MINORITY PREDOCTORAL FELLOWSHIP PROGRAM

病原体必须完成几项任务才能致病，包括分泌毒力因子。然而，革兰氏阴性细菌有两层膜和中间的空间，这对分泌造成了巨大的障碍。自动转运蛋白(ATS)是一个蛋白质家族，其特征是N端有一个信号序列将它们引导到周质空间，C端结构域在外膜上形成一个桶，乘客区通过这个桶转移，为这个问题提供了一个非常简单的解决方案。然而，ATS的外膜转位还不是很清楚。自体转运蛋白还可以进一步划分为亚家族。其中之一是肠杆菌科(Spates)的丝氨酸蛋白酶自转运体。这些都是分泌的毒力因子，在它们的乘客区具有丝氨酸蛋白酶基序。这项建议试图研究ESPP的分泌机制，ESPP是这个亚家族的成员，作为Spates的模型。我们的假设是，ESPP将有一个连接区，帮助通过外膜有效地转移客体结构域。第一个目的是确定该连接子的预测二级结构。第二个目的是研究连接子的结构和功能之间的关系，包括有效的外膜转位和C末端插入外膜。这些研究将有助于实现了解自身转运蛋白分泌的长期目标，以便开发其在活疫苗中用于抗原递呈的潜在用途。它们还将对革兰氏阴性菌的发病机制提供更清晰的理解。

Pathogenic organisms must accomplish several tasks in order to cause disease, including the secretion of virulence factors. Gram-negative bacteria, however, have two membranes and a space in between that creates a formidable obstacle to secretion. The autotransporters (ATs), a family of proteins characterized by a signal sequence at the N-terminus that directs them to the periplasmic space, a C-terminal domain that forms a barrel in the outer membrane, and a passenger domain that is translocated through this barrel, provide a remarkably simple solution for this problem. However, outer membrane translocation by ATs is not well understood. The autotransporters can be further divided into subfamilies. One of these is the Serine Protease Autotransporters of Enterobacteriaceae (SPATES). These are secreted virulence factors that possess a serine protease motif in their passenger domains. This proposal seeks to study the mechanism of secretion of EspP, a member of this subfamily, as a model for the SPATES. Our hypothesis is that EspP will have a linker region that aids in efficient translocation of the passenger domain through the outer membrane. The first aim seeks to define the predicted secondary structure of this linker. The second aim is to study the relationship between linker structure and function both for efficient outer membrane translocation and insertion of the C-terminus into the outer membrane. These studies will help in accomplishing the long-term goals of understanding autotransporter secretion so that its potential use for antigen presentation in live vaccines can be developed. They will also provide a clearer understanding of gram-negative pathogenesis.