Characterization of the Francisella virulence factor RipA

Francisella tularensis is a highly virulent facultative intracellular pathogen; however, the mechanisms by which Francisella virulence factors mediate this virulence are not well defined. We identified the Francisella tularensis LVS FTL_1914 locus in a transposon mutagenesis screen for genes required for intraepithelial replication. Creation and characterization of a F. tularensis LVS DeltaripA strain revealed that the locus was necessary for intracellular replication and we termed the gene ripA (Required for Intracellular Propagation, Factor A). We have studied the Francisella virulence factor RipA in an attempt to define its importance in pathogenesis. RipA is required for Francisella virulence in a mouse model of tularemia. A Francisella DeltaripA mutant fails to replicate after intracellular colonization. The mutant invades at the same rate as wild type, escapes the phagosome, then fails to replicate in the cytoplasm. RipA localizes to the cytoplasmic membrane of Francisella and interacts with the transcriptional regulator IclR. How this interaction impacts virulence phenotypes and IclR transcriptional regulation are currently unknown. RipA is repressed by the Francisella virulence regulator MglA and is suppressed during intracellular growth after replication in the cytosol. MglA is upregulated after cell invasion. Most other virulence factors regulated by MglA are positively impacted by MglA suggesting that other factors act together with MglA to negatively influence gene expression. We screened a transposon library to identify loci that can influence ripA gene expression as identified using a lacZ reporter system. HU form B and NusA were identified as possible regulators that influence ripA expression. RipA may be part of a programmed gene response of Francisella during different stages in its intracellular lifecycle. MglA acting with other regulators may act to positively and negatively impact gene expression during different phases of intracellular growth and in the much more complex setting of in vivo pathogenesis.