Characterization of PdpC, a protein encoded by the Francisella pathogenicity island




Nix, Eli Beauford

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Tularemia is a zoonotic disease caused by the bacterial pathogen Francisella. A major virulence determinant of Francisella is the ability to survive and multiply within macrophages. Previous research identified a genetic element of approximately 30 kb in length, which possessed characteristics typical of a pathogenicity island. In F. novicida, the Francisella pathogenicity island (FPI) is composed of 18 genes. Initial studies revealed that several FPI-encoded genes are required for intramacrophage growth. The FPI contains several homologues of a newly described type six secretion system (T6SS). I developed a chicken embryo infection model to provide a simple, low-cost assay to evaluate the virulence of Francisella strains. The results demonstrate that this assay is able to discriminate large differences in virulence among Francisella strains. Further, this system can facilitate large-scale experiments to quickly survey mutant collections for virulence, while reducing animal suffering. Next, I adapted a genetic technique called co-transformation for use in Francisella. This technique facilitates the introduction of mutant or wild type DNA into the chromosome, without requiring the introduction of antibiotic resistance markers or negative selection markers. I also developed two new Francisella shuttle vectors for use in complementation studies. I demonstrated that these vectors are compatible with other pFNL-10-based Francisella shuttle vectors. They also permit tri-parental mating, allowing researchers to circumvent the restriction modification system in F. novicida. Finally, conjugation removes the need for electroporation equipment, which can create aerosols. These aerosols can represent a potential health risk for researchers studying highly virulent Francisella strains. The FPI gene pdpC was investigated for its role in virulence and intramacrophage growth. We found that pdpC was dispensable for growth in macrophages but required for virulence in two animal models. Microscopy studies using epitope tagged pdpC suggest that the protein may be secreted during macrophage infection. Quantitative microscopy provides evidence that PdpE (the gene immediately downstream of PdpC) is secreted in a T6SS dependent manner. Additional mutations in the pdpC gene revealed an effect upon the expression of the Igl proteins located in the minor FPI operon. The mechanism linking pdpC to iglA-D expression is unknown, but it is unlikely to be post-translational in nature. The genetic basis for this effect has been difficult to define, but we have developed a working hypothesis. We propose that two genetic mutations in pdpC are required; the first consists of a defined deletion in the N-terminal-half of the gene, while the second consists of an undefined region located at the C-terminal end.



Tularemia, virulence, intramacrophage