SummaryColonization of the human small intestine by Vibrio cholerae requires the type 4 toxin co-regulated pilus (TCP). Genes encoding the structure and biogenesis functions of TCP are organized within an operon located on the Vibrio Pathogenicity Island (VPI). In an effort to elucidate the functions of proteins involved in TCP biogenesis, in frame deletions of all of the genes within the tcp operon coding for putative pilus biogenesis proteins have been constructed and the resulting mutants characterized with respect to the assembly and function of TCP. As a result of this analysis, we have identified the product of one of these genes, tcpF , as a novel secreted colonization factor. Chromosomal deletion of tcpF yields a mutant that retains in vitro phenotypes associated with the assembly of functional TCP yet is severely attenuated for colonization of the infant mouse intestine. Furthermore, we have determined that the mechanism by which TcpF is translocated across the bacterial outer membrane requires the TCP biogenesis machinery and is independent of the type II extracellular protein secretion (EPS) system. These results suggest a dual role for the TCP biogenesis apparatus in V. cholerae pathogenesis and a novel mechanism of intestinal colonization mediated by a soluble factor.

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The toxin-coregulated pilus (TCP) of Vibrio cholerae and the soluble TcpF protein that is secreted via the TCP biogenesis apparatus are essential for intestinal colonization. The TCP biogenesis apparatus is composed of at least nine proteins but is largely uncharacterized. TcpC is an outer membrane lipoprotein required for TCP biogenesis that is a member of the secretin protein superfamily. In the present study, analysis of TcpC in a series of strains deficient in each of the TCP biogenesis proteins revealed that TcpC was absent specifically in a tcpQ mutant. TcpQ is a predicted periplasmic protein required for TCP biogenesis. Fractionation studies revealed that the protein is not localized to the periplasm but is associated predominantly with the outer membrane fraction. An analysis of the amount of TcpQ present in the series of tcp mutants demonstrated the inverse of the TcpC result (absence of TcpQ in a tcpC deletion strain). Complementation of the tcpQ deletion restored TcpC levels and TCP formation, and similarly, complementation of tcpC restored TcpQ. Metal affinity pull-down experiments performed using His-tagged TcpC or TcpQ demonstrated a direct interaction between TcpC and TcpQ. In the presence of TcpQ, TcpC was found to form a high-molecular-weight complex that is stable in 2% sodium dodecyl sulfate and at temperatures below 65°C, a characteristic of secretin complexes. Fractionation studies in which TcpC was overexpressed in the absence of TcpQ showed that TcpQ is also required for proper localization of TcpC to the outer membrane.Vibrio cholerae is a gram-negative bacterium that causes the gastrointestinal disease cholera, which is spread via the fecaloral route. Upon passage through the stomach, the bacterium utilizes its single polar flagellum to swim from the lumen through the intestinal mucous layer to reach the epithelial surface in the crypts of the intestine where it colonizes. Toxincoregulated pilus (TCP) serves as the primary factor involved in the colonization and persistence of the bacteria in the small intestine (18,32,39,40). TCP is classified as a type 4 pilus based on the N-terminal homology of the monomeric (pilin) subunit, TcpA. Type 4 pili are long, filamentous appendages expressed by a number of gram-negative bacteria and are classified by sequence homology within the hydrophobic N-terminal region of the mature pilin and a methylated N-terminal residue (10,25,37). There are two subclasses of type 4 pili, namely, 4A and 4B, with TCP representing the latter (35). TCP has been demonstrated to be necessary for the colonization of humans, and the colonization defect of tcp mutants is paralleled in the infant mouse model (12). Upon colonization the bacterium produces cholera toxin, which is an ADP-ribosylating toxin composed of one A subunit and five B subunits encoded by the ctxAB operon. Cholera toxin is secreted by a type II extracellular protein secretion (Eps) system (16,33). The ctxAB operon is part of the genome of the cholera toxin bacteriophage (CTX), a 7-kb lysogenic filamento...

This paper introduces a framework for conducting and disseminating mixed methods research on positive outlier countries that successfully improved their health outcomes and systems. We provide guidance on identifying exemplar countries, assembling multidisciplinary teams, collecting and synthesising pre-existing evidence, undertaking qualitative and quantitative analyses, and preparing dissemination products for various target audiences. Through a range of ongoing research studies, we illustrate application of each step of the framework while highlighting key considerations and lessons learnt. We hope uptake of this comprehensive framework by diverse stakeholders will increase the availability and utilisation of rigorous and comparable insights from global health success stories.

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