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ROLE of in VIVO-INDUCED GENES ILVI and HFQ in the SURVIVAL and VIRULENCE of ACTINOBACILLUS PLEUROPNEUMONIAE By

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ROLE of in VIVO-INDUCED GENES ILVI and HFQ in the SURVIVAL and VIRULENCE of ACTINOBACILLUS PLEUROPNEUMONIAE By ROLE OF IN VIVO-INDUCED GENES ILVI AND HFQ IN THE SURVIVAL AND VIRULENCE OF ACTINOBACILLUS PLEUROPNEUMONIAE By Sargurunathan Subashchandrabose A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Comparative Medicine and Integrative Biology 2011 ABSTRACT ROLE OF IN VIVO-INDUCED GENES ILVI AND HFQ IN THE SURVIVAL AND VIRULENCE OF ACTINOBACILLUS PLEUROPNEUMONIAE By Sargurunathan Subashchandrabose Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a contagious and often fatal disease of pigs. Infection with this bacterium leads to the development of a fulminant pleuropneumonia resulting in severe damage to the lungs. A. pleuropneumoniae genes that are up-regulated during the infection of pig lungs were previously identified in our laboratory and designated as in vivo-induced genes. The role of two such in vivo-induced genes, ilvI and hfq, in the pathobiology of A. pleuropneumoniae is the subject of this dissertation. The gene ilvI encodes an enzyme involved in the biosynthesis of branched-chain amino acids (BCAAs). The leucine-responsive regulatory protein (Lrp) is a transcriptional regulator of the ilvIH operon. BCAA biosynthetic genes are associated with virulence in pathogens infecting the respiratory tract and blood stream. Also, twenty five percent of the A. pleuropneumoniae in vivo-induced genes were up-regulated under BCAA limitation, suggesting that these BCAAs may be found at limiting concentrations in certain sites of the mammalian body such as the respiratory tract. The concentration of amino acids in the porcine pulmonary epithelial lining fluid was determined and BCAAs were found at limiting concentration in the respiratory tract. Further, the virulence of two BCAA auxotrophs, an ilvI mutant and an lrp mutant, was tested in a pig infection model and both were found to be attenuated. Finally, inhibitors of BCAA biosynthesis were found to prevent the growth of A. pleuropneumoniae. Hfq, encoding the Host factor Q-beta (Hfq) in A. pleuropneumoniae, was identified as an in vivo-induced gene and is also up-regulated under BCAA limitation. Since Hfq is a global regulator which is induced under one of the signals found in the porcine respiratory tract, the role of Hfq in A. pleuropneumoniae was analyzed. An A. pleuropneumoniae hfq mutant strain failed to form biofilm. Levels of the pgaC transcript, encoding the biofilm matrix biosynthetic enzyme, were ~14-fold lower in the hfq mutant compared to wild-type strain. The hfq mutant displayed enhanced sensitivity to superoxide stress and tellurite. Hfq was found to regulate two virulence- associated phenotypes, biofilm formation and resistance to oxidative stress, in A. pleuropneumoniae. As Hfq was associated with at least two virulence-associated phenotypes, the effect of Hfq on the virulence of A. pleuropneumoniae was tested in a pig infection model. Infection with the hfq mutant did not result in the development of pleuropneumonia while pigs infected with the wild-type strain exhibited classic signs of pleuropneumonia. Competitive index analysis revealed that the hfq mutant is severely attenuated, compared to the wild-type strain. In summary, studies described in this dissertation have uncovered a new host signal found in the porcine respiratory tract and provide evidence for the potential of inhibitors of BCAA biosynthesis as antibacterial agents. Hfq, regulated by BCAA limitation, was found to regulate biofilm formation, resistance to oxidative stress and virulence in A. pleuropneumoniae. The role of Hfq in the virulence of a bacterial pathogen during infection of lungs has been examined in the studies described in this dissertation. The hfq mutant is highly attenuated and is a potential candidate for the development of a live-attenuated vaccine against porcine pleuropneumonia. ACKNOWLEDGEMENTS I thank my mentor Dr. Martha Mulks for her inspiration, support and guidance throughout my program and for critical reading of this dissertation. Her infectious enthusiasm for science helped me overcome some of the frustrations invariably associated with research. I thank Drs. Cindy Arvidson, Matti Kiupel and Robert Britton for serving on my guidance committee and for their constructive criticism about my research. A special thanks to Dr. Matti Kiupel for teaching me the fundamental principles of necropsy. Dr. Roy Kirkwood deserves a special mention for training me in several techniques used in animal experiments. I wish to recognize the past and present members of Dr. Mulks laboratory who have made it an exciting and fun place to work. I thank Rhiannon LeVeque for teaching me several techniques during rotation and help with major experiments. I thank the Comparative Medicine and Integrative Biology program for providing me a great opportunity to pursue graduate studies and help fulfill my goal to get research experience in microbial pathogenesis. Dr. Vilma Yuzbasiyan-Gurkan’s constant encouragement and unwavering support has definitely made a difference in my experience through graduate school. Further, I would like to thank the College of Veterinary Medicine for providing summer support fellowships. I thank Drs. Robert Hausinger and Walter Esselman in the Department of Microbiology and Molecular Genetics of providing me with teaching opportunity in the Biological Sciences program and in the Microbial Genomics laboratory course. I thank Dr. Rudolph Hugh for his philanthropy in creating the Rudolph Hugh fellowship which has helped me present my work at international meetings. iv I will make use of this opportunity to thank all my teachers who have inspired me, from kindergarten to graduate school. I thank Drs. Sheba and MohanKumar for introducing me to the unique graduate programs offered by the College of Veterinary Medicine at Michigan State University. Last but definitely not the least I thank my family and friends being in my life. It is because of my parents love, support and encouragement that I am able to pursue my goals. Haritha, my wife, has been an angel in my life making difference every day. I just want to mention that I love my family. My friends have always been amazing and I thank them all for just being in my life. v PREFACE Three chapters presenting original research, formatted as manuscripts, are included in this dissertation. Chapter 2, entitled ―Branched-chain amino acids are required for the survival and virulence of Actinobacillus pleuropneumoniae in swine‖, has been published in the journal Infection and Immunity with Ms. Rhiannon LeVeque and Drs. Trevor Wagner, Roy Kirkwood, Matti Kiupel and Martha Mulks as co-authors (Volume 77 (11), Pages 4929-4933, 2009). Contribution of co-authors: Ms. Rhiannon LeVeque and Dr. Trevor Wagner constructed the ilvI and lrp mutants, respectively; Rhiannon LeVeque performed the Q-PCR experiment and helped with animal infection study; Dr. Roy Kirkwood provided veterinary care for the animals; Dr. Matti Kiupel conducted necropsy of pigs and interpreted histopathology results. Chapters 3 and 4 of this dissertation will be combined into one manuscript and submitted to the journal Infection and Immunity with Drs. Roy Kirkwood, Matti Kiupel and Martha Mulks as co-authors. Contribution of co-authors: Dr. Roy Kirkwood provided veterinary care for the animals; Dr. Matti Kiupel conducted necropsy and interpreted histopathology results. vi TABLE OF CONTENTS LIST OF TABLES .......................................................................................................................ix LIST OF FIGURES .....................................................................................................................x CHAPTER I AN INTRODUCTION TO ACTINOBACILLUS PLEUROPNEUMONIAE AND PORCINE PLEUROPNEUMONIA ..............................................................................................................01 Introduction ......................................................................................................................02 Porcine pleuropneumonia ................................................................................................03 Pathological changes in porcine pleuropneumonia ..........................................................04 Virulence factors ..............................................................................................................05 Fimbriae ...........................................................................................................................05 Biofilm .............................................................................................................................06 Iron acquisition ................................................................................................................09 Capsule .............................................................................................................................11 Lipopolysaccharide ..........................................................................................................12 Apx toxins ........................................................................................................................14 Resistance to oxidative stress...........................................................................................15 Functional genomics studies ............................................................................................17 In vivo expression technology .........................................................................................17
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