Mutagenesis and characterization of pdpC in Francisella novicida




Cheung, Karen K. M.

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Francisella tularensis is a highly infectious. Gram-negative coccobacillus that is the etiological agent of the acute. febrile. zoonotic disease tularemia. A ca. 35 kb Francisella pathogenicity island (FM) was previously discovered. Two genes. pdpA and pdpD were shown to be required for virulence. The FP1 gene pdpC encodes a protein that has no significant similarities to any motifs, domains, or homologues of known bacterial proteins. This gene of unknown function may encode a novel virulence factor involved in Francisella infection. The role of PdpC in F. novicida intracellular growth was investigated. Results from this study demonstrated that the erythromycin allelic replacement mutant of pdpC was more attenuated in intracellular growth in the murine macrophage-like J774A.1 cells than in bone marrow-derived macrophages from BALB/c mice and that complementation in trans partially complements this mutation. To further investigate the role of pdpC in virulence. partial deletion mutagenesis in the C-terminus of PdpC was performed which resulted in four mutants that showed slight attenuation in J774A.1 intramacrophage growth but behaved like wildtype F, novicida in bone marrow-derived macrophages. Chicken embryos were infected to evaluate the virulence of these pdpC mutants. The virulence of the Em allelic replacement mutant was significantly more attenuated than wildtype F. novicida and complementation partially restored virulence. Partial deletion mutants of pdpC exhibited greater virulence than the EmR mutant in chicken embryos and were able to cause 100% mortality at day 6. Furthermore, eukaryotic expression of triple FLAG-tagged PdpC in chicken embryo fibroblasts resulted in cells that exhibited different morphologies than uninfected fibroblasts which suggests that PdpC may play a role in cytoskeletal rearrangements by altering host cell signaling pathways.



francisella, tularemia, novicida, infectious disease, virulence, pathogen