Different susceptibility of two animal species infected with isogenic mutants of Mycobacterium bovis identifies phoT as having roles in tuberculosis virulence and phosphate transport

The Mycobacterium tuberculosis complex includes Mycobacterium bovis, which causes tuberculosis in most mammals, including humans. In previous work, it was shown that M. bovis ATCC 35721 has a mutation in its principal sigma factor gene, sigA, causing a single amino acid change affecting binding of SigA with the accessory transcription factor WhiB3. ATCC 35721 is avirulent when inoculated subcutaneously into guinea pigs but can be restored to virulence by integration of wild-type sigA to produce M. bovis WAg320. Subsequently, it was surprising to discover that WAg320 was not virulent when inoculated intratracheally into the Australian brushtail possum (Trichosurus vulpecula), a marsupial that is normally very susceptible to infection with M. bovis. In this study, an in vivo complementation approach was used with ATCC 35721 to produce M. bovis WAg322, which was virulent in possums, and to identify the virulence-restoring gene, phoT There are two point deletions in the PhoT gene of ATCC 35721 causing frameshift inactivation, one of which is also in the phoT of BCG. Knockout of phoT from ATCC 35723, a virulent strain of M. bovis, produced M. bovis WAg758, which was avirulent in both guinea pigs and possums, confirming that phoT is a virulence gene. The effect on virulence of mode of infection versus animal species susceptibility was investigated by inoculating all the above strains by aerosol into guinea pigs and mice and comparing these to the earlier results. Characterization of PhoT indicated that it plays a role in phosphate uptake at low phosphate concentrations. At least in vitro, this role requires the presence of a wild-type sigA gene and appears separate from the ability of phoT to restore virulence to ATCC 35721. This study shows the advantages of using different animal models as tools for the molecular biological investigation of tuberculosis virulence.