The Tad secreton: Dissecting the structure and outer membrane assembly of the Flp-pilus-biogenesis apparatus in Aggregatibacter actinomycetemcomitans

Proteins of the tad (tight adherence) locus are required in the periodontal pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans for formation of the bacterium's characteristically strong biofilms and its virulence. Tad proteins are predicted to assemble to form a complex dedicated to the biogenesis and export of type IVb Flp pili, but the architecture of the system and functions of many of the Tad proteins have not yet been elucidated. In this study, I determined patterns of localization and abundance, as well as potential interactions, of both conserved and unique proteins in the Tad secreton. Specific antisera were generated and used to show that all three rough colony proteins (Rcp proteins) localize to the bacterial outer membrane, and that RcpA is found in detergent-stable multimers, supporting the hypothesis that the RcpA protein forms an oligomeric channel for the egress of Flp pili. RcpA was previously shown to belong to a novel and distinct family within the secretin protein superfamily, and the abundance of RcpB and RcpC—proteins only found in tad loci—was dependent on the presence of the RcpA secretin. Of ten Tad proteins tested, all were localized to or in association with membranes. One inner membrane protein, TadC, was required for wild-type levels of all three Rcp proteins. Finally, production of TadD, a putative outer membrane lipoprotein, was required for RcpA and RcpB abundance and, importantly, for the multimerization and localization of the RcpA secretin. These data imply that TadD mediates both formation and transport of RcpA oligomers, and that TadD may be the newest member of a class of secretin-docking proteins. Further, the characterization presented here provides essential information about assembly and stability of the Tad secreton's outer membrane module, and suggests hypothetical functions for RcpB, as a secretin-gating protein, and RcpC, as a chaperone for FIpl subunits and/or as a facilitator of substrate recognition. My localization studies have helped to define the topology of the secretion apparatus, and I propose that the Tad secretion system is an interconnected, membrane-spanning structure.