CONCERNING Brucella LPS: GENETIC ANALYSIS AND ROLE IN HOST- AGENT INTERACTION A Dissertation by JOSHUA EDWARD TURSE Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2005 Major Subject: Genetics CONCERNING Brucella LPS: GENETIC ANALYSIS AND ROLE IN HOST- AGENT INTERACTION A Dissertation by JOSHUA EDWARD TURSE Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Thomas A. Ficht Committee Members, James Samuel Jon Skare Renée Tsolis Chair of Genetics Faculty, Geoffrey Kapler August 2005 Major Subject: Genetics iii ABSTRACT Concerning Brucella LPS: Genetic Analysis and Role in Host-Agent Interaction. (August 2005) Joshua Edward Turse, B.S., Bates College Chair of Advisory Committee: Dr. Thomas A. Ficht Brucella lipopolysaccharide is an important component of virulence in brucellosis. Recent research in macrophage models has shown that Brucella LPS does not behave like classical LPS by stimulating potent inflammatory responses. The central hypothesis of this work is that O-antigen is dynamic signaling molecular and participates in complex interactions with the host to promote productive infection. A corollary to this is that the host environment is dynamic, and Brucella has evolved mechanisms to cope with changing environments. In an effort to understand the contribution of Brucella LPS to virulence and pathogenesis, the function of a metabolic locus important in the synthesis of LPS has been demonstrated and complemented. The spontaneous loss of LPS expression has been characterized. Contribution of LPS to acquisition of the host environment in tissue culture and mouse models has been explored. This work demonstrated that genes outside the O-antigen biosynthesis (manBA) cluster contribute to LPS biosynthesis. Further high iv frequency mutation involving manBA is partly responsible for observed dissociation of Brucella strains. Finally, work herein attempts to look at the role of LPS in acquisition of the host environment and shows that LPS is important for recruiting particular cell populations within a host model of brucellosis. v DEDICATION …to my biggest fans, my family, …my wife, Carol …Mom & Dad, Jim & Cindy …my brother, Jason …my in-laws, Walter & Vivian …my brother in-law, Gene …and yes, even my dog, Shawna vi ACKNOWLEDGEMENTS I sincerely thank my committee members Dr. Thomas Ficht, Dr. James Samuel, Dr. Jon Skare, and Dr. Renee Tsolis for their guidance and support over the years. Dr. Ficht has been especially patient and understanding as I have tackled the bumpy road that is graduate school. I also thank my labmates, Priscilla, Melissa, Carol, Jenni, Dr. Pei, Dr. Wu, for endless conversations, collaborations, and support. Finally, I thank my wife for her endless understanding of my consuming insanity. vii TABLE OF CONTENTS Page ABSTRACT .........................................................................................................iii DEDICATION .......................................................................................................v ACKNOWLEDGEMENTS....................................................................................vi TABLE OF CONTENTS .....................................................................................vii LIST OF TABLES .................................................................................................x LIST OF FIGURES..............................................................................................xi INTRODUCTION AND LITERATURE REVIEW .................................................. 1 BACKGROUND.......................................................................................... 1 Taxonomy. .......................................................................................... 1 Molecular biology. ............................................................................... 2 The disease......................................................................................... 2 Pathogenesis. ..................................................................................... 2 Threat assessment.............................................................................. 3 Brucella virulence factors. ................................................................... 4 Genetics of Brucella O-antigen synthesis............................................ 6 O-antigen dissociation......................................................................... 8 SPECIFIC AIMS ................................................................................................ 15 ManBA (BMEII0899-0900) IS ESSENTIAL FOR LPS BIOSYNTHESIS............ 16 INTRODUCTION...................................................................................... 16 MATERIALS AND METHODS.................................................................. 19 Bacteria, plasmids, growth conditions and restriction endonucleases.................................................................................. 19 Molecular biology, polymerase chain reaction, and primers.............. 21 Production of hybrid PCR products. .................................................. 22 Transformation and selection of recombinant plasmids. ................... 23 Transformation and selection of Brucella mutants. ........................... 25 Transformation of Brucella strains by conjugation............................. 26 viii Page Molecular detection of LPS................................................................ 27 Phenotypic confirmation of rough mutants. ....................................... 29 Complement mediated killing. ........................................................... 30 RESULTS................................................................................................. 30 Targeting of manBA for deletion by allelic exchange......................... 30 Construction of manBA knockout vector. .......................................... 31 Complementation test confirms restoration of LPS expression in rough Brucella. .................................................................................. 36 Complement mediated killing of rough Brucella strains..................... 39 DISCUSSION ........................................................................................... 41 GENETIC CHANGES AT manBA ARE RESPONSIBLE FOR HIGH FREQUENCY LOSS OF LPS EXPRESSION.................................................... 42 INTRODUCTION...................................................................................... 42 MATERIALS AND METHODS.................................................................. 44 Molecular biology, polymerase chain reaction, and primers.............. 44 Bacteria, plasmids, growth conditions and restriction endonucleases.................................................................................. 44 Rough mutant isolation from smooth colonies................................... 45 Determination of the background mutation rate in Brucella: Antibiotic resistance. ......................................................................... 45 Determination of the background mutation rate in Brucella: Pyrimidine auxotrophy....................................................................... 46 Phenotypic confirmation of rough mutants. ....................................... 49 Genotypic tests for rough mutants..................................................... 49 Recovery of rough isolates from mice. .............................................. 49 In vitro competitive growth................................................................. 50 RESULTS................................................................................................. 51 Dissociation rate determination. ........................................................ 51 Mutation rate estimation.................................................................... 52 Genotypic tests for rough mutants..................................................... 55 Rough B. melitensis grow faster than smooth strains. ...................... 58 Creation of non-dissociative Brucella strains.................................... 58 DISCUSSION ........................................................................................... 61 ix Page LPS PROMOTES ‘PROPER’ INNATE IMMUNE RESPONSE .......................... 68 INTRODUCTION...................................................................................... 68 MATERIALS AND METHODS.................................................................. 70 Bacteria, plasmids, growth conditions and restriction endonucleases.................................................................................. 70 Macrophage infection........................................................................ 70 LDH assay to measure cell death...................................................... 71 Assay to measure release of nitric oxide........................................... 71 Murine infection assay (Long-term)................................................... 72 Murine infection assay (Short-term). ................................................. 72 Cytokine analysis. ............................................................................. 73 RESULTS................................................................................................. 73 Evaluation of infection by rough