Mechanisms of Francisella tularensis virulence as revealed by RipA and IclR

F. tularensis is a Gram-negative coccobacillus that is the etiologic agent of the zoonotic disease tularemia. With a low infectious dose via the inhalational route and the ability to cause a potentially severe disease in humans, F. tularensis is a very successful pathogen. While it is known that F. tularensis depends on intracellular replication and immune suppression of the host, little is known about the specific mechanisms of virulence. Using a screen to identify genes required for intracellular replication, we identified a locus FTL_1914 which was subsequently named ripA. A mutant containing a deletion of ripA (LVSΔripA) escaped the phagosome; however, it failed to replicate intracellularly in the cytoplasm of macrophages and epithelial cells and was attenuated in a mouse model of pulmonary tularemia. Later studies showed that RipA is a conserved cytoplasmic membrane protein with similarity to hypothetical proteins of unknown function in a few randomly-distributed bacterial strains. Therefore, the function of RipA is not known. Investigation into protein-protein interactions involving RipA identified a putative RipA-interacting protein termed IclR (FTL_1364). In studies to determine the biological relevance of the RipA-IclR interaction, we analyzed the role of IclR to the virulence of F. tularensis. Deletion of iclR in F. tularensis LVS and Schu S4 suggested that unlike the non-pathogenic F. novicida, IclR was not required for virulence of human-virulent strains of F. tularensis and therefore, likely not required for RipA function. Further studies into RipA function using LVSΔripA led to a greater understanding of innate immune signaling pathways that are being suppressed by F. tularensis. Finally, biochemical determination of the unique topology of RipA and identification of functional domains revealed new insights into a potentially new family in proteins conserved throughout Prokaryotes. Together, not only do these studies provide general characterization of two F. tularensis proteins, they help elucidate mechanisms of virulence utilized by the highly pathogenic F. tularensis