Structural characterization of outer membrane proteins from Yersinia pestis

当前的研究 我们在NIAID的合作者B.Joseph Hinnebusch博士使用DNA微阵列比较了从鼠疫感染的大鼠中恢复的鼠疫杆菌的表达谱，而不是在摇瓶培养中培养的鼠疫杆菌。使用这些方法，他们已经确定了8种可能的外膜蛋白，这些蛋白在受感染的动物中发现的含量更高。几乎所有这些都是在鼠疫杆菌外膜中发现的蛋白质，因此，人们认为它们可能是很好的疫苗靶标。这是因为这些蛋白质的一部分突出到细菌周围的环境中，在那里它们可以很容易地被感染但已接种疫苗的人的免疫系统检测到。此外，几乎所有的细胞都参与了铁进入细胞的过程。获取铁的能力对包括病原菌在内的大多数细菌的生存至关重要。在鼠疫耶尔森氏菌中，从感染宿主的贫铁环境中获取铁的能力与感染小鼠的毒力和致死性有关。我们的实验室在铁输入途径的基础和结构生物学方面拥有丰富的专业知识。在处理大肠杆菌中类似蛋白质方面获得的经验已经对研究鼠疫耶尔森氏菌蛋白质有价值。 我们已经着手克隆、表达、纯化和结晶这8个蛋白质，总的目的有两个：第一，为疫苗开发研究生产蛋白质；第二，利用多余的材料进行结构研究。到目前为止，我们已经表达并成功纯化了8个蛋白中的5个。 2009年，我们解决了鼠疫杆菌铁转运蛋白的两种结构，其中一种结构具有apo形式，另一种结构具有结合的同源铁质载体。这些结构目前被用于通过将小分子对接到铁载体结合口袋中来进行硅胶药物设计。一旦找到了好的候选分子，他们将被合成并分析竞争结合。这种方法可能导致专门针对鼠疫杆菌的新型抗生素。在相关工作中，我们还解决了一种鼠疫杆菌细菌素的结构，我们设计的这种细菌素可以杀死多种耶尔森菌菌株。这种工程细菌素可能会为鼠疫提供一种替代药物治疗。这项工作已提交出版，目前正在审查中。 在相关工作中，我们最近解决了鼠疫杆菌外膜蛋白的结构，该蛋白参与了与宿主细胞的黏附和逃避宿主补体反应。我们进行了功能实验，以确定这两种活性的结合伙伴，并确定该蛋白在小鼠中产生免疫反应。Hinnebusch实验室目前正在将其作为潜在的疫苗靶标进行研究。 2010年更新：我们已经开发出针对鼠疫杆菌铁转运蛋白的小分子和蛋白质药物一份手稿正在修改中，目前正在进行小鼠实验。我们设计的蛋白质药物已经申请了临时专利。对于我们解决的外膜粘附素，我们确定了黏附和血清耐药靶标，并解决了一个复杂的结构，该结构映射了补体成分之一的结合部位。这份手稿是最近提交的。我们的合作者目前正在研究这种外膜蛋白用于疫苗开发，在小鼠身上的实验已经证明了强大的免疫反应。

Current research Our collaborator at NIAID, Dr. B. Joseph Hinnebusch, has used DNA microarrays to compare expression profiles of Y. pestis recovered from plague infected rats as opposed to Y. pestis that are grown in flask cultures. Using these methods they have identified eight putative outer membrane proteins that are found in higher amounts in infected animals. Almost all of these are proteins found in the outer envelope of Y. pestis and for this reason it is thought that they might make good vaccine targets. This is because portions of these proteins protrude into the surrounding environment of the bacteria where they can be readily detected by the immune system of an infected but vaccinated human being. Additionally almost all are involved in the import of iron into the cell. The ability to acquire iron is essential for the survival of most bacteria including pathogenic bacteria. In Y. pestis the ability to obtain iron from the iron poor environment of the infected host is correlated to virulence and lethality of infection in mice. Our lab has a lot of expertise on the basic and structural biology of iron import pathways E. coli. The experiences gained on the handling of similar proteins in E. coli have already been valuable in the study of Y. pestis proteins. We have proceeded to clone, express, purify and crystallize these 8 proteins with two general aims: first, to generate protein for vaccine development studies and second, to make use of the excess material for structural studies. So far we have expressed and successfully purified 5 out of the 8 proteins. In 2009, we solved two structures of Y. pestis iron transporters, and in one case we have structures of the apo form and a form with bound cognate siderphore. These structures are currently being used to do in silico drug design by docking small molecules into the siderophore binding pocket. Once good candidate molecules are found, they will be synthesized and analyzed for competitive binding. This approach may lead to novel antibiotics specifically targeting Y. pestis. In related work, we also solved the structure of a Y. pestis bacteriocin which we engineered to kill a wide variety of Yersinia strains. The engineered bacteriocin may provide an alternative drug treatment for plague. This work has been submitted for publication and is currently under review. In related work, we recently solved the structure of a Y. pestis outer membrane protein involved in adhesion to host cells and evasion of the host complement response. We carried out functional experiments to identify binding partners for both activities and have determined that this protein produces an immune response in mice. It is currently being investigated as a potential vaccine target by the Hinnebusch lab. 2010 update: We have developed both small molecule and protein drugs targeting a Y. pestis iron transporter one manuscript is under revision and experiments in mice are currently underway. A provisional patent has been filed for the protein drug that we engineered. For the outer membrane adhesin that we solved, we identified adhesion and serum resistance targets and solved a complex structure that maps the binding site for one of the complement components. The manuscript was recently submitted. This outer membrane protein is currently being investigated for vaccine development by our collaborator, and experiments in mice have already demonstrated a robust immune response.