Zhong Zhijun, Wang Yufei, Qiao Feng, Wang Zhoujia, Du Xinying, Xu Jie, Zhao Jin, Qu Qing, Dong Shicun, Sun Yansong, Huang Liuyu, Huang Kehe, Chen Zeliang
Institute of Disease Control and Prevention, Academy of Military Medical Science, Beijing 100071, PR China.
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, PR China.
Microbiology (Reading). 2009 Oct;155(Pt 10):3392-3402. doi: 10.1099/mic.0.030619-0. Epub 2009 Jul 23.
Some Brucella rough mutants cause cytotoxicity that resembles oncosis and necrosis in macrophages. This cytotoxicity requires the type IV secretion system (T4SS). In rough mutants, the cell-surface O antigen is shortened and the T4SS structure is thus exposed on the surface. Cytotoxicity effector proteins can therefore be more easily secreted. This enhanced secretion of effector proteins might cause the increased levels of cytotoxicity observed. However, whether this cytotoxicity is unique to the rough mutant and is mediated by overexpression of the T4SS has not been definitively determined. To test this, in the present study, a virB inactivation mutant (BMDeltavirB) and an overexpression strain (BM-VIR) of a smooth Brucella melitensis strain (BM) were constructed and their cytotoxicity for macrophages and intracellular survival capability were analysed and compared. Cytotoxicity was detected in macrophages infected with higher concentrations of strains BM or BM-VIR, but not in those infected with BMDeltavirB. The quorum sensing signal molecule N-dodecanoyl-dl-homoserine lactone (C(12)-HSL), a molecule that can inhibit expression of virB, inhibited the cytotoxicity of BM and BM-VIR, but not of BMDeltavirB. These results indicated that overexpression of virB is responsible for Brucella cytotoxicity in macrophages. Transcription analysis showed that virB is regulated in a cell-density-dependent manner both in in vitro culture and during macrophage infection. When compared with BM, BM-VIR showed a reduced survival capacity in macrophages and mice, but both strains demonstrated similar resistance to in vitro stress conditions designed to simulate intracellular environments. Taken together, the cytotoxicity of Brucella for macrophages is probably mediated by increased secretion of effector proteins that results from overexpression of virB or an increase in the number of bacterial cells. The observation that both inactivation and overexpression of virB are detrimental for Brucella intracellular survival also indicated that the expression of virB is tightly regulated in a cell-density-dependent manner.
一些粗糙型布鲁氏菌突变体可引起细胞毒性，这种毒性在巨噬细胞中类似于肿胀性坏死。这种细胞毒性需要IV型分泌系统（T4SS）。在粗糙型突变体中，细胞表面的O抗原缩短，T4SS结构因此暴露于表面。因此，细胞毒性效应蛋白能够更容易地被分泌。效应蛋白分泌的增强可能导致观察到的细胞毒性水平增加。然而，这种细胞毒性是否为粗糙型突变体所特有以及是否由T4SS的过表达介导，尚未得到明确确定。为了验证这一点，在本研究中，构建了光滑型羊种布鲁氏菌菌株（BM）的virB失活突变体（BMDeltavirB）和过表达菌株（BM-VIR），并分析和比较了它们对巨噬细胞的细胞毒性和细胞内存活能力。在用高浓度菌株BM或BM-VIR感染的巨噬细胞中检测到细胞毒性，但在用BMDeltavirB感染的巨噬细胞中未检测到。群体感应信号分子N-十二烷酰-DL-高丝氨酸内酯（C(12)-HSL），一种能够抑制virB表达的分子，抑制了BM和BM-VIR的细胞毒性，但未抑制BMDeltavirB的细胞毒性。这些结果表明，virB的过表达是布鲁氏菌在巨噬细胞中产生细胞毒性的原因。转录分析表明，virB在体外培养和巨噬细胞感染过程中均以细胞密度依赖性方式受到调控。与BM相比，BM-VIR在巨噬细胞和小鼠中的存活能力降低，但两种菌株对旨在模拟细胞内环境的体外应激条件表现出相似的抗性。综上所述，布鲁氏菌对巨噬细胞的细胞毒性可能是由virB过表达或细菌细胞数量增加导致的效应蛋白分泌增加所介导的。virB的失活和过表达均对布鲁氏菌细胞内存活不利这一观察结果也表明，virB的表达以细胞密度依赖性方式受到严格调控。
