Russell-Goldman Eleanor, Xu Jiayong, Wang Xiaobing, Chan John, Tufariello JoAnn M
Departments of Medicine, Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Infect Immun. 2008 Sep;76(9):4269-81. doi: 10.1128/IAI.01735-07. Epub 2008 Jun 30.
Resuscitation-promoting factors (Rpfs), apparent peptidoglycan hydrolases, have been implicated in the reactivation of dormant bacteria. We previously demonstrated that deletion of rpfB impaired reactivation of Mycobacterium tuberculosis in a mouse model. Because M. tuberculosis encodes five Rpf paralogues, redundant functions among the family members might obscure rpf single-knockout phenotypes. A series of rpf double knockouts were therefore generated. One double mutant, DeltarpfAB, exhibited several striking phenotypes. Consistent with the proposed cell wall-modifying function of Rpfs, DeltarpfAB exhibited an altered colony morphology. Although DeltarpfAB grew comparably to the parental strain in axenic culture, in vivo it exhibited deficiency in reactivation induced in C57BL/6 mice by the administration of nitric oxide synthase inhibitor (aminoguanidine) or by CD4(+) T-cell depletion. Notably, the reactivation deficiency of DeltarpfAB was more severe than that of DeltarpfB in aminoguanidine-treated mice. A similar deficiency was observed in DeltarpfAB reactivation from a drug-induced apparently sterile state in infected NOS2(-/-) mice upon cessation of antimycobacterial therapy. Secondly, DeltarpfAB showed a persistence defect not seen with the DeltarpfB or DeltarpfA single mutants. Interestingly, DeltarpfAB exhibited impaired growth in primary mouse macrophages and induced higher levels of the proinflammatory cytokines tumor necrosis factor alpha and interleukin 6. Simultaneous reintroduction of rpfA and rpfB into the double-knockout strain complemented the colony morphology and macrophage cytokine secretion phenotypes. Phenotypes related to cell wall composition and macrophage responses suggest that M. tuberculosis Rpfs may influence the outcome of reactivation, in part, by modulating innate immune responses to the bacterium.
复苏促进因子（Rpfs）是一种明显的肽聚糖水解酶，与休眠细菌的重新激活有关。我们之前证明，在小鼠模型中，rpfB的缺失会损害结核分枝杆菌的重新激活。由于结核分枝杆菌编码五种Rpf同源物，家族成员之间的冗余功能可能会掩盖rpf单基因敲除的表型。因此，我们构建了一系列rpf双基因敲除菌株。其中一个双突变体DeltarpfAB表现出几种显著的表型。与Rpfs提出的细胞壁修饰功能一致，DeltarpfAB表现出菌落形态改变。虽然DeltarpfAB在无菌培养中与亲本菌株生长相当，但在体内，通过给予一氧化氮合酶抑制剂（氨基胍）或通过CD4(+) T细胞耗竭诱导C57BL/6小鼠重新激活时，它表现出缺陷。值得注意的是，在氨基胍处理的小鼠中，DeltarpfAB的重新激活缺陷比DeltarpfB更严重。在抗分枝杆菌治疗停止后，从感染的NOS2(-/-)小鼠的药物诱导的明显无菌状态重新激活DeltarpfAB时，也观察到类似的缺陷。其次，DeltarpfAB表现出DeltarpfB或DeltarpfA单突变体未出现的持续存在缺陷。有趣的是，DeltarpfAB在原代小鼠巨噬细胞中的生长受损，并诱导更高水平的促炎细胞因子肿瘤坏死因子α和白细胞介素6。将rpfA和rpfB同时重新引入双基因敲除菌株中，可补充菌落形态和巨噬细胞细胞因子分泌表型。与细胞壁组成和巨噬细胞反应相关的表型表明，结核分枝杆菌Rpfs可能部分通过调节对该细菌的固有免疫反应来影响重新激活的结果。
