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Actin-based motility of a bacterial pathogen

细菌病原体基于肌动蛋白的运动

基本信息

批准号：
9903054
负责人：
JULIE A. THERIOT
金额：
$ 27.76万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9903054
关键词：
Actins Address Antibodies Award Bacteria Bacterial Genes Bacterial Infections Behavior Biochemical Biological Models Biophysical Process Blood - brain barrier anatomy Blood Vessels Brain Bypass Cell Differentiation process Cell Line Cell Wall Cells Cellular Structures Complex Cytoplasm Diffuse Disease Dissection Distal Elderly Endothelial Cells Endothelium Entropy Epithelial Cells Food Poisoning Genetic Goals Gram-Positive Bacteria Growth Human Immune Immune response Immune system Immunocompromised Host Infection Intestines Invaded Lead Listeria monocytogenes Listeriosis Liver Mediating Membrane Membrane Proteins Meningitis Methods Modeling Molecular Molecular Genetics Movement Mus Neoplasm Metastasis Neuraxis Newborn Infant Organism Pathogenesis Pathway interactions Phase Placenta Pregnant Women Process Property Protein Secretion Proteins Research Role Site Small Interfering RNA Spontaneous abortion Staphylococcus aureus Structure Surface System Tail Testing Thick Tissues Virulence Factors Work arm base biophysical properties cell motility crosslink fighting foodborne infection macrophage mathematical model monolayer mouse model nanoscale pathogenic bacteria physical property polarized cell polymerization prevent reconstitution screening single molecule tissue culture

项目摘要

ABSTRACT / PROJECT SUMMARY Request for 5-year extension of Al - 36929 under MERIT award Listeria monocytogenes is a ubiquitous Gram-positive bacterium that can cause serious food-borne infections in pregnant women, newborns, and immunocompromised or older adults. From the initial site of Infection in the intestine, the bacteria are able to spread systemically while avoiding the antibody-mediated arm of the hose immune response. These bacteria grow directly in the cytoplasm of infected host cells and move rapidly throughout and between infected cells using a form of actin-based motility. This remarkable ability allows the bacteria to spread from the intestinal epithelial cells into circulating macrophages, which then carry the bacteria throughout the body and are thought to mediate their spread into distal tissues including the liver, the brain, and the placenta (in pregnant women). The intracellular actin-based motility of L. monocytogenes has served as an important model system for understanding the molecular and biophysical mechanisms of eukaryotic processes driven by actin polymerization, including whole-cell crawling in the immune system and in cancer metastasis. Through our interdisciplinary work involving biochemical reconstitution of motility, biophysical measurement of force-generating processes operating at the bacterial surface, molecular genetic dissection of the bacterial and host contributions to motility, and mathematical modeling of this complex process, we have developed a very detailed understanding of the intracellular motility phase of the infection cycle. In our current work, we are expanding our interdisciplinary analysis to other steps in the infection, including host cell invasion, bacterial growth and surface polarization, and cell-to-cell spread. The L. monocytogenes surface protein, ActA, is expressed in a polarized fashion and interacts with host cell cytoskeletal factors to induce the polymerization of an actin "comet tail" structure that pushes the bacterium through the host cell cytoplasm. The interactions between ActA and host cell cytoplasmic factors have been well-studied, but the behavior of ActA itself is less explored. This is a very large intrinsically disordered protein, whose size is such that it should not be able to diffuse through the nanometer-scale pores in the thick, cross-linked Gram-positive bacterial cell wall. Nevertheless, it does extend through the wall while remaining anchored in the membrane. Very recently, we have developed a conceptual breakthrough that can explain quantitative features of ActA translocation as an entropy-driven process. Our new model is highly relevant for surface presentation of other virulence factors in Gram-positive organisms. Over the next five years of this ongoing project, we propose to use L monocytogenes as a genetic system to identify bacterial genes involved in determination of the physical properties of the cell wall (such as thickness and pore size) that govern entropy-driven protein secretion, and expand our analysis to other Gram-positive virulence factors that are structurally related to ActA, particularly in Staphylococcus aureus. For our studies of invasion and cell-to-cell spread, we are focusing on interactions between L. monocytogenes and the endothelial cells that line blood vessels, as these cells should represent a critical barrier to systemic dissemination of the bacteria. The most likely mechanisms of bypassing the barrier properties of the endothelium include: direct infection of endothelial cells, infection of endothelial cells via cell-to-cell spread from infected circulating immune system cells, and transmigration of infected immune cells across an uninfected endothelium. In tissue culture, we have been able to replicate each of these processes, and have used systematic siRNA screening to identify host cell factors uniquely involved in each step. These initial results have yielded many surprises, and have demonstrated that invasion of endothelial cells is mechanistically distinct from invasion of intestinal epithelial cells. Over the next five years, we propose to continue and expand our molecular dissection of these processes and test the role of the molecules we identify in mouse models of L monocytogenes infection.

摘要/项目摘要根据优异奖要求将Al-36929延长5年单核细胞增多性李斯特菌是一种普遍存在的革兰氏阳性细菌，可导致孕妇、新生儿和免疫低下或老年人的严重食源性感染。从肠道的最初感染部位开始，细菌能够在避开抗体介导的软管免疫反应的同时进行系统传播。这些细菌直接生长在感染宿主细胞的细胞质中，并利用一种基于肌动蛋白的运动形式在感染细胞内和感染细胞之间快速移动。这种非凡的能力使细菌能够从肠道上皮细胞传播到循环中的巨噬细胞，然后巨噬细胞携带细菌遍布全身，被认为是它们传播到远端组织的媒介，包括肝脏、大脑和胎盘(在孕妇中)。单核细胞增生性李斯特氏菌基于肌动蛋白的细胞内运动已成为了解肌动蛋白聚合所驱动的真核过程的分子和生物物理机制的重要模型系统，包括免疫系统中的全细胞爬行和肿瘤转移。通过我们的跨学科工作，包括动力的生化重建，对细菌表面作用力过程的生物物理测量，对细菌和宿主对动力的贡献的分子遗传解剖，以及对这一复杂过程的数学建模，我们已经对感染周期的细胞内动力阶段有了非常详细的理解。在我们目前的工作中，我们正在将我们的跨学科分析扩展到感染的其他步骤，包括宿主细胞入侵、细菌生长和表面极化，以及细胞到细胞的传播。单核细胞增多性李斯特菌表面蛋白ActA以极化方式表达，并与宿主细胞细胞骨架因子相互作用，诱导肌动蛋白“彗星尾”结构聚合，推动细菌穿过宿主细胞细胞质。ActA和宿主细胞胞质因子之间的相互作用已经得到了很好的研究，但ActA本身的行为却很少被探索。这是一种非常大的天生无序的蛋白质，其大小不应该能够通过厚厚的、交联的革兰氏阳性细菌细胞壁中的纳米级毛孔扩散。尽管如此，它确实延伸到了壁上，同时仍固定在膜上。最近，我们在概念上取得了突破，可以将ActA易位的数量特征解释为一个由熵驱动的过程。我们的新模型与革兰氏阳性生物中其他毒力因子的表面呈现高度相关。在这个正在进行的项目的接下来的五年里，我们建议使用L单核细胞增多症作为一个遗传系统来识别参与决定细胞壁物理性质(如厚度和孔径)的细菌基因，这些基因控制着熵驱动的蛋白质分泌，并将我们的分析扩展到其他与ActA结构相关的革兰氏阳性毒力因子，特别是在金黄色葡萄球菌中。在我们对入侵和细胞间传播的研究中，我们专注于单核细胞增多性乳杆菌与血管内皮细胞之间的相互作用，因为这些细胞应该是细菌系统性传播的关键屏障。绕过内皮屏障特性的最可能的机制包括：内皮细胞的直接感染，受感染的循环免疫系统细胞通过细胞间传播感染内皮细胞，以及受感染的免疫细胞跨未感染的内皮细胞的迁移。在组织培养中，我们已经能够复制这些过程中的每一个，并使用系统的siRNA筛选来识别在每个步骤中唯一涉及的宿主细胞因子。这些初步结果已经产生了许多令人惊讶的结果，并证明了内皮细胞的侵袭与肠上皮细胞的侵袭在机制上是不同的。在接下来的五年里，我们建议继续并扩大我们对这些过程的分子解剖，并测试我们识别的分子在L单核细胞增多症小鼠模型中的作用。