Studies on the Bacterial Stress Proteins as Virulence Fctors

小肠结肠炎耶尔森氏菌是一种兼性细胞内病原体，能够抵抗巨噬细胞的杀微生物机制并在吞噬细胞内生长。包括小肠结肠炎耶尔森氏菌在内的一些细菌已被证明通过产生一组也由环境应激诱导的应激蛋白来响应巨噬细胞中的不利环境。为了了解应激蛋白对细菌细胞内存活的作用，我们鉴定并克隆了小肠结肠炎耶尔森氏菌gsrA（global stress requirement）基因。鉴定gsrA基因是因为其通过转座子的插入失活导致生物体不能在升高的温度下生长并且在吞噬作用后不能在巨噬细胞内存活。对gsrA基因进行测序，结果显示其编码一种49500 Da的碱性蛋白。GsrA蛋白显示出与最初在大肠杆菌中鉴定的HtrA应激蛋白显著的氨基酸序列同源性。此外，构建并表征了遗传定义的小肠结肠炎耶尔森氏菌gsrA突变体。gsrA的插入突变导致在39 ℃以上的温度下生长受到抑制，并且对氧化和渗透胁迫的敏感性大大增加。突变体还失去了在巨噬细胞内生存和复制的能力。这些结果共同表明，gsrA基因是小肠结肠炎耶尔森氏菌所采用的保护机制的重要组成部分，使其能够对巨噬细胞中发现的细胞内应激以及细胞外环境应激做出反应。

Yersinia enterocolitica is a facultative intracellular pathogen which is able to resist the microbicidal mechanisms of macrophages and to grow within phagocytic cells. Some bacteria including Y.enterocolitical have been shown to respond to the hostile environment in macrophages by producing a set of stress proteins which are also induced by environmental stresses. To understand the role of stress proteins on the intracellular survival of bacteriawe identified and cloned a Y.enterocolitica gene, celled gsrA (global stress requirement). The gsrA gene was identified because its insertional inactivation by a transposon resulted in the inability of the organism to grow at an elevated temperature and to survive within macrophages after phagocytosis. The gsrA gene was sequenced and shown to encode for a basic, 49500 Da protein. The GsrA protein shows significant amino acid sequence homology to the HtrA stress protein which was originally indentified in Escherichia coli. Furthermore, the genetically defined Y.enterocolitica gsrA mutant was constructed and characterized. The insertional mutation of gsrA resulted in the inhibition of growth at temperatures above 39ﾟC and the grealty increased susceptibility to oxidative and osmotic stresses. The mutant additionally lost the ability to survive and replicate within macrophages. These results, taken together, indicate that the gsrA gene is an essential component of the protection mechanism employed by Y.enterocolitica, allowing it to respond against the intracellular stress found in macrophages as well as extracellular environmental stress.