VopE of Vibrio cholerae mediates mitochondrial dynamics by the Miro GTPase

 描述(由申请人提供)：细菌病原体的成功取决于其避免被宿主天然免疫系统识别的能力。大量保守的病原体相关分子模式被宿主受体识别，包括核酸、脂蛋白、鞭毛蛋白、脂多糖和肽聚糖。为了抵消对细菌中保守结构的识别，理论上，这种识别应该足以诱导抗菌机制和清除任何病原体，革兰氏阴性细菌进化出了依赖于使用物种特异性效应器的策略，这些效应器的目标是诱导先天性免疫反应。由细菌运送这种效应器需要复杂的纳米机械，如III型分泌系统(T3SS)。本课题组研究表明，无霍乱毒素和毒素共调控菌毛的非O1、非O139霍乱弧菌AM-19226的毒力依赖于T3SS的活性。我们发现，包括VopE在内的至少4种不同的效应物是上皮表面破坏和腹泻反应所必需的。然而，这些效应器的靶点和机制还没有得到充分的表征。在这项建议中，我的目标是确定控制霍乱弧菌AM-19226避免先天免疫反应的分子机制。我们的初步数据显示，在感染过程中，效应器VopE针对线粒体。此外，在感染VopE突变体的细胞中，线粒体形态以T3SS依赖的方式发生了剧烈的变化，这明显表现为与野生型感染细胞相比，核周围线粒体聚集的显著增加。线粒体动力学的调节是由于VopE和线粒体Rho GTP酶Miro-1和Miro-2之间的特异性相互作用，并且是线粒体外膜蛋白MAVS聚集和抑制I型干扰素信号所必需的。这些数据表明，VopE活性是抑制保守的病原体识别途径所必需的，这是识别霍乱弧菌AM-19226所必需的。在目标1中，我将鉴定在没有VopE的情况下诱导线粒体聚集的细菌效应物，并将利用显微镜、生物化学和细胞生物学方法确定它们的活性机制(S)。在目标2中，我将结合活细胞显微镜和基因敲除实验来确定Miro-1/2诱导MAV聚集的机制。我的假设是，Miro-1/2通过与MAV调节蛋白(如MFN-2)相互作用而诱导MAV聚集，MFN-2是已知与Miro和MAV相互作用的线粒体融合所必需的蛋白质。在目标3中，我将确定霍乱弧菌释放的细胞因子，并将确定感染期间先天免疫反应的重要性。这些发现将确定霍乱弧菌为避免宿主识别和诱导先天性免疫反应而颠覆的关键信号事件，并可能对后续新型抗菌和抗炎药物的开发产生重大影响。

 DESCRIPTION (provided by applicant): Success of a bacterial pathogen depends on its ability to avoid recognition by the host innate immune system. A plethora of conserved pathogen-associated molecular patterns are recognized by host receptors including nucleic acids, lipoproteins, flagellin, lipopolysaccharides, and peptidoglycans. To counteract recognition of structures conserved in bacteria that, in theory, should be sufficient for the induction of antibacterial mechanisms and clearance of any pathogen, Gram-negative bacteria evolved strategies that rely on a use of species-specific effectors that target induction of the innate immune response. Delivery of such effectors by bacteria requires complex nanomachineries such as Type III Secretion System (T3SS). Our group showed that virulence of non-O1, non-O139 Vibrio cholerae strain AM-19226 that lacks cholera toxin and toxin-coregulated pilus relies on the activity of T3SS. We showed that at least 4 different effectors, including VopE, are required for epithelial surface disruption and diarrheal response. However, targets and mechanisms of these effectors are not fully characterized. In this proposal I aim to identify molecular mechanisms that govern avoidance of innate immune responses by V. cholerae AM-19226. Our preliminary data showed that the effector VopE is targeted to mitochondria during infection. Furthermore, mitochondrial morphology was drastically changed in cells infected with the vopE mutant in a T3SS-dependent manner, which was apparent by a significant increase in perinuclear mitochondrial clustering compared to the wild-type-infected cells. Modulation of mitochondrial dynamics was due to a specific interaction between VopE and mitochondrial Rho GTPases Miro-1 and Miro-2, and was required for clustering of the mitochondrial outer membrane protein MAVS and inhibition of type I interferon signaling. These data indicate that VopE activity is required for inhibition of a conserved pathogen-recognition pathway that is essential for recognition of V. cholerae AM-19226. In Aim 1, I will identify bacterial effectors tht induce clustering of mitochondria in the absence VopE and will determine mechanism(s) of their activity utilizing microscopy, biochemistry, and cell biology approaches. In Aim 2, I will determine the mechanism by which Miro-1/2 induces clustering of MAVS using a combination of live-cell microscopy and knockdown experiments. My hypothesis is that Miro-1/2 induces clustering of MAVS through interaction with MAVS-regulating proteins such as Mfn-2, a protein essential for mitochondrial fusion that is known to interact both with Miro and MAVS. In Aim 3, I will determine the cytokines that are released in response to V. cholerae, and will define the importance of the innate immune response during infection. These findings will define key signaling events that V. cholerae subvert to avoid host recognition and induction of the innate immune response, and can have a large impact on subsequent development of novel antibacterial and anti-inflammatory agents.