RipA, a Cytoplasmic Membrane Protein Conserved amongFrancisellaSpecies, Is Required for Intracellular Survival

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Abstract
Francisella tularensisis a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of theF. tularensislive vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequencedFrancisellaspecies yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele intrans. Based on the deletion mutant phenotype, FTL_1914 was termedripA(required forintracellularproliferation, factorA). Following uptake by J774.A1 cells,F. tularensisLVSΔripAcolocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of theF. tularensisLVS ΔripAmutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. TheF. tularensisLVSΔripAmutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved amongFrancisellaspecies that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence.