Phillips R W, Elzer P H, Roop RM I I
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71190-3932, USA.
Microb Pathog. 1995 Nov;19(5):227-84.
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 degrees 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 < 0.001) to killing by H2O2 and puromycin than the parental strain, and a significant reduction (P < 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 timepoints 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.
高温需求A（HtrA）家族的细菌应激反应蛋白是丝氨酸蛋白酶，它们似乎在细胞内氧化损伤的蛋白质达到毒性水平之前将其清除的过程中发挥重要作用。通过基因置换从强毒力的羊种布鲁氏菌16M构建了一个同源htrA缺失突变体，命名为RWP5，以确定羊种布鲁氏菌HtrA蛋白是否作为应激反应蛋白发挥作用，并评估该蛋白对毒力的贡献。与亲本菌株不同，RWP5在40℃的固体培养基上不能形成孤立菌落，在无血补充的 Schaedler琼脂上也不能生长。与16M相比，RWP5在肉汤培养中的生长也很差。羊种布鲁氏菌htrA突变体对过氧化氢和嘌呤霉素杀伤的敏感性显著高于亲本菌株（P < 0.001），并且在感染后1周，与16M相比，从实验感染的BALB/c小鼠脾脏和肝脏中回收的RWP5数量显著减少（P < 0.001）。然而，在感染后3周及之后直至感染后20周，从实验感染小鼠的脾脏和肝脏中回收的RWP5和16M水平相似。对感染后4周和16周从小鼠脾脏中重新分离得到的RWP5进行体外和体内评估，结果表明小鼠传代并未显著改变RWP5的这些体外和体内特性。这些结果支持羊种布鲁氏菌HtrA蛋白具有应激反应功能，并表明该蛋白在感染早期对羊种布鲁氏菌的致病机制有贡献。目前尚不清楚在感染小鼠后期时间点RWP5恢复的基础；然而，此处呈现的结果表明这不是由小鼠传代导致的稳定遗传变化引起的。
