Identifying Wolbachia effectors that facilitate host infection

识别促进宿主感染的沃尔巴克氏体效应子

• 批准号: 10132974
• 负责人: Irene Newton
• 金额: $ 38.61万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-10 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10132974
• 关键词: Actins; Aedes; Alphaproteobacteria; Animals; Arbovirus Infections; Bacteria; Bioinformatics; Biological Assay; Biology; Cell Line; Cell physiology; Cells; Cellular biology; Collaborations; Communicable Diseases; Coupling; Culicidae; Data; Dengue Virus; Disease Vectors; Drosophila genus; Drosophila melanogaster; Environment; Evolution; Future; Gene Expression; Genetic; Genome; Goals; Growth; Immunoprecipitation; In Vitro; Infection; Insecta; Invaded; Investigation; Mass Spectrum Analysis; Mediating; Modeling; Modification; Molecular; Oocytes; Parasites; Phenotype; Planet Earth; Planets; Proteins; Proteomics; RNA Viruses; Regulation; Reproduction; Research; Specificity; Symbiosis; System; Transgenic Organisms; Type IV Secretion System Pathway; Vaccines; Viral; Virus; Virus Replication; Wolbachia; Work; comparative genomics; differential expression; disorder control; fly; genetic manipulation; host colonization; human disease; infancy; interest; mortality; mosquito-borne pathogen; nanomachine; next generation; non-Native; overexpression; pathogen; prevent; programs; symbiont; transmission process; vector competence; vector control

PROJECT SUMMARY/ABSTRACT Wolbachia pipientis is an obligate intracellular alpha-proteobacterium that infects 40-60% of insect species on the planet. Wolbachia infection inhibits RNA virus replication in insects, a phenomenon known as pathogen blocking. Therefore, Wolbachia infected mosquitos are being released in many parts of the world to control the spread of human diseases. Importantly, although the mechanism behind Wolbachia’s virus inhibition is not known, Wolbachia must colonize the host and be efficiently maternally transmitted in order for pathogen blocking to work. Our long-term goals are to identify the mechanisms used by Wolbachia to establish infection. To that end, we focus on the type IV secretion system (T4SS), a molecular nanomachine used by Wolbachia to inject proteins, termed effectors, into the host cellular environment. Via these secreted effectors, the host cell is modified, allowing Wolbachia to invade and persist. Our previous work identified and characterized the first secreted effector in Wolbachia (WalE1) and established an important correlation between T4SS gene expression and expression of secreted effectors. Wolbachia effectors and the T4SS are upregulated during host pupation. Strikingly, overexpression of WalE1 in Wolbachia-infected flies facilitated colonization of the developing oocyte and maternal transmission. These results led to our central hypothesis that Wolbachia uses secreted effectors to establish and maintain host colonization. Towards this hypothesis, we have identified Wolbachia effectors across the genus, and used a large-scale growth screen to further identify Wolbachia effectors. We have discovered that many Wolbachia effectors are clade specific, but many are shared between the insect associated strains. In collaboration with Peter Christie (UTH), we developed a heterologous secretion assay for Wolbachia effectors, allowing us to confirm interactions between the type IV coupling protein VirD4 and candidate Wolbachia effectors. Guided by strong preliminary data, we propose to pursue three Specific Aims to identify and characterize Wolbachia effectors, and their host targets, using the power of the Drosophila system. We will (1) determine the Wolbachia secretome, (2) predict effectors and their conservation across Wolbachia infecting insects, and (3) identify host targets and effectors that facilitate infection by Wolbachia. Studies of Wolbachia - host interactions are still in their infancy despite the recognized contributions of endosymbiotic associations to insect reproduction and evolution, and the ability to alter vector competence. These proposed studies will significantly advance our understanding of how Wolbachia employs its T4SS to establish infection, a necessary prerequisite to pathogen blocking.

项目概要/摘要 Wolbachia pipientis 是一种专性细胞内 α 变形菌，可感染 40-60% 的昆虫物种 这个星球。沃尔巴克氏体感染抑制昆虫中 RNA 病毒的复制，这种现象称为病原体 阻塞。因此，世界许多地方都释放了感染沃尔巴克氏体的蚊子来控制 人类疾病的传播。重要的是，尽管沃尔巴克氏体病毒抑制背后的机制尚不清楚 已知，沃尔巴克氏体必须定植于宿主并有效地母系传播才能传播病原体 阻碍工作。我们的长期目标是确定沃尔巴克氏体用于建立感染的机制。 为此，我们重点关注 IV 型分泌系统（T4SS），这是沃尔巴克氏菌用来 将称为效应物的蛋白质注入宿主细胞环境。通过这些分泌的效应子，宿主细胞 修改，允许沃尔巴克氏体入侵并持续存在。 我们之前的工作鉴定并表征了沃尔巴克氏体 (WalE1) 中的第一个分泌效应子，并建立了 T4SS 基因表达与分泌效应子表达之间的重要相关性。沃尔巴克氏体 效应子和 T4SS 在宿主化蛹期间上调。引人注目的是，WalE1 在 感染沃尔巴克氏体的果蝇促进了正​​在发育的卵母细胞的定植和母体传播。这些 结果得出了我们的中心假设：沃尔巴克氏体利用分泌的效应器来建立和维持宿主 殖民化。针对这一假设，我们已经确定了整个属的沃尔巴克氏体效应子，并使用了 大规模生长筛选以进一步鉴定沃尔巴克氏体效应子。我们发现许多沃尔巴克氏体 效应器是进化枝特异性的，但许多效应器在昆虫相关品系之间共享。与合作 Peter Christie (UTH)，我们开发了一种针对沃尔巴克氏体效应子的异源分泌测定法，使我们能够 证实 IV 型偶联蛋白 VirD4 和候选沃尔巴克氏体效应子之间的相互作用。指导者 强有力的初步数据，我们建议追求三个具体目标来识别和表征沃尔巴克氏体 效应器及其宿主目标，利用果蝇系统的力量。我们将 (1) 确定 沃尔巴克氏体分泌组，(2) 预测沃尔巴克氏体感染昆虫的效应子及其保守性，以及 (3) 识别促进沃尔巴克氏体感染的宿主靶标和效应器。 尽管沃尔巴克氏体与宿主相互作用的研究已得到公认的贡献，但仍处于起步阶段 内共生与昆虫繁殖和进化的关联，以及改变载体能力的能力。 这些拟议的研究将显着增进我们对沃尔巴克氏体如何利用其 T4SS 来 建立感染是阻断病原体的必要先决条件。