Ex Vivo Virulence Studies 
Changes in the regulatory potential of a pathogen are often accompanied by altered virulence.
In a first attempt to determine the virulence of the complemented H37Ra knock-in strains relative to wild-type H37Ra and H37Rv, bone marrow-derived murine macrophages (BMM) were infected with the different strains at a multiplicity of infection (MOI) of 1:1 and 10:1 bacteria per cell.
As depicted in Figure 1, macrophages were able to control ex vivo growth of H37Ra, but failed to control growth of H37Rv, confirming results from Freeman and colleagues [20].
When H37Ra knock-ins were tested, important differences in their ex vivo growth characteristics were found.
Whereas H37Ra::fadE5 (Figure 1) and H37Ra::rpsL (unpublished data) showed very little or no growth over the 7-d period, the H37Ra::phoP mutant grew more vigorously, with a 7.5-fold increase in colony-forming units (CFU) over the 7-d period.
From these experiments we concluded that complementation of H37Ra with the phoP wild-type copy partially restored its virulence, but not to the extent of the fully virulent H37Rv strain.
