Leucine-Responsive Regulatory Protein (Lrp) Acts as a Virulence Repressor in Salmonella enterica Serovar Typhimurium
Leucine-Responsive Regulatory Protein (Lrp) Acts as a Virulence Repressor in Salmonella enterica Serovar Typhimurium
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DOI：
10.1128/jb.01142-08
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发表时间：
2009-02-15
影响因子：
3.2
通讯作者：
Curtiss, Roy, III
中科院分区：
文献类型：
--
作者：
Baek, Chang-Ho;Wang, Shifeng;Curtiss, Roy, III
亮氨酸响应调节蛋白（Lrp）是一种全局基因调控因子，它影响包括大肠杆菌和沙门氏菌中毒力相关基因在内的大量基因的表达。在沙门氏菌中，尚未有关于Lrp对毒力基因调控的系统性研究报道。我们在此报道，Lrp的组成型表达[lrp(Con)]显著减弱沙门氏菌的毒力，而lrp缺失（Δlrp）突变则增强毒力。lrp(Con)突变体产生多效性影响，包括侵袭、细胞毒性和定植方面的缺陷，而Δlrp突变体在这些活动方面比野生型菌株更熟练。我们提供的证据表明，Lrp通过直接结合其启动子区域P - hilA、P - invF和P - ssrA，抑制沙门氏菌致病性岛1（SPI - 1）和2（SPI - 2）中关键毒力调节基因hilA、invF和ssrA的转录。此外，蛋白质印迹分析表明，SPI - 1效应蛋白SipA的表达在lrp(Con)突变体中降低，在Δlrp突变体中增强。计算分析揭示了位于P - hilA、P - invF和P - ssrA中的假定Lrp结合共有DNA基序。这些结果表明，Lrp结合到共有基序并调节相关基因的表达。亮氨酸在体外增强了Lrp与P - invF的结合，并且向生长培养基中添加亮氨酸降低了invF转录水平。然而，亮氨酸对hilA和ssrA的表达或Lrp的细胞水平没有影响。此外，Lrp似乎是一种抗毒力基因，因为缺失突变体在BALB/c小鼠中显示出增强的细胞侵袭、细胞毒性和高毒力。
Leucine-responsive regulatory protein (Lrp) is a global gene regulator that influences expression of a large number of genes including virulence-related genes in Escherichia coli and Salmonella. No systematic studies examining the regulation of virulence genes by Lrp have been reported in Salmonella. We report here that constitutive expression of Lrp [lrp(Con)] dramatically attenuates Salmonella virulence while an lrp deletion (Delta lrp) mutation enhances virulence. The lrp(Con) mutant caused pleiotropic effects that include defects in invasion, cytotoxicity, and colonization, whereas the Delta lrp mutant was more proficient at these activities than the wild-type strain. We present evidence that Lrp represses transcription of key virulence regulator genes hilA, invF, and ssrA-in Salmonella pathogenicity island 1 (SPI-1) and 2 (SPI-2), by binding directly to their promoter regions, P-hilA, P-invF, and P-ssrA. In addition, Western blot analysis showed that the expression of the SPI-1 effector SipA was reduced in the lrp(Con) mutant and enhanced in the Delta lrp mutant. Computational analysis revealed putative Lrp-binding consensus DNA motifs located in P-hilA, P-invF, and P-ssrA. These results suggest that Lrp binds to the consensus motifs and modulates expression of the linked genes. The presence of leucine enhanced Lrp binding to P-invF in vitro and the addition of leucine to growth medium decreased the level of invF transcription. However, leucine had no effect on expression of hilA and ssrA or on cellular levels of Lrp. In addition, Lrp appears to be an antivirulence gene, since the deletion mutant showed enhanced cell invasion, cytotoxicity, and hypervirulence in BALB/c mice.