Date of Degree
PhD (Doctor of Philosophy)
Bradley D. Jones
F. tularensis is an intracellular pathogen, and is the causative agent of tularemia in humans. The ability of F. tularensis to parasitize host cells is largely dependent upon genes within a pathogenicity island (FPI), including those in the iglABCD operon. Specific mechanisms and gene products involved in regulation of the FPI are not well understood. I initiate the study of this regulatory system by creating an efficient Tn5-based mutagenesis system optimized for use in F. tularensis, and utilize this system to construct a lacZ reporter library. I identify genes differentially regulated in response to growth on two different media, including those in the iglABCD and fslABCD operons, and identify iron availability as a factor contributing to the differential regulation. One of these reporter strains, carrying a chromosomal iglB-lacZ fusion, is used as the basis for a secondary transposon mutagenesis to identify mutations that affect iglABCD expression. One such mutation is in FTL_1542 (migR), a hypothetical protein, and reduces expression of the iglABCD approximately 8-fold. The effect of this mutation on igl expression is likely through its effect on another known virulence regulator, fevR, as demonstrated by data from RT-PCR experiments. I compare the phenotypes of LVS fevR and migR mutant strains in primary macrophage and epithelial cell lines and in neutrophils. The mutation in migR effects growth and intracellular trafficking in macrophages but not epithelial cells, and reverses the ability of wild type F. tularensis to block the respiratory burst in neutrophils. When similar mutations were examined in the human virulent F. tularensis strain Schu S4, migR retained its regulatory role, but did not impair replication in macrophages. The migR mutation in Schu S4 did however have an attenuating effect when administered to mice intranasally. Comparison of LVS and Schu S4 in primary human airway epithelial cell infections revealed an inability of LVS to replicate within these cells, which is in contrast to the robust replication of LVS in cultured epithelial cell lines. Together, this work contributes to the understanding of regulatory mechanisms governing virulence gene expression in F. tularensis and highlights differences between LVS and Schu S4 strains.
Copyright 2009 Blake Wade Buchan