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
基本信息
- 批准号:10416972
- 负责人:
- 金额:$ 24.15万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2022
- 资助国家:美国
- 起止时间:2022-03-07 至 2024-02-28
- 项目状态:已结题
- 来源:
- 关键词:5&apos Splice SiteAffectAttenuatedBacteriaBacterial Attachment SiteBiochemical PathwayCRISPR/Cas technologyCell LineCell membraneCell physiologyCell secretionCellsDiseaseDockingDown-RegulationEbola virusFeedbackGene ActivationGene ExpressionGenesGenomicsGlycine decarboxylaseGoalsHaploid CellsHaploidyHigh-Throughput Nucleotide SequencingHospitalsHuman Cell LineImmuneImmune systemInfectionInjectionsInsertion MutationIntegration Host FactorsLife StyleLightMediatingMolecularMolecular ConformationMolecular MachinesMutagenesisMutationNeedlesPathogenicityPathway interactionsPerforationPhospholipasePlasma CellsPlayProcessProductionProteinsPseudomonas aeruginosaResearchResistanceRoleSiteStructureSyringesSystemToxinType III Secretion System PathwayUrsidae FamilyWorkbasecytotoxicdelivery vehicledesigndrug developmentexperimental studygain of functiongain of function mutationgenome-wideloss of function mutationmortalitymutantnoveloverexpressionpathogenpromotersecretion processsensorsmall molecule inhibitor
项目摘要
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的突变,而且还可以识别功能获得的突变。
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
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Arne Rietsch其他文献
Arne Rietsch的其他文献
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{{ truncateString('Arne Rietsch', 18)}}的其他基金
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 - 财政年份:2022
- 资助金额:
$ 24.15万 - 项目类别:
Development of recombinase-based tools to study established infections
开发基于重组酶的工具来研究已确定的感染
- 批准号:
10201472 - 财政年份:2020
- 资助金额:
$ 24.15万 - 项目类别:
Development of recombinase-based tools to study established infections
开发基于重组酶的工具来研究已确定的感染
- 批准号:
10040615 - 财政年份:2020
- 资助金额:
$ 24.15万 - 项目类别:
Type III Secretion Translocon Structure and Function
III型分泌易位子的结构和功能
- 批准号:
8701601 - 财政年份:2014
- 资助金额:
$ 24.15万 - 项目类别:
P. aeruginosa type III secreted effectors in corneal disease
角膜疾病中铜绿假单胞菌 III 型分泌效应子
- 批准号:
8404007 - 财政年份:2012
- 资助金额:
$ 24.15万 - 项目类别:
P. aeruginosa type III secreted effectors in corneal disease
角膜疾病中铜绿假单胞菌 III 型分泌效应子
- 批准号:
8962457 - 财政年份:2012
- 资助金额:
$ 24.15万 - 项目类别:
P. aeruginosa type III secreted effectors in corneal disease
角膜疾病中铜绿假单胞菌 III 型分泌效应子
- 批准号:
8217524 - 财政年份:2012
- 资助金额:
$ 24.15万 - 项目类别:
P. aeruginosa type III secreted effectors in corneal disease
角膜疾病中铜绿假单胞菌 III 型分泌效应子
- 批准号:
8597435 - 财政年份:2012
- 资助金额:
$ 24.15万 - 项目类别:
P. aeruginosa type III secreted effectors in corneal disease
角膜疾病中铜绿假单胞菌 III 型分泌效应子
- 批准号:
9115162 - 财政年份:2012
- 资助金额:
$ 24.15万 - 项目类别:
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