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.

抽象的 III型分泌系统（T3SS）是复杂的分子机器，允许革兰氏阴性病原体 直接将效应蛋白注入宿主细胞。注射效应子蛋白的武器库之间有所不同 病原体并支持多种致病生活方式，核心设备得到了保守。一个关键功能 T3SS的效应器易位是由宿主电池触点触发的。为了注入蛋白质，细菌 附着在宿主细胞上，并使用T3S将孔形成孔的转运蛋白插入宿主细胞等离子体 膜。针的尖端被停靠到该孔，形成了转运。宿主细胞接触通过 传播到设备底部的孔中的构象变化。虽然有足够的证据 该转运组件和功能由宿主单元调节，宿主细胞涉及哪些细胞函数，并且 他们影响的易位过程中的哪一步知之甚少。 我们提议在单倍体人细胞中执行基因组广泛的饱和诱变选择 识别铜绿假单胞菌III型效应子易位的细胞途径 分泌系统。我们将采用一个转座子，该转座也具有构成启动子和剪接供体网站， 这将使我们不仅可以识别功能突变的丧失，还可以识别出功能突变的增益 来自下游基因的人工产生。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 细胞触发以开始效应子分泌。这将使我们能够隔离触发效应器的宿主细胞过程 分泌。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 选择对铜绿假单胞菌T3SS的转座子插入突变以及 识别候选命中所需的高吞吐量测序和分析。在AIM 2中，我们将验证突变 我们确定。 综上 宿主细胞对转运功能的贡献。通过识别功能突变体的损失和增益，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 迄今为止。实验的设计还允许我们特别确定有助于触发的因素 宿主细胞接触的效应子分泌，这是III型分泌系统的标志性特征，仍然是一个谜 这一天。