A Brucella melitensis high temperature requirement A (htrA) deletion mutant demonstrates a stress response defective phenotype in vitro and transient attenuation in the BALB/c mouse model

Bacterial stress response proteins of the high temperature requirement A (HtrA) family are serine proteases which appear to play an important role in scavenging oxidatively damaged proteins from the cell before they reach toxic levels. An isogenic htrA deletion mutant, designated RWP5, was constructed from virulent Brucella melitensis 16M via gene replacement to determine whether the B. melitensis HtrA protein functions as a stress response protein, and to evaluate the contribution of this protein to virulence. Unlike the parental strain, RWP5 would not form isolated colonies on solid media at 40°C or grow on Schaedler agar without blood supplementation. RWP5 also grew poorly in broth culture in contrast to 16M. The B. melitensis htrA mutant was significantly more sensitive (P \u3c 0.001) to killing by H2O2 and puromycin than the parental strain, and a significant reduction (P \u3c 0.001) in the number of RWP5 recovered from the spleens and livers of experimentally infected BALB/c mice was observed at one week post infection compared to 16M. However, by 3 weeks post-infection and continuing thereafter through to 20 weeks post-infection, the levels of RWP5 and 16M recovered from the spleens and livers of experimentally infected mice were similar. In vitro and in vivo evaluation of RWP5 reisolates obtained from the spleens of mice at 4 and 16 weeks post-infection demonstrated that mouse passage did not significantly alter these characteristic in vitro and in vivo properties of RWP5. These results support a stress response function for the B. melitensis HtrA protein and suggest that this protein contributes to the pathogenesis of B. melitensis early in infection. The basis for the recovery of RWP5 at later time points in infected mice is presently unknown; however, the results presented here suggest that it is not caused by a stable genetic change resulting from mouse passage