Functional analysis of VopF activity required for colonization in Vibrio cholerae.
Functional analysis of VopF activity required for colonization in Vibrio cholerae.
复制标题
霍乱弧菌定植所需的 VopF 活性的功能分析。
DOI：
10.1128/mbio.00289-10
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发表时间：
2010
期刊：
影响因子：
6.4
通讯作者：
Mekalanos,JohnJ
中科院分区：
文献类型：
--
作者：
Tam,VincentC;Suzuki,Masato;Coughlin,Margaret;Saslowsky,David;Biswas,Kuntal;Lencer,WayneI;Faruque,ShahM;Mekalanos,JohnJ
霍乱弧菌是一种革兰氏阴性兼性致病菌，是霍乱的病原体。我们以前的特点是临床分离，AM-19226，易位的III型分泌系统（T3 SS）的效应蛋白与肌动蛋白成核活性，VopF，进入宿主细胞。从比较基因组研究中，我们确定了一个不同的T3 SS岛在额外的分离物，具有VopF同源，VopN。与VopF介导的突起形成不同，VopN定位于宿主细胞中的应力纤维，类似于VopL，其存在于副溶血性弧菌的大流行菌株中。嵌合体和酵母双杂交研究表明，VopF和VopN蛋白的氨基末端区域与不同的宿主细胞因子相互作用。我们确定AM-19226感染的细胞被阻滞在细胞周期的S期，并且VopF/VopN是抗凋亡因子。为了了解VopF如何参与AM-19226的发病机制，我们在体外极化上皮模型和体内成年兔回肠模型中检测了VopF的作用。在T3 SS致病性岛内是VopE，其是来自耶尔森氏菌的YopE的同源物，已显示其使紧密连接松动。在极化的肠上皮细胞中，VopF和VopE通过诱导皮质肌动蛋白解聚和紧密连接蛋白ZO-1的异常定位而损害紧密连接的完整性。在成年兔腹泻模型中的致病性测定表明，这些效应物参与引起感染兔的腹泻反应。重要性霍乱弧菌是一种细菌病原体，导致腹泻病霍乱，这仍然是许多发展中国家的主要公共卫生问题。虽然流行性霍乱弧菌菌株的主要毒力因子已被表征，但霍乱弧菌的新临床菌株已经出现，使用未知的致病机制引起散发性霍乱样疾病。此前，我们在一种新的临床霍乱弧菌菌株中发现了III型分泌系统，并鉴定了一种效应蛋白VopF，该蛋白被注射到宿主细胞中并诱导肌动蛋白细胞骨架的变化。在这项工作中，我们确定了一个同源的VopF，导致一个独特的细胞表型和效应器和宿主蛋白之间的相互作用。我们还发现，这两种效应物都能阻止感染细胞中细菌诱导的细胞死亡。在我们的组织培养和动物模型中，我们表明VopF有助于破坏上皮完整性和炎症反应。
Vibrio cholerae, a Gram-negative facultative pathogen, is the etiologic agent for the diarrheal disease cholera. We previously characterized a clinical isolate, AM-19226, that translocates a type III secretion system (T3SS) effector protein with actin-nucleating activity, VopF, into the host cells. From comparative genomic studies, we identified a divergent T3SS island in additional isolates which possess a VopF homolog, VopN. Unlike the VopF-mediated protrusion formation, VopN localizes to stress fiber in host cells similarly to VopL, which is present in the pandemic strain of Vibrio parahaemolyticus. Chimera and yeast two-hybrid studies indicated that the amino-terminal regions of VopF and VopN proteins interact with distinct host cell factors. We determined that AM-19226-infected cells are arrested at S phase of the cell cycle and that VopF/VopN are antiapoptotic factors. To understand how VopF may contribute to the pathogenesis of AM-19226, we examined the effect of VopF in anin vitropolarized-epithelial model and anin vivoadult rabbit diarrheal model. Within the T3SS pathogenicity island is VopE, a homolog of YopE fromYersinia, which has been shown to loosen tight junctions. In polarized intestinal epithelia, VopF and VopE compromised the integrity of tight junctions by inducing cortical actin depolymerization and aberrant localization of the tight-junction protein ZO-1. An assay for pathogenicity in the adult rabbit diarrhea model suggested that these effectors are involved in eliciting the diarrheal response in infected rabbits.IMPORTANCEVibrio cholerae is a bacterial pathogen that causes the diarrheal disease cholera, which remains a major public health problem in many developing countries. While the major virulence factors of the pandemic V. cholerae strains have been characterized, new clinical strains of V. cholerae have arisen, causing sporadic cholera-like diseases using unknown pathogenic mechanisms. Previously, we discovered the type III secretion system in a new clinical strain of V. cholerae and also identified an effector protein, VopF, which is injected into the host cells and induces changes in the actin cytoskeleton. In this work, we identified a homolog of VopF that causes a distinct cellular phenotype and interactions between the effectors and host proteins. We also discovered that both effectors prevent bacterium-induced cell death in infected cells. In our tissue culture and animal models, we showed that VopF contributes to the disruption of epithelial integrity and the diarrheal response.