Investigation of the biogenesis of Type V protein secretion in Gram-negative bacteria

V型分泌途径是致病性革兰氏阴性菌分泌毒力因子的途径之一，它至少由两个分支组成，称为自体转运体和两个伙伴分泌途径。自体转运蛋白由两个结构域组成：C端的β结构域和N端的乘客结构域，其中包含毒力功能。最初，C-末端的β结构域本身被认为足以调节其N-末端客体结构域的分泌。最近，由大肠杆菌中5个BAMA-E亚基组成的用于组装β-桶外膜蛋白的蛋白质复合体--β-桶组装机械(BAM)被发现位于自体转运蛋白附近，这表明BAM可能参与了自体转运蛋白的生物发生。此外，另一台名为搬运和组装模块()的机器也被提议参与自动运输器的组装。目前，BAM和在自体转运蛋白的生物发生中的作用尚不清楚。通过使用一个多功能的体外生化系统，概括了细菌β-桶外膜蛋白的易位(自体转运体和两个伙伴分泌)和功能整合，申请人想要研究BAM是如何介导自体转运体的生物发生，特别是每个BAM亚单位在这一过程中的功能，并确定在自体转运体的生物发生中的确切功能，即与生物转运蛋白合作还是只负责特定的自体转运体的生物发生？两个伙伴的分泌途径由两种蛋白质组成：一种TPSA蛋白，通过嵌入外膜的β-桶TPSB蛋白分泌。在这项提案中，申请人希望具体研究TPSB蛋白是如何被靶向并组装到外膜中的。此外，申请人还希望通过使用特定部位的光交联策略来绘制自动转运体、BAM和机制之间的相互作用以及TPSB与BAM、TPSB和TPSA之间的相互作用。

The Type V secretion pathway, which is composed of at least two branches called the autotransporter and the two-partner-secretion pathways, is one of the pathways that pathogenic Gram-negative bacteria use to secrete virulence factors. Autotransporters are composed of two domains: a C-terminal beta-domain and an N-terminal passenger domain that harbours the virulence function. Originally the C-terminal beta-domain by itself was believed to be sufficient to mediate the secretion of its N-terminal passenger domain. Recently, the beta-barrel assembly machinery (BAM), a protein complex that is composed of the five BamA-E subunits in E. coli and is used to assemble beta-barrel outer membrane proteins, was found in a close proximity to autotransporters thus suggesting that the BAM might be involved in autotransporter biogenesis. In addition, another machine called Translocation and Assembly Module (TAM) was proposed to be involved in the assembly of autotransporters, too. Currently, it is unclear how BAM and TAM contribute to the biogenesis of autotransporters. By use of a versatile in vitro biochemical system that recapitulates the translocation (autotransporter and two-partner-secretion) and functional integration of bacterial beta-barrel outer membrane proteins, the applicant wants to investigate how the BAM mediates the biogenesis of autotransporters, particularly the function of each BAM subunits in this process, and to determine the exact function of TAM on the biogenesis of autotransporters, i.e. cooperation with BAM or responsible only for the biogenesis of specific autotransporters? Two-partner-secretion pathway is composed of two proteins: a TpsA protein that is secreted via a beta-barrel TpsB protein that is embedded within the outer membrane. In this proposal, the applicant wants to specifically study how TpsB protein is targeted and assembled into the outer membrane. Furthermore, the applicant also wants to map the interactions among autotransporters, BAM and TAM machineries as well as interactions between TpsB and BAM, TpsB and TpsA by use of site-specific photo-cross linking strategy.