Identifying Wolbachia effectors that facilitate host infection

项目概要/摘要 Wolbachia pipientis是一种专性细胞内α-变形杆菌，感染40-60%的昆虫物种， 木星的远近沃尔巴克氏体感染抑制RNA病毒在昆虫中的复制，这种现象被称为病原体 阻挡。因此，世界上许多地方正在释放受沃尔巴克氏体感染的蚊子，以控制蚊子的数量。 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不是 已知，沃尔巴克氏体必须定殖宿主并有效地通过母体传播，以便病原体 阻止工作。我们的长期目标是确定Wolbachia用于建立感染的机制。 为此，我们专注于IV型分泌系统（T4 SS），这是Wolbachia使用的分子纳米机器， 将称为效应子的蛋白质注入宿主细胞环境。通过这些分泌的效应物，宿主细胞被 经过修饰，使沃尔巴克氏体得以入侵并持续存在。 我们以前的工作确定并表征了Wolbachia中的第一个分泌效应子（WalE 1），并建立了 T4 SS基因表达和分泌效应子表达之间的重要相关性。Wolbachia 效应子和T4 SS在宿主化蛹期间上调。令人惊讶的是，WalE 1的过度表达在 沃尔巴克氏体感染的苍蝇促进了发育中的卵母细胞的定植和母体传播。这些 结果导致我们的中心假设，沃尔巴克氏体使用分泌的效应子来建立和维持宿主 殖民化对于这一假设，我们已经确定了整个属的沃尔巴克氏体效应子，并使用了一个 大规模的生长筛选，以进一步确定沃尔巴克氏体效应。我们发现许多沃尔巴克氏体 效应子是进化枝特异性的，但许多在昆虫相关菌株之间是共享的。协同 Peter Christie（UTH），我们开发了一种用于Wolbachia效应子的异源分泌测定，使我们能够 证实IV型偶联蛋白VirD 4和候选沃尔巴克氏体效应子之间的相互作用。指导 强有力的初步数据，我们建议追求三个具体目标，以确定和表征沃尔巴克氏体 效应器和它们的宿主目标，利用果蝇系统的力量。我们将（1）确定 Wolbachia分泌组，（2）预测效应子及其在Wolbachia感染昆虫中的保守性，以及（3） 鉴定促进沃尔巴克氏体感染的宿主靶标和效应物。 沃尔巴克氏体与宿主相互作用的研究仍处于起步阶段，尽管 昆虫繁殖和进化的内共生关系，以及改变媒介能力的能力。 这些拟议的研究将大大推进我们对沃尔巴克氏体如何利用其T4 SS 建立感染，这是病原体阻断的必要前提。

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.