Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection
Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection
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DOI：
10.1099/mic.0.023754-0
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发表时间：
2009-03-01
期刊：
影响因子：
2.8
通讯作者：
Roberts, Mark
中科院分区：
文献类型：
--
作者：
Lewis, Claire;Skovierova, Henrieta;Roberts, Mark
HtrA 是许多细菌病原体成功引起感染所需的双功能应激蛋白。肠沙门氏菌鼠伤寒血清型 (S. Typhimurium) htrA 突变体在巨噬细胞内存活方面存在缺陷，并且在小鼠中高度减毒。 htrA 在大肠杆菌中的转录由依赖于 sigma(E) (RpoE) 的单个启动子控制。 S. Typhimurium htrA 还具有 sigma(E) 依赖性启动子；然而，我们发现 sigma(E) 的缺失对鼠伤寒沙门氏菌 HtrA 的产生几乎没有影响。这表明额外的启动子控制鼠伤寒沙门氏菌中 htrA 的表达。我们鉴定了三个鼠伤寒沙门氏菌 htrA 启动子。只有最近的启动子 htrAp3 是 sigma(E) 依赖性的。其他启动子 htrAp1 和 htrAp2 可能被主要 sigma 因子 sigma(70) 识别。这两个启动子组成型表达，但也受到热休克的轻微诱导。因此htrA的表达在鼠伤寒沙门氏菌和大肠杆菌中是不同的。 HtrA 的作用是处理周质中错误折叠/损坏的蛋白质。它可以通过降解（蛋白酶活性）或折叠/捕获（伴侣/隔离、C/S、活性）异常蛋白质来实现这一点。我们在体外和体内研究了这些功能中哪些对鼠伤寒沙门氏菌很重要。编码变体 HtrA 分子的 htrA 点突变体或缺失突变体已在先前的研究中用于研究 HtrA 不同区域在 C/S 和蛋白酶活性中的作用。这些 htrA 变体置于鼠伤寒沙门氏菌 htrAP123 启动子的控制下，并在鼠伤寒沙门氏菌 htrA 突变体 GVB1343 中表达。野生型HtrA和缺乏蛋白酶活性的HtrA（HtrA S210A）都使GVB1343能够在高温（46℃）下生长。这两种分子还显着增强了感染期间小鼠肝脏和脾脏中 GVB1343 的生长/存活。然而，野生型 HtrA 的表达使 GVB1343 能够生长到比 HtrA S210A 的表达高得多的水平。因此，HtrA 的蛋白酶和 C/S 功能在感染期间在体内发挥作用，但蛋白酶功能可能更重要。任何一个 PDZ 结构域的缺失都完全消除了 I 在体外或体内补充 GVB1343 生长缺陷的能力。
HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on sigma(E) (RpoE). S. Typhimurium htrA also possesses a sigma(E)-dependent promoter; however, we found that the absence of sigma(E) had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S.Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was sigma(E) dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor sigma(70). These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 degrees C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of I to complement the growth defects of GVB1343 in vitro or in vivo.