The Role of Francisella Phagosomal Transporters FptA and FptF in the Pathogenesis of Francisella tularensis LVS and Their Potential as Targets for Live Attenuated Vaccine Development

Abstract

Francisella tularensis is a Gram-negative, facultative intracellular bacterium that is a Tier 1 Select Agent of concern for biodefense for which there is no licensed vaccine. A subfamily of 9 Francisella phagosomal transporter (fpt) genes belonging to the Major Facilitator Superfamily of transporters was identified as critical to pathogenesis and potential targets for attenuation and vaccine development. We hypothesized that fptA and fptF are critical for the virulence of F. tularensis, and that deletion of one or both may limit pathogenic potential and result in a vaccine strain that is safe and protective against lethal challenge. fptA and fptF deletion mutants were generated in the F. tularensis Live Vaccine Strain (LVS). We demonstrated that the LVSΔfptA and LVSΔfptF mutant strains exhibited reduced intracellular replication versus parental LVS within primary macrophages from C57BL/6 mice. LVSΔfptA and LVSΔfptF were highly attenuated in the C57BL/6J mouse model of respiratory tularemia with LD50 values >20 times that of LVS when administered intranasally and vaccination with these mutant strains conferred 100% protection against lethal challenge with LVS. The bacterial burdens of LVSΔfptA and LVSΔfptF mutant strains at 6-days post-infection were significantly reduced compared to that of LVS in murine spleens, lungs and livers. Further studies demonstrated that infection with the LVSΔfptA and LVSΔfptF mutant strains resulted in reduced pathology and tissue destruction in mouse lungs than infection with LVS. The immune responses to LVSΔfptA and LVSΔfptF were characterized by decreased levels of IL-10 and IL-1β in the bronchoalveolar lavage fluid versus LVS, and increased numbers of B cells, αβ and γδ T cells, NK cells, DCs, and CD11b- macrophages in lungs versus LVS. These results support a fundamental requirement for FptA and FptF in the pathogenesis of F. tularensis and the modulation of the host immune response and support their further consideration as targets for the development of live attenuated vaccines against virulent F. tularensis.