Functional impact of mutational activation on the Listeria monocytogenes central virulence regulator PrfA
Functional impact of mutational activation on the Listeria monocytogenes central virulence regulator PrfA
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DOI：
10.1099/mic.0.2008/021063-0
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发表时间：
2008-11-01
期刊：
影响因子：
2.8
通讯作者：
Freitag, Nancy E.
中科院分区：
文献类型：
--
作者：
Miner, Maurine D.;Port, Gary C.;Freitag, Nancy E.
转录激活因子PrfA是表达单核细胞增生李斯特菌致病所必需的毒力因子所必需的。PrfA被认为在L.单核细胞增多症进入感染的宿主细胞的胞质溶胶，导致靶基因的诱导，其产物是细菌细胞内生长和细胞间传播所需的。已经鉴定出几种似乎将PrfA锁定在其高度活化的胞质形式中的突变（称为prfA* 突变）。在这项研究中，PrfA和五个PrA* 突变蛋白表现出不同程度的活动进行了纯化和分析，以确定不同方面的PrfA活性的突变的影响。基于有限的蛋白水解消化，检测到的PrfA* 突变体蛋白相比，野生型PrfA的构象变化。除了一个突变体（PrfA Y 63 C），电泳迁移率变动测定法测定的DNA结合亲和力似乎与PrfA突变激活水平直接相关，因此高活性突变体表现出DNA结合亲和力的最大增加，中度激活突变体表现出更温和的增加。令人惊讶的是，PrfA和PrfA* 突变体在溶液中形成晚餐的能力似乎与PrfA依赖性基因表达水平负相关。基于与PrfA家族成员Crp和CooA的蛋白质活性和结构相似性的比较，prfA* 突变改变了PrfA活性的不同方面，包括DNA结合和蛋白质-蛋白质相互作用。
The transcriptional activator PrfA is required for the expression of virulence factors necessary for Listeria monocytogenes pathogenesis. PrfA is believed to become activated following L. monocytogenes entry into the cytosol of infected host cells, resulting in the induction of target genes whose products are required for bacterial intracellular growth and cell-to-cell spread. Several mutations have been identified that appear to lock PrfA into its highly activated cytosolic form (known as prfA* mutations). In this study PrfA and five PrA* mutant proteins exhibiting differing degrees of activity were purified and analysed to define the influences of the mutations on distinct aspects of PrfA activity. Based on limited proteolytic digestion, conformational changes were detected for the PrfA* mutant proteins in comparison to wild-type PrfA. For all but one mutant (PrfA Y63C), the DNA binding affinity as measured by electophoretic mobility shift assay appeared to directly correlate with levels of PrfA mutational activation, such that the high-activity mutants exhibited the largest increases in DNA binding affinity and moderately activated mutants exhibited more moderate increases. Surprisingly, the ability of PrfA and PrfA* mutants to form dinners in solution appeared to inversely correlate with levels of PrfA-dependent gene expression. Based on comparisons of protein activity and structural similarities with PrfA family members Crp and CooA, the prfA* mutations modify distinct aspects of PrfA activity that include DNA binding and protein-protein interactions.