Characterization of the Direct Interaction between Hybrid Sensor Kinases PA1611 and RetS That Controls Biofilm Formation and the Type III Secretion System in Pseudomonas aeruginosa
Characterization of the Direct Interaction between Hybrid Sensor Kinases PA1611 and RetS That Controls Biofilm Formation and the Type III Secretion System in Pseudomonas aeruginosa
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DOI：
10.1021/acsinfecdis.6b00153
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发表时间：
2017-02-01
影响因子：
5.3
通讯作者：
Duan, Kangmin
中科院分区：
文献类型：
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作者：
Bhagirath, Anjali Y.;Pydi, Sai P.;Duan, Kangmin
囊性纤维化（CF）患者发病率和死亡率的主要原因之一是铜绿假单胞菌的肺部感染，并且CF中肺部感染的病理生理学受这种微生物的生活方式的影响。RetS-GacS/A-RsmA是铜绿假单胞菌中决定细菌生活方式选择的关键调节途径。以前，我们确定了PA 1611，一种混合传感器激酶，作为一个新的球员在这条途径中，与RetS相互作用，影响生物膜形成和III型分泌系统。在这项研究中，我们探讨了PA 1611和RetS之间相互作用的结构和机制基础。我们通过分子建模确定了对PA 1611 RetS相互作用至关重要的氨基酸残基。然后靶向这些残基进行定点诱变。在PA 1611中的7个关键位置和RetS中的6个相应关键位置进行氨基酸取代。通过细菌双杂交试验分析了PA 1611中这些取代对相互作用的影响。我们对这些突变体在铜绿假单胞菌中的功能进行了分析，以了解它们对特定表型的影响。发现位于PA 1611的组氨酸激酶A和组氨酸激酶样ATP酶结构域中的两个残基F269和E276在PA 1611 RetS相互作用中起关键作用，并且对表型具有深远的影响。RetS中相应的突变也显示了类似的结果。我们进一步证实了PA 1611中的这些突变通过GacS/GacA-RsmY/Z信号通路起作用。总的来说，我们的研究结果提供了一个非同源传感器激酶直接相互作用模型的信号通路，关键的生活方式选择在铜绿假单胞菌，并针对这种相互作用可能作为一种新的方式控制感染铜绿假单胞菌。
One of the leading causes of morbidity and mortality in cystic fibrosis (CF) patients is pulmonary infection with Pseudomonas aeruginosa, and the pathophysiology of pulmonary infection in CF is affected by the lifestyle of this micro-organism. RetS-GacS/A-RsmA is a key regulatory pathway in P. aeruginosa that determines the bacterium's lifestyle choice. Previously, we identified PA1611, a hybrid sensor kinase, as a new player in this pathway that interacts with RetS and influences biofilm formation and type III secretion system. In this study, we explored the structural and mechanistic basis of the interaction between PA1611 and RetS. We identified the amino acid residues critical for PA1611 RetS interactions by molecular modeling. These residues were then targeted for site-directed mutagenesis. Amino acid substitutions were carried out at seven key positions in PA1611 and at six corresponding key positions in RetS. The influence of such substitutions in PA1611 on the interaction was analyzed by bacterial two-hybrid assays. We carried out functional analysis of these mutants in P. aeruginosa for their effect on specific phenotypes. Two residues, F269 and E276, located within the histidine kinase A and histidine kinase-like ATPase domains of PA1611 were found to play crucial roles in the PA1611 RetS interaction and had profound effects on phenotypes. Corresponding mutations in RetS demonstrated similar results. We further confirmed that these mutations in PA1611 function through the GacS/GacA-RsmY/Z signaling pathway. Collectively, our findings provide a noncognate sensor kinase direct interaction model for a signaling pathway, key for lifestyle selection in P. aeruginosa, and targeting such interaction may serve as a novel way of controlling infections with P. aeruginosa.