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）是复杂的分子机器， 将效应蛋白直接注入宿主细胞。虽然注射的效应蛋白的武库在不同的 病原体和支持各种各样的病原体的生活方式，核心设备是保守的。一个关键特征 T3SSs的一个重要特征是效应子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，并利用T3SS将成孔转运蛋白插入宿主细胞浆中 膜的针尖与这个孔对接，形成易位子。宿主细胞接触由 传播到装置底部的孔中的构象变化。虽然有充分的证据表明 易位子组装和功能由宿主细胞调节，其中涉及细胞功能，并且 它们影响易位过程中的哪一步还知之甚少。 我们建议在人类单倍体细胞中进行全基因组饱和诱变选择 鉴定影响铜绿假单胞菌III型效应子易位的细胞途径 分泌系统我们将使用一种转座子，它也含有一个组成型启动子和剪接供体位点， 这将使我们不仅能够识别功能丧失突变，而且能够识别由此产生的功能突变的获得 从下游基因的人工生产。此外，我们将利用铜绿假单胞菌系统 并且用野生型细菌或不需要特定宿主的突变菌株进行选择 细胞触发以开始效应物分泌。这将使我们能够分离宿主细胞的过程，触发效应 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： 选择赋予对铜绿假单胞菌T3SS的抗性的转座子插入突变，以及 鉴定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。 总之，这项工作将揭示一个未充分研究的方面的III型分泌过程： 宿主细胞对易位子功能的贡献。通过鉴定功能突变体的丧失和获得，并通过 在单倍体细胞系中进行选择，该项目将比进行的任何研究都更全面 迄今实验的设计还使我们能够具体地识别有助于触发的因素。 在宿主细胞接触时的效应子分泌，III型分泌系统的标志性特征，仍然是个谜， 今日