protein Of Analysis of Genes Encoding Outer Membrane Protein of Leptospira interrogans and Its Modulation Due to Host Factors: An Approach for Understanding Host-Pathogen Crosstalk A thesis Submitted in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY by KARUKRITI KAUSHIK GHOSH Under the supervision of Dr. Manish Kumar Department of Biosciences and Bioengineering Indian Institute of Technology Guwahati Guwahati-781039, Assam, India. February, 2019 TH-2331_136106014 TH-2331_136106014 Analysis of Genes Encoding Outer Membrane Protein of Leptospira interrogans and Its Modulation Due to Host Factors: An Approach for Understanding Host-Pathogen Crosstalk by Karukriti Kaushik Ghosh IIT Guwahati, 2019 Doctoral Committee Dr. Manish Kumar (Department of Biosciences and Bioengineering) Supervisor Dr. Anil Mukund Limaye (Department of Biosciences and Bioengineering) Chairperson Dr. Sachin Kumar (Department of Biosciences and Bioengineering) Member Dr. Debasis Manna (Department of Chemistry) Member TH-2331_136106014 TH-2331_136106014 DEDICATION I dedicate this work to my grandparents and parents for their selfless sacrifices and belief in my abilities. They are my inspiration and pillars of strength. TH-2331_136106014 TH-2331_136106014 DECLARATION I hereby declare that the matter embodied in this thesis entitled “Analysis of Genes Encoding Outer Membrane Protein of Leptospira interrogans and Its Modulation Due to Host Factors: An Approach for Understanding Host-Pathogen Crosstalk” is the result of investigations carried out by me in the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam, India under the supervision of Dr. Manish Kumar. In keeping with the general practice of reporting scientific observations, due acknowledgments have been made wherever the work of other investigators are referred. Date: 19.06.19 Karukriti Kaushik Ghosh Roll no. 136106014 TH-2331_136106014 TH-2331_136106014 CERTIFICATE This is to certify that work described in the thesis entitled “Analysis of Genes Encoding Outer Membrane Protein of Leptospira interrogans and Its Modulation Due to Host Factors: An Approach for Understanding Host-Pathogen Crosstalk” by Ms. Karukriti Kaushik Ghosh (Roll No: 136106014), submitted to the Indian Institute of Technology Guwahati, India for the award of the degree of Doctor of Philosophy, is an authentic record of the results obtained from the research work carried out under my supervision at the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, India and this work has not been submitted elsewhere for a degree. Dr. Manish Kumar (Thesis supervisor) TH-2331_136106014 TH-2331_136106014 ACKNOWLEDGMENTS I would like to express my heartfelt gratitude and sincere regards to Dr. Manish Kumar, for providing me the opportunity to work in his laboratory. I feel privileged to be the first graduate student of his lab and will cherish the memories of working with him in the lab during the initial years of my research. I would also like to thank him for his valuable guidance, suggestions and his patience throughout the duration of this study. His organized way of working has always inspired me and I hope to look upon him as a guide throughout my life. I would like to convey my sincere thanks to the esteemed members of my doctoral committee, Dr. Anil Mukund Limaye, Dr. Sachin Kumar, Dr. Debasis Manna, and Prof. Vikash Kumar Dubey, for their valuable inputs and timely suggestions during the course of this study. I would like to thank Dr. Vinayagamurthy Balamurugan for providing us with the precious serum samples which helped us a lot in our studies. My thanks are also due to RMRC (ICMR), Port Blair for providing us with the Leptospira cultures to pursue our studies. I am indebted to Dr. Rajeev Kumar Sharma and the Department of Microbiology, College of Veterinary Science, Guwahati for their help in the generation of the polyclonal antibodies. I would also like to thank Dr. David Haake (Division of Infectious Diseases, Los Angeles, California, USA) for his generous gift of the antibodies used in this study. I also acknowledge the DBT program support facility available in the Department of Biosciences and Bioengineering, IIT Guwahati, for generating the Circular dichroism data. TH-2331_136106014 Acknowledgements I owe my heartfelt gratitude to Prof. Latha Rangan, Head of the Department and former Heads of the Department, Prof. K. Pakshirajan and Prof. V. V. Dasu, for providing me the departmental facilities to carry out my research work. My thanks are also due to the technical and non-technical staffs of my department for their warm and generous behavior towards me throughout my study tenure. Among the many people to whom I owe my gratitude, I would like to record my heartfelt thanks to Aman Prakash for his helping hand during my research work and thesis writing. I also record my sincere thanks to Prateek Shrivastav, Gary Fernandes, Saurabh Bora, Rishabh Singh, Bhuvan Dixit, Vineet Anand, Anusua Dhara, Md. Saddam Hussain, Juhi Rajesh Rathod, and all the past and present lab members for their constant help and support during the entire research period. I am also thankful to my friends Reshmi Das, Neha Arora, Amrendra Kumar, Rakesh Kumar, Ashish Prabhu A. and Ankit Gangrade for making the past five years of stay a pleasant and joyful experience. I shall always be indebted to my family members for their love, continuous support and innumerable sacrifices. I would like to thank my parents for their constant motivation. I would also like to express my gratitude to my grandparents, whom I lost during the course of my study, for believing in me from childhood. Last but not the least; I am grateful to the Almighty, who gave me the courage and support to complete this study. Karukriti Kaushik Ghosh TH-2331_136106014 TABLE OF CONTENTS CHAPTER 1 Introduction and literature review ............................................................... 1 1.1 Historical aspects........................................................................................................ 3 1.2 Leptospirosis .............................................................................................................. 3 1.2.1 Epidemiology ...................................................................................................... 4 1.2.2 Modes of transmission of Leptospira ................................................................. 6 1.2.3 Pathogenesis of Leptospira ................................................................................. 8 1.2.4 Host factors implicated in the pathogenesis of the bacteria ................................ 9 1.2.5 Clinical manifestations...................................................................................... 19 1.2.6 Laboratory diagnosis of leptospirosis ............................................................... 20 1.2.7 Treatment .......................................................................................................... 24 1.2.8 Prevention ......................................................................................................... 24 1.2.9 Vaccines against leptospirosis .......................................................................... 25 1.3 Leptospira, the etiological agent of leptospirosis .................................................... 27 1.3.1 Taxonomy and Classification ........................................................................... 27 1.3.2 Nutritional requirements, growth, and cultivation of Leptospira ..................... 29 1.3.3 Cell Biology ...................................................................................................... 30 1.3.4 Genome of Leptospira ...................................................................................... 30 1.3.5 Gene content in Leptospira ............................................................................... 31 1.3.6 Outer membrane proteins (OMPs) of Leptospira ............................................. 32 (I) Transmembrane proteins ............................................................................................ 33 (II) Lipoproteins .............................................................................................................. 33 (III) Peripheral membrane proteins ................................................................................. 35 1.3.7 Hypothetical membrane proteins ...................................................................... 36 1.3.8 Role of OMPs in the pathogenesis .................................................................... 37 1.3.9 Role of OMPs in diagnosis ............................................................................... 37 i TH-2331_136106014 Table of Contents CHAPTER 2 In silico based selection of genes of L. interrogans encoding hypothetical membrane proteins and extracellular proteins .................................................................. 41 2.1 Abstract .................................................................................................................... 41 2.2 Introduction .............................................................................................................. 42 2.3 Materials and Methods ............................................................................................. 44 2.3.1 Leptospira nucleic acid and protein sequence retrieval .................................... 44 2.3.2 Identification of hypothetical proteins of L. interrogans
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