Pei Jianwu, Turse Joshua E, Wu Qingmin, Ficht Thomas A
Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843-4467, USA.
Infect Immun. 2006 May;74(5):2667-75. doi: 10.1128/IAI.74.5.2667-2675.2006.
Previous studies suggest that smooth Brucella organisms inhibit macrophage apoptosis. In contrast, necrotic cell death of macrophages infected with rough Brucella organisms in vitro has been reported, which may in part explain the failure of some rough organisms to thrive. To characterize these potential macrophage killing mechanisms, J774.A1 murine macrophages were infected with Brucella abortus S2308-derived rough mutant CA180. Electron microscopic analysis and polyethylene glycol protection assays revealed that the cells were killed as a result of necrosis and oncosis. This killing was shown to be unaffected by treatment with carbenicillin, an inhibitor of bacterial cell wall biosynthesis and, indirectly, replication. In contrast, chloramphenicol treatment of macrophages infected at multiplicities of infection exceeding 10,000 prevented cell death, despite internalization of large numbers of bacteria. Similarly, heat-killed and gentamicin-killed CA180 did not induce cytopathic effects in the macrophage. These results suggested that killing of infected host cells requires active bacterial protein synthesis. Cytochalasin D treatment revealed that internalization of the bacteria was necessary to initiate killing. Transwell experiments demonstrated that cell death is not mediated by a diffusible product, including tumor necrosis factor alpha and nitric oxide, but does require direct contact between host and pathogen. Furthermore, macrophages preinfected with B. abortus S2308 or pretreated with B. abortus O polysaccharide did not prevent rough CA180-induced cell death. In conclusion, Brucella rough mutant infection induces necrotic and oncotic macrophage cell death that requires bacterial protein synthesis and direct interaction of bacteria with the target cells.
先前的研究表明，光滑型布鲁氏菌可抑制巨噬细胞凋亡。相比之下，已有报道称体外感染粗糙型布鲁氏菌的巨噬细胞会发生坏死性细胞死亡，这可能部分解释了一些粗糙型菌株难以存活的原因。为了阐明这些潜在的巨噬细胞杀伤机制，用流产布鲁氏菌S2308衍生的粗糙型突变体CA180感染J774.A1小鼠巨噬细胞。电子显微镜分析和聚乙二醇保护试验表明，细胞因坏死和胀亡而死亡。结果显示，用羧苄青霉素（一种细菌细胞壁生物合成抑制剂，间接抑制细菌复制）处理并不会影响这种杀伤作用。相反，当感染复数超过10000时，用氯霉素处理感染的巨噬细胞可防止细胞死亡，尽管此时大量细菌已内化。同样，热灭活和庆大霉素灭活的CA180也不会在巨噬细胞中诱导细胞病变效应。这些结果表明，感染宿主细胞的杀伤需要细菌进行活跃的蛋白质合成。细胞松弛素D处理表明，细菌的内化是启动杀伤作用所必需的。Transwell实验表明，细胞死亡不是由包括肿瘤坏死因子α和一氧化氮在内的可扩散产物介导的，但确实需要宿主与病原体之间的直接接触。此外，预先感染流产布鲁氏菌S2308或用流产布鲁氏菌O多糖预处理的巨噬细胞并不能阻止粗糙型CA180诱导的细胞死亡。总之，布鲁氏菌粗糙型突变体感染可诱导巨噬细胞发生坏死性和胀亡性细胞死亡，这需要细菌蛋白质合成以及细菌与靶细胞的直接相互作用。
