Wolbachia disrupts eukaryotic endolysosomal membrane dynamics

项目摘要 盘尾丝虫科（Onchocerediae）的丝状线虫引起使人衰弱的疾病，例如淋巴结炎。 丝虫病由于目前约有1.5亿人感染这些线虫， 病原体生物学的深入知识将有助于解决全球健康问题。众所周知， 线虫马来丝虫（Brugia malayi）具有沃尔巴克氏体属（Wolbachia genus）的细胞内共生细菌，并且该 这种关系是必不可少的;用抗生素清除线虫中的沃尔巴克氏体， 死亡了解沃尔巴克氏体在线虫体内维持细胞内存活的机制 因此可能为控制病原线虫种群提供重要途径， 布鲁吉亚氏菌和沃尔巴克氏菌都不适合基因操作。重要发现 因此，细菌与线虫在分子水平上的相互作用被证明是极其困难的。 在这项提议中，我们的目标是利用沃尔巴克氏体的蛋白质， 酵母内溶酶体膜动力学的保守途径。这些分泌的“效应”蛋白是 已知改变宿主过程以支持细菌在真核宿主中的存活， 确保其自身繁殖，因此是影响真核生物生理学的有效试剂。为此， 我的实验室采用了芽殖酵母，酿酒酵母（Sce），作为一个模型系统， 发现调节真核细胞生物学的细菌蛋白质，重点是那些 抑制细胞内膜融合和蛋白运输途径。 在先前的候选wBm分泌效应蛋白的筛选中，我们已经鉴定了 有能力操纵真核生物的wBM。在这项工作中，我们发现了一种这样的蛋白质， wBm 0152，强烈抑制内体：空泡运输途径在体外。这种抑制似乎是由 调节保守的ESCRT复合物。由于已知wBm在其宿主中改变膜动力学， 它的共生，再加上事实上，膜动力学的调节是强烈保守的整个 真核生物，本提案中进行的详细遗传、分子和生物化学研究将适用于 到wBM：B。马来人之间的相互作用，从而导致人类丝虫病。最后，利用我们实验室的优势 在微生物学、细胞生物学和蛋白质/脂质生物化学中，我们将仔细地详细介绍 这种沃尔巴克氏体衍生的ESCRT调节剂，并确定了酵母中的重要调节剂和结合伴侣， 很可能是布鲁吉亚的保守基因这项工作将开始描述迄今未知的wBM：B。马来互动， 从而不仅为布鲁日丝虫生理学提供了新的见解，而且为ESCRT依赖性 真核生物的活动。

PROJECT SUMMARY Filarial nematodes of the family Onchocercidae cause debilitating human diseases, such as lymphatic filariasis. As approximately 150 million individuals are currently infected with these nematodes, obtaining in- depth knowledge of pathogen biology will serve to address a global health issue. It is known that the filarial nematode, Brugia malayi, harbors an intracellular endosymbiotic bacterium of the Wolbachia genus, and this relationship is essential; clearance of Wolbachia from the nematode with antibiotics leads to eventual nematode death. Understanding the mechanisms by which Wolbachia maintains its intracellular survival within nematodes would therefore likely provide an important avenue towards controlling pathogenic nematode populations, but both Brugia and Wolbachia are not amenable to genetic manipulations. Discoveries of important bacterium:nematode interactions at the molecular level, therefore, have proven exceedingly difficult. In this proposal, our goals are to utilize proteins from Wolbachia to genetically and biochemically dissect conserved pathways of endolysosomal membrane dynamics in yeast. These secreted “effector” proteins are known to alter host processes in order to support the survival of the bacterium in the eukaryotic host and to ensure its own reproduction, and are therefore potent reagents that impact eukaryotic physiology. To this end, my laboratory has employed the budding yeast, Saccharomoyces cerevisiae (Sce), as a model system towards the discovery of bacterial proteins that modulate eukaryotic cellular biology, with a focus on those proteins which inhibit intracellular membrane fusion and protein trafficking pathways. In a previous screen of candidate wBm secreted effector proteins, we have already identified proteins from wBm that have the ability to manipulate eukaryotic biology. In this work, we show that one such protein, wBm0152, strongly inhibits endosome:vacuole trafficking pathways in vitro. This inhibition appears to result from modulation of the conserved ESCRT complex. As wBm is known to alter membrane dynamics in its host during its symbiosis, and coupled with the fact that regulation of membrane dynamics is strongly conserved throughout eukaryotes, the detailed genetic, molecular, and biochemical studies carried out in this proposal will be applicable to wBm:B. malayi interactions, and thus, human filarial diseases. Finally, leveraging our laboratory's strengths in microbiology, cellular biology, and protein/ lipid biochemistry, we will carefully detail the biochemical activity of this Wolbachia-derived ESCRT modulator and identify important regulators and binding partners in yeast, which are likely conserved in Brugia. This work will begin to describe heretofore unknown wBm:B. malayi interactions, thus providing novel insight into not only Brugia physiology, but also provide new insight into ESCRT-dependent activities in eukaryotes.