Structure of Proteins Involved in Bacterial Pathogenesis

描述(申请人提供)：许多革兰氏阴性细菌的粘附素被结合到通过伴侣/引导者途径组装的杂多聚体纤维中。这些纤维的形态从FGS(F1G1短)伴侣亚家族成员组装的复合毛到FGL(F1G1长)伴侣亚家族成员组装的纤维囊样结构。在这里，我们建议建立在我们对P和1型菌毛的工作基础上，它已经为在不同的革兰氏阴性生物体中组装数百个毒力纤维提供了一个范例。利用生物化学、遗传学、电子显微镜和X射线结晶学，我们详细地确定了周质伴侣的结构和作用机制。我们已经解决了与TIP接头亚单位PAPK和主要TIP亚单位PAPE形成的复合体中PAPD周质伴侣的结构。我们还解决了PapE亚基与PapK N端延伸的复合体的结构，以及PapG粘附素的受体结合域的结构及其宿主碳水化合物配体的结构。菌毛伴侣蛋白由两个免疫球蛋白(Ig)样结构域组成。这些结构表明，Pilin亚单位也有一个类似Ig的折叠，但它们缺少第七条(G)链，从而暴露了疏水核心。在我们称之为供体-链互补的过程中，伴侣的G1链充当毛细蛋白的第七条链，催化亚基的折叠，防止非生产性亚基聚集。在引座处，菌毛的组装是通过供体-链交换进行的，其中伴侣的G1链被存在于每个亚单位上的N-末端延伸所取代，并暴露在进入的伴侣-亚单位复合体上。菌毛亚单位随后经历拓扑转变，触发其凹槽的关闭，将其邻居的NTE作为其自身Ig折叠的一部分。因此，最终的菌毛结构是一系列Ig样结构域，每个结构域都由两个单独的亚单位单体的一部分形成。每个亚基对其他相互作用的亚基有不同的特异性，在菌毛组装中也有不同的作用。我们建议扩大我们对粘附素的结构的了解，并进一步研究两个这样的组装系统的亚基结构和组装机制：泌尿系致病大肠杆菌的原型FGS P菌毛系统和沙门氏菌的Saf FGL系统。此外，我们还将对抗伴侣化合物进行结构研究。

DESCRIPTION (provided by applicant): Many of the adhesins of Gram-negative bacteria are incorporated into heteropolymeric fibers assembled by the chaperone/usher pathway. These fibers range in morphology from composite pili assembled by members of the FGS (F1G1 short) subfamily of chaperones, to fibrous capsule-like structures assembled by the FGL (F1G1 long) subfamily of chaperones. Here we propose to build on our work on P and type 1 pili, which has provided a paradigm for the assembly of hundreds of virulence fibers in diverse Gram-negative organisms. Using biochemistry, genetics, electron microscopy and X-ray crystallography, we have defined the structure and mechanism of action of the periplasmic chaperones in significant detail. We have solved the structure of the PapD periplasmic chaperone in complex with the tip adaptor subunit, PapK, and with the major tip subunit, PapE. We have also solved the structure of the PapE subunit in complex with the N-terminal extension of PapK and the structure of the receptor binding domain of the PapG adhesin with and without its host carbohydrate ligand. Pilus chaperones are comprised of two immunoglobulin (Ig)-like domains. These structures showed that pilin subunits also have an Ig-like fold, but they are missing their seventh (G) strand, thus exposing the hydrophobic core. In a process we termed donor-strand complementation, the chaperone's G1 strand serves as the pilin's seventh strand, catalyzing the folding of the subunit and preventing non-productive subunit aggregation. At the usher, pilus assembly occurs by donor-strand exchange, in which the G1 strand of the chaperone is replaced by an N-terminal extension that is present on every subunit and exposed on incoming chaperone-subunit complexes. The pilus subunit then undergoes a topological transition that triggers the closure of its groove, cementing its neighbor's NTE as part of its own Ig fold. Thus, the final pilus structure is a series of Ig-like domains, each of which is formed from parts of two individual subunit monomers. Each subunit has distinct specificity for other interactive subunits and distinct roles in pilus assembly. We propose to expand our knowledge of the structures of the pilus adhesins and further study the structure of the subunits and assembly machinery of two such assembly systems: the prototypical FGS P pilus system of uropathogenic Escherichia coli and the Saf FGL system of Salmonella. In addition, we will perform structural studies on anti-chaperone compounds.