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

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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.