Construction and Characterization of an Attenuated Purine Auxotroph in a Francisella tularensisLive Vaccine Strain

ABSTRACTFrancisella tularensisis a facultative intracellular pathogen and is the etiological agent of tularemia. It is capable of escaping from the phagosome, replicating to high numbers in the cytosol, and inducing apoptosis in macrophages of a variety of hosts. F. tularensishas received significant attention recently due to its potential use as a bioweapon. Currently, there is no licensed vaccine against F. tularensis, although a partially protective live vaccine strain (LVS) that is attenuated in humans but remains fully virulent for mice was previously developed. An F. tularensisLVS mutant deleted in the purMCDpurine biosynthetic locus was constructed and partially characterized by using an allelic exchange strategy. The F. tularensisLVS ΔpurMCDmutant was auxotrophic for purines when grown in defined medium and exhibited significant attenuation in virulence when assayed in murine macrophages in vitro or in BALB/c mice. Growth and virulence defects were complemented by the addition of the purine precursor hypoxanthine or by introduction of purMCDNin trans. The F. tularensisLVS ΔpurMCDmutant escaped from the phagosome but failed to replicate in the cytosol or induce apoptotic and cytopathic responses in infected cells. Importantly, mice vaccinated with a low dose of the F. tularensisLVSΔ purMCDmutant were fully protected against subsequent lethal challenge with the LVS parental strain. Collectively, these results suggest that F. tularensismutants deleted in the purMCDbiosynthetic locus exhibit characteristics that may warrant further investigation of their use as potential live vaccine candidates.