Host cell factors controlling type III secretion effector translocation

控制III型分泌效应器易位的宿主细胞因子

• 批准号: 10586145
• 负责人: Arne Rietsch
• 金额: $ 20.13万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-07 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10586145
• 关键词: 5' Splice Site; Affect; Attenuated; Bacteria; Bacterial Attachment Site; Biochemical Pathway; CRISPR/Cas technology; Cell Line; Cell membrane; Cell physiology; Cell secretion; Cells; Disease; Docking; Down-Regulation; Ebola virus; Experimental Designs; Feedback; Gene Activation; Gene Expression; Genes; Genomics; Goals; Haploid Cells; Haploidy; High-Throughput Nucleotide Sequencing; Hospitals; Human Cell Line; Immune; Immune system; Infection; Injections; Insertion Mutation; Integration Host Factors; Life Style; Mediating; Molecular; Molecular Conformation; Molecular Machines; Mutagenesis; Mutagens; Mutation; Needles; Pathogenicity; Pathway interactions; Perforation; Phospholipase; Plasma Cells; Play; Process; Production; Proteins; Pseudomonas aeruginosa; Research; Resistance; Role; Site; Structure; Syringes; System; Toxin; Type III Secretion System Pathway; Work; base; candidate identification; cytotoxic; drug development; experimental study; gain of function; gain of function mutation; genome-wide; loss of function mutation; mortality; mutant; novel; overexpression; pathogen; promoter; secretion process; sensor; small molecule inhibitor

Abstract Type III secretion systems (T3SS) are intricate molecular machines that allow Gram-negative pathogens to directly inject effector proteins into host cells. While the arsenal of injected effector proteins varies among pathogens and supports a wide variety of pathogenic lifestyles, the core apparatus is conserved. A key feature of T3SSs is that effector translocation is triggered by host-cell contact. In order to inject proteins, the bacterium attaches to the host cell and uses the T3SS to insert pore-forming translocator proteins into the host cell plasma membrane. The tip of the needle is docked to this pore, forming the translocon. Host cell contact is sensed by a conformational change in the pore that is propagated to the base of the apparatus. While there is ample evidence that translocon assembly and function are modulated by the host cell, which cellular functions are involved, and what step in the translocation process they influence is poorly understood. We are proposing to perform a genome wide saturation mutagenesis selection in a haploid human cell line to identify cellular pathways that impact effector translocation by the Pseudomonas aeruginosa type III secretion system. We will employ a transposon that also harbors a constitutive promoter and splice donor site, which will allow us to not only identify loss of function mutations, but also gain of function mutations resulting from artificial production of a downstream gene. Moreover, we will take advantage of the P. aeruginosa system and perform the selection with either wild type bacteria or a mutant strain that does not require a specific host cell trigger to commence effector secretion. This will allow us to isolate the host cell process that triggers effector secretion. To achieve these goals, the application is divided into two specific aims. Aim 1 encompasses the selection for transposon insertion mutations that confer resistance to the P. aeruginosa T3SS, as well as the high throughput sequencing and analysis needed to identify candidate hits. In Aim 2 we will validate mutations we identify. Taken together this work will shed light on an understudied aspect of the type III secretion process: the contribution of the host cell to translocon function. By identifying both loss- and gain of function mutants, and by performing the selection in a haploid cell line, this project will be more comprehensive than any study performed to date. The design of the experiments also allows us to specifically identify factors contributing to triggering of effector secretion on host cell contact, a hallmark feature of type III secretion systems that remains a mystery to this day.

摘要 III型分泌系统(T3SS)是一种复杂的分子机器，它允许革兰氏阴性病原体 将效应蛋白直接注入宿主细胞。虽然注射的效应器蛋白的武器库在 病原体和支持多种致病的生活方式，核心器官是保守的。一项关键功能 T3SS的一个特点是效应器易位是由宿主细胞接触触发的。为了注入蛋白质，细菌 附着在宿主细胞上，并使用T3SS将形成孔的转位蛋白插入宿主细胞质中 薄膜。针尖与这个毛孔对接，形成转位。宿主细胞接触由一个 孔道中的构象变化，这种变化传播到导管的底部。在有充分证据的时候 转运子的组装和功能是由宿主细胞调节的，涉及哪些细胞功能，以及 人们对它们影响易位过程的哪一步知之甚少。 我们建议在单倍体人类细胞中进行全基因组饱和突变选择 鉴定影响铜绿假单胞菌III型效应分子转位的细胞通路 分泌系统。我们将使用一个转座子，该转座子也含有一个构成启动子和剪接供体位点， 这将使我们不仅能够识别功能突变的丢失，而且还可以识别由此导致的功能突变的获得 来自人工生产的下游基因。此外，我们将利用铜绿假单胞菌系统 并用野生型细菌或不需要特定宿主的突变菌株进行选择 细胞触发开始效应器分泌。这将使我们能够分离触发效应器的宿主细胞进程 分泌物。为了实现这些目标，应用程序分为两个具体目标。目标1包括 筛选对铜绿假单胞菌T3SS产生抗性的转座子插入突变，以及 识别候选命中所需的高通量测序和分析。在目标2中，我们将验证突变 我们认同。 综上所述，这项工作将揭示III型分泌过程中一个未被充分研究的方面： 宿主细胞对转位蛋白功能的贡献。通过鉴定功能突变体的损失和获得，并通过 在单倍体细胞系中进行选择，这个项目将比任何进行的研究都更全面 到目前为止。实验的设计还使我们能够明确地确定触发 宿主细胞接触上的效应器分泌，这是III型分泌系统的一个显著特征，至今仍是一个谜 这一天。