MINORITY PREDOCTORAL FELLOWSHIP PROGRAM

致病生物必须完成几项任务才能引起疾病，包括分泌毒力因子。然而，革兰氏阴性细菌有两层膜，中间有一个空间，这对分泌产生了巨大的障碍。自转运蛋白（AT）是一个蛋白质家族，其特征在于N端的信号序列将其引导到周质空间，C端结构域在外膜中形成桶，以及通过该桶易位的乘客结构域，为这个问题提供了非常简单的解决方案。然而，AT的外膜易位还不清楚。自动转运蛋白可以进一步分为亚科。其中之一是肠杆菌科丝氨酸蛋白酶自身转运蛋白（SPATES）。这些是分泌的毒力因子，在其乘客结构域中具有丝氨酸蛋白酶基序。该建议旨在研究该亚家族成员EspP的分泌机制，作为SPATES的模型。我们的假设是，EspP将有一个接头区域，有助于乘客结构域通过外膜的有效易位。第一个目的是试图定义该接头的预测二级结构。第二个目的是研究连接体结构和功能之间的关系，用于有效的外膜易位和C-末端插入外膜。这些研究将有助于实现理解自身转运蛋白分泌的长期目标，以便开发其在活疫苗中抗原呈递的潜在用途。它们还将提供对革兰氏阴性致病机理的更清楚的理解。

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.