Molecular Interactions between phage and the catfish pathogen Edwardsiella ictaluri and Comparative Genomics of Epidemic strains of Aeromonas hydrophila by Mohammad Jahangir Hossain A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama December 8, 2012 Keywords: Bacteriophages, Edwardsiella ictaluri, Epidemic Outbreak, Aeromonas hydrophila, Comparative Genomics, Channel Catfish Copyright 2012 by Mohammad Jahangir Hossain Approved by Mark R. Liles, Chair, Associate Professor of Biological Sciences Jeffery S. Terhune, Associate Professor of Fisheries and Allied Aquaculture Sharon R. Roberts, Associate Professor of Biological Sciences John F. Murphy, Professor of Plant Pathology Abstract Edwardsiella ictaluri causes Enteric Septicemia of Catfish (ESC) which is responsible for significant economic loss of the catfish aquaculture industry in the southeastern United States. A better diagnostic tool is required for the identification of Edw. ictaluri from ESC-affected catfish. Bacteriophages ΦeiAU and ΦeiDWF are specifically lytic to Edw. ictaluri strains and have tremendous potential to be used as diagnostic agents for the identification of Edw. ictaluri from ESC-affected catfish. However, bacteriophages ΦeiAU and ΦeiDWF demonstrate varying degree of lytic activity to different Edw. ictaluri strains. To identify the basis for variation and understand the mechanisms of phage-host interactions, phage resistant Edw. ictaluri mutants were generated by transposon mutagenesis of wild type Edw. ictaluri strains. Characterization of phage resistant Edw. ictaluri mutants revealed different host factors including integral membrane proteins, molecular chaperon, protein involved in LPS biosynthesis, proteins with proteolytic and regulatory activities, and proteins of unknown functions contribute to phage infection. Since outer membrane porin protein LC (OmpLC) is predictably located on the outermost layer of bacterial cells and deletion of the ompLC from Edw. ictaluri abolished phage infectivity, we investigated the role of OmpLC in varying degree of phage susceptibility. Phage binding assay with Edw. ictaluri and an ompLC mutant demonstrated that OmpLC is the receptor for phage attachment and infection to Edw. ictaluri strains. The prediction of the three- dimensional structure of the OmpLC protein revealed a typical porin structure with 16 antiparallel β strands and 8 extracellular loops. Site-directed mutagenesis and deletion of loop 8 ii of the OmpLC protein demonstrated that OmpLC protein is the modulating factor for phage susceptibility nature in different Edw. ictaluri strains. Mutant phages with enhanced lytic activity to phage resistant Edw. ictaluri strains were isolated by serial passaging and phage factors involved in broader host range were investigated. Pairwise comparison of the whole genome sequences of wild type and mutant phages with enhanced host lytic activity revealed that mutant phages accumulated several point mutations in six different open reading frames (ORFs). Recombineering experiment carried out with genomic DNA of wild type phage and PCR amplicons of mutated ORFs from mutant phages followed by the generation of recombinant phages demonstrated that phage host specificity protein (HspP) is responsible for the broader host susceptibility to Edw. ictaluri strain. This study highlights the importance of both the OmpLC protein of Edw. ictaluri and the ΦeiDWF phage protein HspP in defining the nature of phage susceptibility in different Edw. ictaluri strains. Generation of recombinant phages through directed mutagenesis of HspP protein will provide bacteriophages with enhanced infectivity for phage-resistant Edw. ictaluri strains and these bacteriophages with broader lytic activity could be used as diagnostic agents to identify Edw. ictaluri from ESC-affected catfish. In addition to ESC, the recent epidemic outbreak of motile Aeromonas septicemia (MAS) of catfish caused by a highly virulent and emerging strain of Aeromonas hydrophila is a major threat to the catfish industry in the southeastern United States. The lack of complete genome sequence of epidemic A. hydrophila hampered efforts to understand the highly virulent nature of this emerging pathogen. To investigate the pathogenic nature of these epidemic strains, a total of 12 A. hydrophila isolates including six recent epidemic isolates and six reference isolates were sequenced at >160-fold coverage. Genome-wide comparisons were carried out to identify iii epidemic-associated genomic regions exclusively present within the epidemic isolates. Comparative genomics revealed that the epidemic A. hydrophila isolates are highly clonal whereas reference isolates are greatly diverse. These epidemic isolates share 54 unique genetic regions comprising 326 kb with 307 predicted genes that putatively encode prophage elements, virulence factors, and gene clusters predicted to be involved in pilus biogenesis and myo-inositol utilization, along with many predicted genes of unknown functions. Five different novel O- antigen biosynthesis gene clusters were identified within the genome of sequenced A. hydrophila isolates, and all epidemic strains shared a single type of O-antigen biosynthesis gene cluster. A large percentage of the epidemic-associated unique genomic regions were present within genomic islands which was suggestive of possible events of lateral genes transfer for the acquisition of those unique regions. An in vitro growth assay demonstrated that epidemic A. hydrophila isolates were able to use myo-inositol as a sole carbon source and that this phenotypic property could be used to test field isolates for detection of epidemic A. hydrophila strains. Our comparative genomic study revealed new insights into the evolutionary changes that have occurred in A. hydrophila strains associated with epidemic outbreaks in channel catfish and provides a foundation for studying the specific molecular determinants of virulence in this emerging pathogen. iv Acknowledgments I thank Dr. Mark R. Liles for the opportunity to conduct research under his guidance. His constant support and contagious enthusiasm helped me greatly to finish my dissertation work and for which I am greatly indebted to him. I am very thankful to my co-mentor Dr. Jeffery S. Terhune for his constant support and guidance to conduct my dissertation research. I am indebted to my advisory committee member Dr. John F. Murphy, who always has highly positive thoughts about me, for his valuable advice and help in my graduate study. I am very thankful to my advisory committee member Dr. Sharon R. Roberts for her thoughtful advice and support. I am very grateful to Dr. Covadonga R. Arias for serving as University Reader. I am thankful to Dr. Paul Cobine and Dr. Sang-Jin Suh for stimulating discussions during my graduate study. I would like to thank my colleagues in Dr. Liles lab including Nancy Capps, Ran Chao, Malachi Williams, Dr. Abel Carrias, Dr. Larissa Parsley, Dr. Kavida Kakirde, Dr. Molli Newman, Erin Consuegra, Ann Marie Goode, Joshua Stough, Lauren Ulrich, Paul Bergen, Charles Thurlow, Jinglie Zhou, Alysa Moore, Erin Schmale for their constant support and encouragement. I am highly pleased and very thankful to my friends including Lipon, Ovee, Mary Sandage, Kiril, Jessica Scoffield, Paul, Hayaa, Katherine Vest and Dennis Donegan who made my graduate school experience pleasant. I am very thankful and indebted to my mother-in-law Mahmuda Akhter for helping my family during the critical moments of my life. I am also very thankful to my loving brothers Jahid and Jamil, and sister Jasmin for their inspiration and encouragement. I thank my parents Mohammad Altaf Hossain and Jahanara Nargis for their unconditional and constant supports for carrying my dissertation research abroad. Without their support and encouragement it could have been difficult to cross this long path. I would like to express my sincere love and utmost gratitude to my lovely wife Shamima Nasrin and my beloved daughter Sarina Naureen. They made great personal sacrifices, supported and inspired me to push beyond boundaries that would have been difficult to cross without their great help and sacrifices. I would like to dedicate this dissertation to my father-in-law Abdus Sukur Sikder who I wish were with us to be able to share with what I have achieved in my life. v Table of Contents Abstract ......................................................................................................................................... ii Acknowledgments ........................................................................................................................ v List of Tables ............................................................................................................................... ix List of Figures .............................................................................................................................. xi Chapter I: Introduction and Literature Review ........................................................................... 1 1.1 Introduction ................................................................................................................. 1 1.2 Objective of the Study ................................................................................................ 4 2. Literature Review .........................................................................................................