Pore Formation by Cholesterol Dependent Cytolysins

胆固醇依赖性溶细胞素形成孔

• 批准号: 10584602
• 负责人: Rodney K. Tweten
• 金额: $ 47.52万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10584602
• 关键词: Address; Anti-Bacterial Agents; Area; Award; Bacteria; Bacterial Toxins; Bacteroides; Basic Science; Binding; Cholesterol; Complex; Cytolysins; Data; Diatoms; Disease; Environment; Epitopes; Exhibits; Foundations; Genes; Goals; Grant; Human Microbiome; Infection; Instruction; Intestines; Listeria monocytogenes hlyA protein; Membrane; Molecular; Nature; Oral; Pathogenesis; Pathway interactions; Proteins; Publishing; Research; Specificity; Structure; Thermodynamics; Toxin; VDAC1 gene; Vaccines; Work; beta barrel; fungus; gut microbiome; insight; monomer; novel; oral microbiome; sensor; translational applications

Our work over the course of nearly 25 years of this grant have revealed insights onto the pore-forming mechanism of the cholesterol-dependent cytolysins (CDCs), which have laid the foundations to understand the basis by which this large class of toxins make the transition from soluble monomers to large oligomeric membrane-embedded pore complexes. In the next 5 years of the merit award we propose to address three specific aims, which will continue our work on the CDCs but will also open up new major fields of research in related areas. In the first aim we will address outstanding issues in the mechanism of the CDC pore-forming mechanism. The first of these is to understand how the β-barrel pore assembles and inserts into the membrane bilayer. This is a question that applies to all β-barrel pore-forming proteins but is fraught with complicating thermodynamic factors. Using our previous discoveries of the transition state barriers to pore formation and the ability to change the nature of the β-hairpins we will determine the mechanism of β-barrel assembly and membrane insertion. These studies will also address a contentious issue in the CDC-mechanism, which is whether incomplete oligomers (rather than complete rings) can insert a partial β-barrel into the membrane. In this aim we will also reassess the basis by which the listeriolysin 0 pH sensor functions, as we have recent published data and preliminary studies, which suggest that our original hypothesis made in 1995 is not the mechanism of pH sensing. In the second aim we plan to pursue the study of the CDC-like (CDCL) proteins that we have determined to be ancient relatives of the CDCs (perhaps the forerunners of the CDCs), the genes for which are widespread in numerous bacterial species (>300 so far) from at least 6 different phyla, as well as a few species of diatoms and fungi. Our primary goals are to (1) understand the pore-forming mechanism of these toxins, as some preliminary data suggest there are some fundamental differences with the CDCs, and (2) to identify the prokaryotic and/or eukaryotic targets of several of these proteins isolated from species from the oral and intestinal human microbiomes and from terrestrial environments, as none of the CDCL binding domains exhibit any similarity to the canonical binding domain of the CDCs. In the third aim we plan to study the mechanism of bacterial killing by a novel class of anti-bacterial toxins that are produced by a few species of the Provotella and Bacteroides found in the oral and intestinal microbiome of humans. Our preliminary studies suggest that they are proteolytically-activated toxins that form higher order complexes (possibly pore-forming toxins), which target various species of bacteria. Our preliminary studies suggest that they are a unique class of antibacterial toxin, as there are no known class of prokaryotic or eukaryotic toxins that exhibit any similarity to the primary structure of these toxins.

我们在这笔赠款的近25年中所做的工作揭示了对气孔形成机制的见解。 胆固醇依赖的细胞溶血素(CDCs)，它们奠定了理解 这一大类毒素使可溶单体向大分子寡聚体膜包埋过渡 毛孔复合体。在未来5年的优异奖中，我们建议解决三个具体目标，这将 继续我们在疾控中心方面的工作，但也将在相关领域开辟新的主要研究领域。第一个目标是 我们将解决疾控中心成孔机制中的突出问题。其中第一个是 了解β-Barrel孔是如何组装并插入膜双层的。这是一个适用于 对所有β-Barrel成孔蛋白都有作用，但充满了复杂的热力学因素。使用我们以前的 孔道形成过渡态障碍的发现和改变β-发夹性质的能力 我们将确定β桶组装和膜插入的机制。这些研究还将涉及 CDC-机制中有争议的问题是不完全齐聚物(而不是完整环) 可以将部分β桶插入膜中。在这个目标中，我们还将重新评估李斯特菌溶血素的基础 酸碱度传感器的功能，因为我们最近公布的数据和初步研究表明，我们最初的 1995年提出的假说并不是pH传感的机制。在第二个目标中，我们计划继续研究 我们已经确定的CDC样蛋白(CDCL)是CDC的远亲(可能是 CDC的前身)，其基因在许多细菌物种(&gt；300到目前为止)中广泛存在。 至少6个不同的门，以及几种硅藻和真菌。我们的主要目标是(1)了解 这些毒素的成孔机制，因为一些初步数据表明，有一些基本的 与CDC的差异，以及(2)鉴定其中几种蛋白质的原核和/或真核靶标 从口腔和肠道人类微生物群以及陆地环境中分离出来的物种，作为无 的CDCL结合域与CDCs的典型结合域有任何相似之处。第三个目标 我们计划研究一种新型的抗菌毒素的杀菌机制，这种毒素是由一种由一种 在人类口腔和肠道微生物群中发现的普罗沃特氏菌和类杆菌很少。我们的 初步研究表明，它们是蛋白质分解激活的毒素，形成更高阶的复合体。 (可能是形成毛孔的毒素)，目标是各种细菌。我们的初步研究表明他们 是一种独特的抗菌毒素，因为目前还没有已知的原核生物或真核生物毒素 与这些毒素的一级结构有任何相似之处。