Pore Formation by Cholesterol Dependent Cytolysins

我们在近25年的研究中揭示了孔隙形成机制 胆固醇依赖性溶细胞素（CDC），这奠定了基础，了解的基础， 这一大类毒素从可溶性单体转变为大的寡聚膜包埋 孔隙复合体在未来5年的优异奖，我们建议解决三个具体目标，这将 继续我们在疾病预防控制中心的工作，但也将在相关领域开辟新的主要研究领域。在第一个目标中 我们将解决悬而未决的问题，在该机制的疾病控制中心成孔机制。其中第一个是 了解B-桶孔如何组装并插入膜双层。这是一个适用于 所有的B-桶孔形成蛋白质，但充满了复杂的热力学因素。使用我们以前的 发现了孔形成的过渡态障碍和改变B-发夹性质的能力 我们将确定B-筒组装和膜插入的机制。这些研究还将探讨 CDC机制中有争议的问题，即不完整的低聚物（而不是完整的环） 可以将部分B筒插入膜中。在这一目标中，我们也将重新评估的基础上， 0 pH传感器功能，因为我们最近公布的数据和初步研究，这表明我们的原始 1995年提出的假说不是pH传感的机制。在第二个目标中，我们计划继续研究 CDC样（CDCL）蛋白，我们已经确定是CDC的古老亲戚（也许是 CDC的前身），其基因广泛存在于许多细菌物种（迄今为止超过300种）中， 至少有6个不同的门，以及一些硅藻和真菌。我们的主要目标是（1）了解 这些毒素的孔形成机制，因为一些初步数据表明，有一些基本的 与CDC的差异，以及（2）鉴定这些蛋白质中的几种的原核和/或真核靶 从口腔和肠道人类微生物组以及陆地环境中的物种中分离， 的CDCL结合结构域表现出与CDC的典型结合结构域的任何相似性。第三个目标 我们计划研究一种新型抗菌毒素的杀菌机制， 在人类的口腔和肠道微生物组中发现的Provote/la和拟杆菌属的几个物种。我们 初步研究表明，它们是蛋白水解激活的毒素， （可能是成孔毒素），其针对各种细菌。我们的初步研究表明， 是一类独特的抗菌毒素，因为没有已知的一类原核或真核毒素表现出 与这些毒素的一级结构有任何相似之处 相关性（参见说明）： 我们提出的工作仍然是对胆固醇依赖性溶细胞素的基础研究 但这通常导致平移应用（即，疫苗），并支持在 这些毒素导致疾病的发病机制。这些研究使我们发现了一种 大类古老的CDC亲属，已蔓延整个细菌王国，并存在于 人类微生物组的许多主要物种。最后，这些研究将揭示分子机制 一种独特的抗菌毒素，可能用于治疗感染。

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 B-barrel pore assembles and inserts into the membrane bilayer. This is a question that applies to all B-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 B-hairpins we will determine the mechanism of B-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 B-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 Provote/la 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. RELEVANCE (See instructions): Our proposed work continues to be fundamentally basic research on the cholesterol-dependent cytolysins but which often leads to translational applications (i.e., vaccines) and supports the efforts in the field of pathogenesis where these toxins contribute to disease. These studies have led us to the discovery of a large class of ancient CDC relatives that have spread throughout the Bacteria kingdom and are present in many major species of the human microbiome. Finally, these studies will reveal the molecular mechanism of a unique class of anti-bacterial toxins, which could potentially be used in therapies for infections.