Wang Yufei, Chen Zeliang, Qiao Feng, Ying Tianyi, Yuan Jing, Zhong Zhijun, Zhou Lei, Du Xinying, Wang Zhoujia, Zhao Jin, Dong Shicun, Jia Leili, Yuan Xitong, Yang Ruifu, Sun Yansong, Huang Liuyu
Institute of Disease Control and Prevention, Academy of Military Medical Science, Beijing, China.
PLoS One. 2009;4(4):e5368. doi: 10.1371/journal.pone.0005368. Epub 2009 Apr 29.
Brucella melitensis is a facultative, intracellular, pathogenic bacterium that replicates within macrophages. The type IV secretion system encoded by the virB operon (virB) is involved in Brucella intracellular survival. However, the underlying molecular mechanisms, especially the target proteins affected by the virB, remain largely unclear.
METHODOLOGY/PRINCIPAL FINDINGS: In order to define the proteins affected by virB, the proteomes of wild-type and the virB mutant were compared under in vitro conditions where virB was highly activated. The differentially expressed proteins were identified by MALDI-TOF-MS. Forty-four down-regulated and eighteen up-regulated proteins which exhibited a 2-fold or greater change were identified. These proteins included those involved in amino acid transport and metabolism, lipid metabolism, energy production, cell membrane biogenesis, translation, post-translational modifications and protein turnover, as well as unknown proteins. Interestingly, several important virulence related proteins involved in intracellular survival, including VjbR, DnaK, HtrA, Omp25, and GntR, were down-regulated in the virB mutant. Transcription analysis of virB and vjbR at different growth phase showed that virB positively affect transcription of vjbR in a growth phase dependent manner. Quantitative RT-PCR showed that transcription of these genes was also affected by virB during macrophage cell infection, consistent with the observed decreased survival of the virB mutant in macrophage.
CONCLUSIONS/SIGNIFICANCE: These data indicated that the virB operon may control the intracellular survival of Brucella by affecting the expression of relevant proteins.
羊种布鲁氏菌是一种兼性胞内致病菌，可在巨噬细胞内复制。由virB操纵子（virB）编码的IV型分泌系统参与布鲁氏菌在细胞内存活。然而，其潜在的分子机制，尤其是受virB影响的靶蛋白，仍不清楚。
方法/主要发现：为了确定受virB影响的蛋白质，在virB高度激活的体外条件下比较野生型和virB突变体的蛋白质组。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）鉴定差异表达的蛋白质。鉴定出44种下调和18种上调的蛋白质，其变化倍数为2倍或更高。这些蛋白质包括参与氨基酸转运和代谢、脂质代谢、能量产生、细胞膜生物合成、翻译、翻译后修饰和蛋白质周转的蛋白质，以及未知蛋白质。有趣的是，一些参与细胞内存活的重要毒力相关蛋白，包括VjbR、DnaK、HtrA、Omp25和GntR，在virB突变体中下调。对virB和vjbR在不同生长阶段的转录分析表明，virB以生长阶段依赖的方式正向影响vjbR的转录。定量逆转录聚合酶链反应（qRT-PCR）表明，在巨噬细胞感染期间，这些基因的转录也受virB影响，这与观察到的virB突变体在巨噬细胞中的存活率降低一致。
结论/意义：这些数据表明，virB操纵子可能通过影响相关蛋白质的表达来控制布鲁氏菌在细胞内存活。
