Wolbachia disrupts eukaryotic endolysosomal membrane dynamics

沃尔巴克氏菌破坏真核细胞内溶酶体膜动力学

• 批准号: 10667824
• 负责人: Vincent Joseph Starai
• 金额: $ 18.42万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-15 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10667824
• 关键词: Address; Adult; Antibiotics; Bacteria; Bacterial Proteins; Binding; Biochemical; Biological Models; Biology; Biotinylation; Brugia; Brugia malayi; Cellular biology; Cessation of life; Complex; Coupled; Data; Development; Disease; Dissection; Elephantiasis; Endosomes; Ensure; Eukaryota; Family; Filarial Elephantiases; Filariasis; Genes; Genetic; Goals; Human; In Vitro; Individual; Intracellular Membranes; Ivermectin; Knowledge; Laboratories; Lipid Biochemistry; Membrane; Membrane Fusion; Membrane Proteins; Microbiology; Modeling; Molecular; Nematoda; Ocular Onchocerciasis; Organism; Outcome; Pathogenicity; Pathway interactions; Physiology; Population; Process; Proteins; Publishing; Reagent; Regulation; Reporting; Reproduction; Resistance; Saccharomycetales; Symbiosis; System; Technology; Testing; Tissues; Vacuole; Wolbachia; Work; Yeasts; endosymbiont; genetic manipulation; global health; human disease; human pathogen; in vivo; inhibitor; insight; novel; pathogen; protein degradation; protein function; protein protein interaction; protein transport; receptor; tool; trafficking; yeast protein

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.

项目总结 杀螺螺科的丝状线虫会导致人类的疾病，如淋巴疾病 丝虫病。由于目前约有1.5亿人感染了这些线虫，获得了- 对病原体生物学的深入了解将有助于解决一个全球健康问题。众所周知，丝虫病 线虫，马来丝虫，拥有一种沃尔巴克氏菌属的细胞内内共生细菌，这 关系是必不可少的；用抗生素清除线虫中的沃尔巴克氏菌最终会导致线虫 死亡。了解沃尔巴克氏杆菌在线虫体内维持其细胞内生存的机制 因此很可能为控制致病线虫种群提供了重要途径，但 布鲁吉亚和沃尔巴克氏菌都不受基因操纵的影响。重要的发现 细菌：因此，在分子水平上线虫的相互作用被证明是极其困难的。 在这项提案中，我们的目标是利用沃尔巴克氏菌的蛋白质从遗传和生物化学上进行解剖 酵母内溶酶体膜动力学的保守途径。这些分泌的“效应器”蛋白是 已知可以改变宿主过程以支持细菌在真核宿主中的生存并 确保其自身繁殖，因此是影响真核生理的有效试剂。为此， 我的实验室采用萌芽酵母Saccharomyces cerevisiae(SCE)作为模型系统，用于 调节真核细胞生物学的细菌蛋白质的发现，重点是那些能够 抑制细胞内膜融合和蛋白质转运途径。 在先前对候选WBM分泌的效应蛋白的筛选中，我们已经确定了蛋白质 来自有能力操纵真核生物学的WBM。在这项工作中，我们展示了一种这样的蛋白质， WBm0152，在体外强烈抑制内体：空泡运输途径。这种抑制似乎是由于 对保守的ESCRT复合体的调节。因为已知WBM会改变宿主体内的膜动力学 它的共生性，再加上膜动力学的调节在整个过程中都是非常保守的 对于真核生物，本提案中进行的详细的遗传、分子和生化研究将适用。 对于WBM：B.Malayi相互作用，因此，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/脂类生物化学中，我们将详细介绍 这种沃尔巴克氏菌衍生的ESCRT调节剂，并确定酵母中重要的调节因子和结合伙伴，这 很可能保存在布鲁贾。这项工作将开始描述迄今未知的WBM：B.Malayi相互作用， 从而不仅为布鲁氏菌的生理学提供了新的见解，而且也为ESCRT依赖提供了新的见解 真核生物中的活动。