The Vibrio cholerae minor pilin TcpB mediates uptake of the cholera toxin phage CTXϕ

Virulent strains of the bacterial pathogen Vibrio cholerae cause the diarrheal disease cholera by releasing cholera toxin into the small intestine. V. cholerae acquired its cholera toxin genes by lysogenic infection with the filamentous bacteriophage CTX?. CTX? uses its minor coat protein pIII, located in multiple copies at the phage tip, to bind to the V. cholerae toxin-coregulated pilus (TCP). However, the molecular details of this interaction and the mechanism of phage internalization are not well-understood. The TCP filament is a polymer of major pilins, TcpA, and one or more minor pilin, TcpB. TCP are retractile, with both retraction and assembly initiated by TcpB. Consistent with these roles in pilus dynamics, we hypothesized that TcpB controls both binding and internalization of CTX?. To test this hypothesis, we determined the crystal structure of the C-terminal half of TcpB and characterized its interactions with CTX? pIII. We show that TcpB is a homotrimer in its crystallographic form as well as in solution and is present in multiple copies at the pilus tip, which likely facilitates polyvalent binding to pIII proteins at the phage tip. We further show that recombinant forms of TcpB and pIII interact in vitro, and both TcpB and anti-TcpB antibodies block CTX? infection of V. cholerae. Finally, we show that CTX? uptake requires TcpB-mediated retraction. Our data support a model whereby CTX? and TCP bind in a tip-to-tip orientation, allowing the phage to be drawn into the V. cholerae periplasm as an extension of the pilus filament.