Organization and function of Shigella IpaB within plasma membrane translocon pores.
质膜转运孔内志贺氏菌 IpaB 的组织和功能。
基本信息
- 批准号:10321532
- 负责人:
- 金额:$ 7.09万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-08-01 至 2023-07-31
- 项目状态:已结题
- 来源:
- 关键词:Amino AcidsBacterial ProteinsBindingCause of DeathCell membraneCellsCysteineCytosolDataDiseaseDockingEffector CellEnteralGoalsHumanIn VitroInfectionIntermediate FilamentsInvadedLateralMammalian CellMembraneMembrane ProteinsMembrane Structure and FunctionModelingMolecular BiologyMolecular ConformationMutagenesisNatureNeedlesPathogenesisPlantsPore ProteinsPositioning AttributeProtein BiochemistryProtein SecretionProtein translocationProteinsPublishingResearch PersonnelShigellaShigella InfectionsShigella flexneriStructureSystemTestingTissuesTransmembrane DomainType III Secretion System PathwayVirulenceWorkattenuationbasecareercrosslinkdiarrheal diseaseenteric pathogenhuman pathogeninsightmonomerpathogenpathogenic bacteria
项目摘要
Shigella spp., the leading cause of death associated with diarrheal illness, are intracellular pathogen that
require a type 3 secretion system (T3SS) to invade host cells. The T3SS is highly conserved among enteric
bacterial pathogens and is essential for pathogenesis; the loss of T3SS leads to an attenuation of virulence.
During S. flexneri infection, the T3SS secretes the bacterial proteins IpaB and IpaC, which form a pore in the
plasma membrane (the translocon pore) that is predicted to be hetero-oligomeric. S. flexneri attaches (“docks”)
onto the pore and secretes virulence proteins (effectors) through the pore into the host cell. Interactions
between IpaB and IpaC in a natively delivered translocon pore have been postulated but never demonstrated.
IpaB and IpaC monomers have been shown to oligomerize in vitro under specific conditions, suggesting similar
interactions may be required for translocon pore function in the context of infection. My preliminary data
establish approaches to investigate the translocon pore proteins when they are natively delivered during S.
flexneri infection of mammalian cells. These approaches have demonstrated that the conformation of the
translocon pore is dynamic and have identified specific IpaC residues that are differentially positioned in pores
that are incompetent for docking and pores that are competent for docking. How IpaB is involved in the shift
from a docking incompetent state to a docking competent state and the implications of IpaB involvement on
pore organization are unknown. My overall hypothesis is that a functional translocon pore is formed when IpaB
and IpaC oligomerize through interactions in their transmembrane spans in such a way that IpaB lines the
majority of the pore channel, and that these interactions change during shifts in pore conformation. I propose to
test aspects of this hypothesis with the following aims:
(I) Determine the topology of IpaB in the context of natively-delivered translocon pores.
(II) Define the organization of and interactions among IpaB monomers and between IpaB and IpaC
in the context of natively delivered translocon pores.
Using S. flexneri T3SS as a model, this proposal is highly likely to generate mechanistic insights into how the
translocon pore contributes to T3SS function. Due to the highly conserved nature of the T3SS and its near
ubiquitous requirement for the virulence of gram-negative enteric pathogens, the work proposed here is highly
likely to have a broad impact on our understanding of bacterial pathogenesis. These studies will provide me with
new expertise in molecular biology and protein biochemistry and place me in an ideal position for reaching my
career goal of becoming an independent researcher.
志贺氏菌是与腹泻相关的死亡的主要原因,是一种细胞内病原体,
需要3型分泌系统(T3SS)才能侵入宿主细胞。 T3SS 在肠溶菌中高度保守
细菌病原体,对于发病机制至关重要; T3SS 的缺失会导致毒力减弱。
在福氏链球菌感染期间,T3SS 分泌细菌蛋白 IpaB 和 IpaC,它们在
质膜(转运孔)预计为异源寡聚体。 S. 福氏附着(“码头”)
到孔上并通过孔分泌毒力蛋白(效应物)进入宿主细胞。互动
已假设 IpaB 和 IpaC 在天然传递的易位子孔中存在,但从未得到证实。
IpaB 和 IpaC 单体已被证明在特定条件下会在体外寡聚,这表明类似的
在感染的情况下,易位子孔功能可能需要相互作用。我的初步数据
建立方法来研究易位孔蛋白在 S.
福氏杆菌感染哺乳动物细胞。这些方法已经证明了
translocon pore 是动态的,并已识别出位于孔中不同位置的特定 IpaC 残基
不能对接的孔和能够对接的孔。 IpaB 如何参与这一转变
从无能力对接国家到有能力对接国家以及 IpaB 参与的影响
孔隙组织未知。我的总体假设是,当 IpaB 存在时,就会形成功能性易位子孔。
和 IpaC 通过跨膜跨度的相互作用进行寡聚化,使得 IpaB 排列在
孔通道的大部分,并且这些相互作用在孔构象变化期间发生变化。我建议
测试该假设的各个方面,目的如下:
(I) 在天然传递的易位子孔的背景下确定 IpaB 的拓扑结构。
(II) 定义IpaB单体之间以及IpaB和IpaC之间的组织和相互作用
在天然传递的易位子孔的背景下。
使用 S. flexneri T3SS 作为模型,该提案很可能产生关于如何
易位子孔有助于 T3SS 功能。由于 T3SS 及其近邻的高度保守性
对革兰氏阴性肠道病原体的毒力的普遍要求,这里提出的工作是高度
可能会对我们对细菌发病机制的理解产生广泛的影响。这些研究将为我提供
分子生物学和蛋白质生物化学方面的新专业知识使我处于实现我的目标的理想位置
成为一名独立研究员的职业目标。
项目成果
期刊论文数量(0)
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科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('Poyin Chen', 18)}}的其他基金
Organization and function of Shigella IpaB within plasma membrane translocon pores.
质膜转运孔内志贺氏菌 IpaB 的组织和功能。
- 批准号:
10455601 - 财政年份:2020
- 资助金额:
$ 7.09万 - 项目类别:
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