Protection and Attachment of Vibrio cholerae Mediated by the Toxin-Coregulated Pilus in the Infant Mouse Model

Abstract

Colonization of the human small intestine by Vibrio cholerae is an essential step in pathogenesis that requires the type IV toxin-coregulated pilus (TCP). To date, three functions of TCP have been characterized: it serves as the CTX⌽ receptor, secretes the colonization factor TcpF, and functions in microcolony formation by mediating bacterium-bacterium interactions. Although type IV pili in other pathogenic bacteria have been characterized as playing a major role in attachment to epithelial cells, there are very few studies to suggest that TCP acts as an attachment factor. Taking this into consideration, we investigated the function of TCP in attachment to Caco-2 cells and found that mutants lacking TCP were defective in attachment compared to the wild type. Overexpression of ToxT, the activator of TCP, significantly increased attachment of wild-type V. cholerae to Caco-2 cells. Using field-emission scanning electron microscopy (FESEM), we also observed TCP-mediated attachment to the small intestines of infected infant mice by using antibodies specific to TCP and V. cholerae. Remarkably, we also visualized matrices comprised of TCP appearing to engulf V. cholerae during infection, and we demonstrated that these matrices protected the bacteria from a component of bile, disclosing a possible new role of this pilus in protection of the bacterial cells from antimicrobial agents. This study provides new insights into TCP's function in V. cholerae colonization of the small intestine, describing additional roles in mediating attachment and protection of V. cholerae bacterial cells.Vibrio cholerae is the etiological agent of the acute diarrheal disease cholera and is characterized as a motile, Gram-negative, bacillus-shaped bacterium. Pathogenesis of V. cholerae O1 critically depends on production of the cholera toxin and the toxin-coregulated pilus (TCP), which are coordinately regulated by the toxR virulence regulon (24, 36). The cholera toxin is an ADP-ribosylating toxin that constitutively activates adenylate cyclase in host cells (reviewed in reference 17), causing copious amounts of diarrhea. Delivery of the cholera toxin requires successful colonization of the small intestine by V. cholerae, which is mediated by TCP, as demonstrated in the infant mouse cholera model (37), the new infant rabbit cholera model (29), and human volunteer studies (15).Biogenesis of TCP is dependent on the tcp operon, which contains a large cluster of 12 genes (37). The first gene in the operon, tcpA, encodes the 20-kDa major pilin subunit of TCP. TcpA is expressed as a preprotein that is cleaved to its mature form by a prepilin peptidase, encoded by tcpJ (22). ToxT, an AraC family member, activates expression of tcp through signals from a transcriptional cascade called the toxR regulon. The remaining genes in the operon, with the exception of tcpF, encode components of the apparatus that are responsible for biogenesis of TCP. This apparatus is thought to span the inner membrane, periplasm, and outer membrane for the energydependent pro...

Zebrafish as a Natural Host Model for Vibrio cholerae Colonization and Transmission

“…V. cholerae cells were visualized in sections of fixed zebrafish by fluorescence microscopy using a primary polyclonal antibody directed against V. cholerae and a secondary monoclonal antibody carrying the fluorescent tag. The contact between V. cholerae and the intestinal epithelial surface observed in zebrafish very closely resembles the interaction between V. cholerae and the epithelial surface observed in mammalian models (36,37).…”

Type IV Pilin Proteins: Versatile Molecular Modules

“…In E. coli, remodeling of bundled pili into even thicker bundles via the action of the retraction ATPase BfpF was required for microcolony dispersal (232), suggesting that the particular mode of pilus-pilus interaction affects the subsequent behavior of bacteria. A recent study of V. cholerae binding to intestinal epithelia revealed that aggregates of T4P enveloped the bacteria, potentially protecting them during the infection process (238). In S. Typhi, bundling of pili is negatively controlled by expression of one of two variants of the minor pilin, PilV (note that this is a different component than the Neisseria protein of the same name).…”

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

“…V. cholerae uses its OmpU outer membrane protein to adhere to the brush border of fully differentiated Caco-2 cells (410). The type IV TCP of V. cholerae has three functions: it acts as a receptor for CTXPhi, the lysogenic filamentous bacteriophage that carries the CT genes in epidemic V. cholerae strains; it secretes the colonization factor TcpF and contributes to microcolony formation by mediating bacterium-bacterium interactions; and it acts as an attachment factor for binding to fully differentiated Caco-2 cells, since attachment was defective in mutants lacking TCP compared to in the wild type (411).…”