VopE of Vibrio cholerae mediates mitochondrial dynamics by the Miro GTPase

霍乱弧菌的 VopE 通过 Miro GTPase 介导线粒体动力学

• 批准号: 8834059
• 负责人: Olga V Danilchanka
• 金额: $ 2.48万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8834059
• 关键词: Anti-Bacterial Agents; Anti-Inflammatory Agents; Antiviral Agents; Bacteria; Bacterial Infections; Binding; Biochemistry; Biological Assay; C Type Lectin Receptors; Cell Line; Cells; Cellular biology; Cholera; Cholera Toxin; Complement Factor B; Complex; Data; Detection; Development; Elements; Epithelial; Event; Flagellin; Generations; Genes; Gram-Negative Bacteria; Guanosine Triphosphate Phosphohydrolases; Human; Immune; Immune response; Immune system; Infection; Inflammatory; Interferon Type I; Interferons; Life; Lipopolysaccharides; Lipoproteins; Mediating; Microscopy; Mitochondria; Mitochondrial Membrane Protein; Mitogen-Activated Protein Kinase Kinases; Mitogens; Modification; Molecular; Morphology; Motor; Natural Immunity; Nuclear; Nucleic Acids; Nucleotides; Outcome; Outer Mitochondrial Membrane; Pathway interactions; Pattern; Pattern recognition receptor; Peptidoglycan; Pilum; Play; Process; Protein Inhibition; Proteins; Reactive Oxygen Species; Receptor Signaling; Role; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Staining method; Stains; Structure; Surface; System; Toll-like receptors; Toxin; Transfection; Tretinoin; Type III Secretion System Pathway; Vibrio cholerae; Virulence; Work; antimicrobial; blind; chemokine; cytokine; interest; killings; mutant; novel; overexpression; pathogen; pathogenic bacteria; protein activation; public health relevance; receptor; research study; response; rho GTP-Binding Proteins; success; theories; trafficking

 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.

 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先天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。为了抵消细菌中保守结构的识别，理论上，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依赖于使用靶向诱导先天免疫应答的物种特异性效应物的策略。通过细菌递送这种效应物需要复杂的纳米机器，例如III型分泌系统（T3 SS）。我们的研究表明，非O 1，非O 139霍乱弧菌菌株AM-19226，缺乏霍乱毒素和毒素协同调节菌毛的毒力依赖于T3 SS的活性。我们发现，至少有4种不同的效应，包括VopE，需要上皮表面破坏和炎症反应。然而，这些效应物的靶点和机制尚未完全表征。在这项提案中，我的目标是确定控制避免先天性免疫反应的分子机制，由霍乱弧菌AM-19226。我们的初步数据表明，效应子VopE在感染期间靶向线粒体。此外，线粒体形态急剧变化的细胞感染的vopE突变体的T3 SS依赖性的方式，这是明显的核周线粒体簇相比，野生型感染的细胞显着增加。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的聚集和I型干扰素信号传导的抑制所需的。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，该途径对于识别霍乱弧菌AM-19226至关重要。在目的1中，我将确定细菌效应物，诱导聚集的线粒体在缺乏VopE，并将确定其活动的机制（S）利用显微镜，生物化学和细胞生物学方法。在目标2中，我将确定米罗-1/2诱导MAVS聚类的机制，使用活细胞显微镜和敲除实验的组合。我的假设是Miro-1/2通过与MAVS调节蛋白（如Mfn-2，一种已知与Miro和MAVS相互作用的线粒体融合所必需的蛋白）相互作用诱导MAVS聚集。在目标3中，我将确定响应于霍乱弧菌而释放的细胞因子，并将定义感染期间先天免疫应答的重要性。这些发现将定义霍乱弧菌破坏以避免宿主识别和诱导先天免疫反应的关键信号事件，并可能对新型抗菌和抗炎药物的后续开发产生重大影响。