This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Francisella tularensis is a highly infectious Category A bacterial pathogen that causes tularemia, a potentially life-threatening disease in humans. Due to the ease of aerosol dissemination of this organism and the minimal inoculum (d10 bacteria) necessary to cause severe disease, F. tularensis has been weaponized for use in biowarfare. Critical to Francisella's pathogenesis are its ability to replicate within macrophages and to subvert the host immune response. We recently employed a powerful global in vivo negative selection screen in mice to identify genes required for the pathogenesis of F. novicida, a subspecies of Francisella that causes disease in mice but not humans. This approach resulted in the identification of 164 genes that are required for virulence, 44 of which appear to encode novel virulence factors. We found that 65 of the 164 genes are required for replication in macrophages in vitro. One of the genes with the strongest phenotypes that was essential for replication in macrophages encodes a novel protein of unknown function. We set out to determine how the protein encoded by this gene contributes to Francisella pathogenesis and the evasion of macrophage defenses at the molecular level. Construction and testing of a clean deletion mutant lacking the protein confirmed that this gene (which we refer to as fpeA - Francisella phagosome escape A) is required for F. novicida replication in macrophages as well as virulence in vivo. Furthermore, complementation of the fpeA mutant strain with a wild-type copy of the gene restored the ability to replicate within macrophages. Electron microscopy revealed that the mutant strain is defective for escape from the macrophage phagosome, explaining its attenuated replication phenotype.