The V antigen of Pseudomonas aeruginosa is required for assembly of the functional PopB/PopD translocation pore in host cell membranes
The V antigen of Pseudomonas aeruginosa is required for assembly of the functional PopB/PopD translocation pore in host cell membranes
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DOI：
10.1128/iai.72.8.4741-4750.2004
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发表时间：
2004-08-01
影响因子：
3.1
通讯作者：
Attree, I
中科院分区：
文献类型：
--
作者：
Goure, J;Pastor, A;Attree, I
铜绿假单胞菌通过III型分泌易位系统（TTSS）的活性有效地毒害真核细胞。易位操纵子pcrGVH-popBD内的基因缺失消除了具有巨噬细胞和TTSS依赖性溶血的铜绿假单胞菌菌株的孔形成活性。在这里，我们调查的要求PcrV，PopB，和PopD孔形成分析特定的突变体，使用红细胞（RBC）和成纤维细胞表达绿色荧光蛋白融合肌动蛋白。同时分泌三种蛋白质，PopB，PopD和PcrV，需要实现野生型溶血和效应易位。在细胞毒性菌株中删除pcrV不影响PopB和PopD的分泌，但废除了溶血活性和效应子易位到成纤维细胞中。值得注意的是，PcrV缺陷型突变体不能将PopD插入宿主细胞膜，而PopB和PopD，而不是PcrV，很容易在野生型感染的RBC的膜内发现。通过使用脂质体模型的孔组件进行的免疫沉淀实验揭示了PopD和PopB之间的直接相互作用，但不是PopD和PcrV之间。因此，PcrV是PopB/D易位复合物的功能组装所必需的，但不直接与成孔Pop蛋白相互作用。
Pseudomonas aeruginosa efficiently intoxicates eukaryotic cells through the activity of the type III secretion-translocation system (TTSS). Gene deletions within the translocation operon pcrGVH-popBD abolish pore-forming activity of P. aeruginosa strains with macrophages and TTSS-dependent hemolysis. Here we investigated the requirements for PcrV, PopB, and PopD in pore formation by analyzing specific mutants using red blood cells (RBCs) and fibroblasts expressing green fluorescent protein fused to actin. Simultaneous secretion of three proteins, PopB, PopD, and PcrV, was required to achieve wild-type hemolysis and effector translocation. Deletion of pcrV in a cytotoxic strain did not affect secretion of PopB and PopD but abolished hemolytic activity and translocation of effectors into fibroblasts. Notably, the PcrV-deficient mutant was not capable of inserting PopD into host cell membranes, whereas PopB and PopD, but not PcrV, were readily found within membranes of wild-type-infected RBCs. Immunoprecipitation experiments performed by using a liposome model of pore assembly revealed a direct interaction between PopD and PopB but not between PopD and PcrV. Consequently, PcrV is necessary for the functional assembly of the PopB/D translocon complex but does not interact directly with pore-forming Pop proteins.