Structural characterization of outer membrane proteins from Yersinia pestis

2011年更新： Y.鼠疫FyuA和pesticin使我们能够设计一种针对革兰氏阴性病原体的新型噬菌体治疗药物： 我们解决了FyuA的结构，一个TonB依赖的铁转运蛋白所需的毒力在腺鼠疫，有和没有其同源铁载体，铁耶尔森菌素。与此同时，我们确定了一种名为pesticin的细菌素的结构，它使用FyuA穿过外膜。一旦进入周质，pesticin通过降解肽聚糖层杀死细胞。从我们的结构中，我们发现pesticin的杀伤结构域类似于噬菌体T4溶菌酶，因此我们工程化了含有细菌靶向结构域（针对FyuA）和噬菌体杀伤结构域的杂合细菌-噬菌体毒素。我们表明，杂合赖氨酸逃避天然保护机制的产毒菌株和杀死所有耶尔森氏菌菌株测试，在体外和体内（小鼠模型的腺鼠疫）。这是第一次证明噬菌体治疗革兰氏阴性病原体，因为到目前为止，没有人知道如何将毒素运输穿过外膜。这项工作正在《自然》杂志上进行修订，等待动物实验的最终统计数据。 Y.鼠疫Ail揭示了细菌外膜蛋白如何与宿主细胞结合： 在我们致力于针对革兰氏阴性病原体的疫苗和药物设计的过程中，我们在过去的九年里一直致力于Y。鼠疫菌外膜蛋白似乎对疾病进展很重要。Y.鼠疫外膜蛋白是Ail，一种小的外膜蛋白，其功能是结合宿主细胞，并且还通过干扰补体级联来逃避宿主免疫应答。在未发表的工作中，我们发现Ail具有免疫原性，并在腺鼠疫小鼠模型中提供保护，我们正在继续努力基于这项工作配制蛋白质组分疫苗。由于Ail是如此重要，我们解决了它的结构和表征粘附到靶细胞。我们确定Ail选择性地结合细胞外基质蛋白层粘连蛋白和肝素，并显示称为LG 4 -5的层粘连蛋白的40 kDa结构域特异性地结合Ail。我们还评估了层粘连蛋白对耶尔森氏菌外蛋白（Yops）向Hep-2细胞递送的贡献。我们解决了Ail单独和与肝素类似物复合的晶体结构，提供了细菌外膜蛋白如何使用多价方法结合宿主细胞的结构描述。这份手稿正在结构出版社出版。

2011 update: Our structures of Y. pestis FyuA and pesticin allowed us to engineer a novel phage therapy drug against a Gram-negative pathogen: We solved the structure of FyuA, a TonB-dependent iron transporter required for virulence in bubonic plague, with and without its cognate siderophore, ferric yersiniabactin. At the same time, we determined the structure of a bacteriocin called pesticin that uses FyuA to cross the outer membrane. Once inside the periplasm, pesticin kills the cell by degrading the peptidoglycan layer. From our structure we discovered that the killing domain of pesticin resembles phage T4 lysozyme, so we engineered a hybrid bacterial-phage toxin that contains a bacterial targeting domain (to FyuA) and a phage killing domain. We showed that the hybrid lysine evades the natural protection mechanism of toxin-producing strains and kills all Yersinia strains tested, both in vitro and in vivo (mouse model of bubonic plague). This is the first demonstration of phage therapy for Gram-negative pathogens because until now, no one knew how to transport the toxin across the outer membrane. This work is under revision at Nature, awaiting final statistics for the animal experiments. Structures of Y. pestis Ail reveal how a bacterial outer membrane protein binds to host cells: In our efforts to contribute to vaccine and drug design against Gram-negative pathogens, we have worked for the past nine years on Y. pestis outer membrane proteins that appear important for disease progression. One of the most highly expressed Y. pestis outer membrane proteins is Ail, a small outer membrane protein that functions to bind host cells and also to evade the host immune response by interfering with the complement cascade. In unpublished work, we found that Ail is immunogenic and confers protection in a mouse model of bubonic plague, and we are continuing efforts to formulate a protein component vaccine based on this work. Because Ail is so important, we solved to structures of it and characterized adhesion to target cells. We determined that Ail selectively binds the extracellular matrix proteins laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yersinia outer proteins (Yops) to Hep-2 cells. We solved crystal structures of Ail alone and in complex with a heparin analogue, providing a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells. This manuscript is in press at Structure.