Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis
Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis
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DOI：
10.1128/mbio.02250-16
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发表时间：
2017-01-01
期刊：
影响因子：
6.4
通讯作者：
Attree, Ina
中科院分区：
文献类型：
--
作者：
Basso, Pauline;Ragno, Michel;Attree, Ina
缺乏III型分泌系统基因的铜绿假单胞菌临床菌株使用一种毒素，溶出素（ExlA）来破坏宿主细胞膜。在这里，我们证明了ExlA的输出需要一个预测的外膜蛋白ExlB，这表明ExlA和ExlB定义了P. aeruginosa一个新的活跃的双伴侣分泌（TPS）系统。除了TPS信号外，ExlA还包含几个不同的结构域，包括一个血凝素结构域、五个精氨酸-甘氨酸-天冬氨酸（RGD）基序和一个缺乏任何可识别序列基序的c端区域。然而，这个c端区域对毒性活性很重要，因为它的删除消除了宿主细胞的裂解。利用脂质囊泡和真核细胞，包括红细胞，我们证明了ExlA在有核细胞的细胞膜破坏之前具有成孔活性。最后，我们开发了一种基于细胞的高通量活死实验，并使用它来筛选产生exla的铜绿假单胞菌临床菌株的转座子突变文库，以寻找exla介导毒性所需的细菌因子。筛选结果确定了参与IV型菌毛形成的蛋白质，这些蛋白质是ExlA通过促进细菌与宿主细胞之间的密切接触来发挥其细胞毒性活性所必需的。这些发现代表了在宿主细胞中毒中，TPS家族的成孔毒素和表面附属物之间合作的第一个例子。铜绿假单胞菌临床分离株急性致毒感染的过程和结果依赖于两种毒力策略之一的部署：通过众所周知的III型分泌系统递送效应器或最近发现的双伴侣分泌毒素exolysin的细胞溶解活性。在这里，我们描述了由溶解素介导的哺乳动物细胞中毒过程的几个特征。我们发现溶解素需要外膜蛋白ExlB才能输出到细胞外培养基中。通过体外重组蛋白和离体实验，我们证明了溶解素的成孔活性。在一种产生溶解素的临床菌株中对转座子突变文库进行细胞毒性筛选，发现IV型菌毛是溶解素毒性功能所需的细菌附属物。这项工作揭示了铜绿假单胞菌临床菌株缺乏III型分泌系统所使用的新型毒力因子活性的分子机制，包括产毒细菌附着在靶细胞上诱导细胞溶解的要求，并确定了开发抗毒策略的新靶点。
Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa. In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycineaspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication.IMPORTANCE The course and outcome of acute, toxigenic infections by Pseudomonas aeruginosa clinical isolates rely on the deployment of one of two virulence strategies: delivery of effectors by the well-known type III secretion system or the cytolytic activity of the recently identified two-partner secreted toxin, exolysin. Here, we characterize several features of the mammalian cell intoxication process mediated by exolysin. We found that exolysin requires the outer membrane protein ExlB for export into extracellular medium. Using in vitro recombinant protein and ex vivo assays, we demonstrated a pore-forming activity of exolysin. A cellular cytotoxicity screen of a transposon mutant library, made in an exolysin-producing clinical strain, identified type IV pili as bacterial appendages required for exolysin toxic function. This work deciphers molecular mechanisms underlying the activity of novel virulence factors used by P. aeruginosa clinical strains lacking the type III secretion system, including a requirement for the toxin-producing bacteria to be attached to the targeted cell to induce cytolysis, and defines new targets for developing antivirulence strategies.