Characterization of PdpD, a Francisella pathogenicity island protein.




Ludu, Jagjit Singh

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Although its highly infectious nature has led to its classification as a potential bio-terror threat, very little is known about the pathogenesis of Francisella. A complete understanding of the mechanisms employed by Francisella to gain residence and replicate within macrophages will provide valuable insight into the means by which F. tularensis, and other intracellular pathogens such as M. tuberculosis and L. pneumophila, invade host cells, secrete effectors, alter phagosome biogenesis and disrupt vesicle traficking. The overall theme of this dissertation is the analysis of genes encoded within a recently identified Francisella pathogenicity island (FPI). In particular, the chapters will focus on the identification, mutagenesis, and phenotypic analysis of Pathogenicity determinant protein D (pdpD), a ~135 kDa protein encoded within the FPI. Chapter 2 addresses the identification of the Francisella pathogenicity island, and the intramacrophage growth of several mutants found within this loci. One of the greatest strengths in determining the roles of putative virulence genes is the ability of researchers to alter and amplify nucleic acids in a highly developed model platform and subsequently introduce the altered genetic material into a pathogen. Although genetic transformation has been well developed and optimized in E. coli, where it is regularly used in cloning experiments, the introduction of DNA into Francisella has been a major deterrent in the mutagenesis of putative virulence factors. Chapter 3 focuses on engineered genetic elements and methods for transformation, antibiotic selection, deletion mutagenesis, and complementation in Francisella strains. The chromosomes of F. tularensis strains carry two identical copies of the Francisella pathogenicity island, and the FPI of North American-specific biotypes contain two genes, anmK and pdpD, that are not found in biotypes distributed over the entire Northern Hemisphere. Furthermore, unlike other known intracellular pathogens, F. tularensis lacks a functional type III or type IV secretion system, which are necessary for other bacterium to arrest maturation of their respective phagosomes. Chapter 4 focuses on the virulence contribution of anmK and pdpD using F. novicida, which is very closely related to F. tularensis but carries only one copy of the FPI. In addition, the outer membrane localization of PdpD is examined in deletions of FPI genes encoding proteins that are homologues of known components of Type VI secretion systems. Although each chapter is a continuum of research related to the Francisella pathogenicity island, each will be treated as a distinct work consisting of an introduction, materials and methods, results, and a discussion. Chapter 5 of this dissertation will consist of an overall conclusion section which will tie the 3 research chapters together as well as focus on future studies.



Francisella, Pathogenesis, Virulence Genes, Protein Secretion