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DISSERTATION THE SURFACE PROTEOME OF FRANCISELLA TULARENSIS Submitted by Jeffrey Craig Chandler Department of Microbiology, Immunology and Pathology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2011 Doctoral Committee: Advisor: John T. Belisle Robert D. Gilmore Lawrence D. Goodridge Jeannine M. Petersen i ABSTRACT THE SURFACE PROTEOME OF FRANCISELLA TULARENSIS The surface associated lipids, polysaccharides, and proteins of bacterial pathogens often have significant roles in environmental and host-pathogen interactions. Lipopolysaccharide and an O-antigen polysaccharide capsule are the best defined Francisella tularensis surface molecules, and are important virulence factors that also contribute to the phenotypic variability of Francisella species, subspecies, and populations. In contrast, little is known regarding the composition and contributions of surface proteins in the biology of Francisella, or what roles they have in the documented phenotypic variability of this genus. A sufficient understanding of the Francisella surface proteome has been hampered by the few surface proteins identified and the inherent difficulty of characterizing new surface proteins. Thus, the objective of this dissertation was to provide an enhanced definition of F. tularensis surface proteome and evaluate how surface proteins relate to aspects of F. tularensis physiology, specifically humoral immunity and phenotypic variability of subspecies and populations. Analyses of the F. tularensis live vaccine strain surface proteome resulted in the identification of 36 proteins, 28 of which were newly described to the surface of this bacterium. Bioinformatic comparisons of surface proteins to their homologs in other Francisella species, subspecies, and populations revealed numerous differences that may contribute variable phenotypes, including significant alterations in the ChiA chitinase (FTL_1521). ii Given the utility of surface proteins in vaccines effective against other bacterial pathogens, the F. tularensis surface proteins recognized by three forms of vaccination were determined in a murine model. Immune serum derived from the most effective F. tularensis vaccine, F. tularensis live vaccine strain, recognized a small set of proteins, of which the majority of antigens were surface localized. In comparison, sera collected from mice vaccinated with two less effective subunit vaccines containing F. tularensis membrane and surface proteins recognized a much greater number of antigens. Although surface proteins were also recognized in response to subunit vaccinations, the majority of antigens were not surface associated. These data suggest that a targeted humoral response to a select set of identified surface proteins offers the greatest protective effect. Finally, the research presented in this dissertation provided the first biochemical characterization of the surface localized ChiA chitinase that was predicted to contribute to the phenotypic variability of Francisella biotypes. Multiple chitinases are often produced by a single organism which synergistically depolymerize chitin in nature. Thus, biochemical evaluations were extended to other F. tularensis and F. novicida chitinases, ChiB (FTT_1768), ChiC (FTW_0313), and ChiD (FTT_0066), that were identified by in silico analyses of Francisella genomes. Differences were noted between the chitinase genes and chitinase activities of Francisella species, subspecies, and populations. The chitinase activities observed for F. tularensis strains were predominantly associated with whole cell lysates, while the chitinase activities of F. novicida localized to the culture supernatant. The overall level of chitinase activity differed among the subpopulations of F. tularensis, and between F. tularensis and F. novicida. Recombinant production of the putative chitinases and enzymatic evaluations revealed ChiA, ChiB, ChiC, and ChiD possessed dissimilar chitinase activities. These iii biochemical studies coupled with bioinformatic analyses and the evaluation of chiA and chiC knockouts in F. tularensis A1 and A2 populations, respectively, provided a molecular basis to explain the differential chitinase activities observed among the species and subpopulations of Francisella. iv ACKNOWLEDGEMENTS I would like to extend my gratitude to the members of my doctoral graduate committee, Drs. John T. Belisle, Robert D. Gilmore, Lawrence D. Goodridge, and Jeannine M. Petersen for their support and guidance. A special thank you is in order to my major professor Dr. John T. Belisle for all his help and patience, and to my friend Dr. Claudia R. Molins who contributed to all aspects of this dissertation. I would also like to acknowledge Drs. Jeannine M. Petersen and Martin E. Schriefer for allowing me the opportunity to perform much of this work at the Bacterial Diseases Branch of the Centers for Disease Control and Prevention. v TABLE OF CONTENTS Chapter I Literature Review Part I: The Genus Francisella 1.1 The history of tularemia ..................................................................................... 1 1.2 Taxonomic classification ................................................................................... 4 1.3 Epidemiology and transmission ......................................................................... 8 1.4 Disease manifestations ................................................................................... 12 1.5 Treatment ........................................................................................................ 13 1.6 Vaccines .......................................................................................................... 15 1.7 Physiology of Francisella ................................................................................. 18 1.7.1 Genomics and proteomics ...................................................................... 19 1.7.2 Lipids ...................................................................................................... 20 1.8 Virulence factors .............................................................................................. 22 1.8.1 Envelope ................................................................................................. 22 1.8.1.1 Capsule ....................................................................................... 23 1.8.1.2 Lipopolysaccharide ..................................................................... 24 1.8.1.3 Envelope proteins ....................................................................... 27 1.8.2 The Francisella pathogenicity island ...................................................... 28 1.8.3 Chaperone proteins ................................................................................ 30 1.8.4 Metabolic proteins .................................................................................. 31 1.9 Host-pathogen interactions ............................................................................. 32 1.9.1 Pathogenesis .......................................................................................... 32 1.9.2 Innate immunity ...................................................................................... 33 vi 1.9.3 Adaptive immunity .................................................................................. 35 1.10 Literature cited ................................................................................................. 37 Chapter II Literature Review Part II: The Surface Proteome of F. tularensis 2.1 Introduction ...................................................................................................... 52 2.2 Bioinformatic signatures of F. tularensis surface proteins ............................... 53 2.3 Surface proteins of F. tularensis ...................................................................... 53 2.4 Gram-negative protein secretion and the secretome of F. tularensis .............. 56 2.5 Analyses of F. tularensis membrane fractions ................................................ 63 2.6 Type IV pili of Francisella species ................................................................... 65 2.7 Possible variations in the surface proteome of Francisella biotypes ............... 66 2.8 Differential production of F. tularensis surface proteins .................................. 70 2.9 Research objectives ........................................................................................ 73 2.10 Literature cited ................................................................................................. 75 Chapter III Expanded Characterization of the F. tularensis Surface Proteome 3.1 Introduction ...................................................................................................... 82 3.2 Materials and methods .................................................................................... 84 3.2.1 Strains and culture conditions ................................................................ 84 3.2.2 Surface protein labeling and label localization ....................................... 85 3.2.3 Biotinylated protein purification ............................................................... 85 3.2.4 Isolation of MPF ...................................................................................... 86 3.2.5 SDS-PAGE ............................................................................................. 86 3.2.6 Western blotting .....................................................................................
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