Structural basis for the toxin-coregulated pilus–dependent secretion of Vibrio cholerae colonization factor

Colonization of the host intestine is the most important step in Vibrio cholerae infection. The toxin-coregulated pilus (TCP), an operon-encoded type IVb pilus (T4bP), plays a crucial role in this process, which requires an additional secreted protein, TcpF, encoded on the same TCP operon; however, its mechanisms of secretion and function remain elusive. Here, we demonstrated that TcpF interacts with the minor pilin, TcpB, of TCP and elucidated the crystal structures of TcpB alone and in complex with TcpF. The structural analyses reveal how TCP recognizes TcpF and its secretory mechanism via TcpB-dependent pilus elongation and retraction. Upon binding to TCP, TcpF forms a flower-shaped homotrimer with its flexible N terminus hooked onto the trimeric interface of TcpB. Thus, the interaction between the minor pilin and the N terminus of the secreted protein, namely, the T4bP secretion signal, is key for V. cholerae colonization and is a new potential therapeutic target.

The figure was generated with ESPript (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi ).(TcpB) and Glu83 (TcpA), and between Arg26 (TcpA) and Glu83 (TcpA).(c) Close-up views of the electrostatic interactions between the amine of Val1 (TcpB) and Glu5 (TcpA) and between the amine of Met1 (TcpA) and Glu5 (TcpA).(d) Electrostatic surface potential representations of the interaction interface between one TcpB-domain 1 and the interacting TcpA, as calculated using the APBS tool in PyMOL (http://www.pymol.org/pymol).In addition to the interaction between the positively charged amine of the N-terminal and Glu5 of two α1s, the TcpB-TcpA interaction is presumably promoted by the shape and charge complementarity of globular domains, such as the electrostatic interaction between Arg26 in α1 of TcpB and Glu83 in α/β-loop of TcpA, corresponding to the Arg26-Glu83 pairwise interaction previously shown as critical in the TcpA filament.

Fig. S2 .
Fig. S2.Crystal structure of TcpB.(a) Ribbon model of the crystal structure of the apo TcpB homotrimer observed in the asymmetric unit.(b) Structural comparison between a TcpB monomer (left panel) and CofB monomer (right panel, pdb code: 5ax6).Each disulfide bond bridge is shown as a yellow stick model.

Fig. S4 .
Fig. S4.Crystal structure of TcpB-TcpF.Front view (left panel) and side view (right panel) of the structure with the TcpB trimer (cyan, magenta, and green) and the bound TcpF trimer (blue, yellow, salmon pink) shown as ribbon models.

Fig. S6 .
Fig. S6.Multiple alignments of the mature TcpF protein sequences among pathogenic and environmental V. cholerae strains.Strains used for the alignment were 569B (O1, classical,

Fig. S9 .
Fig. S9.TCP filament model.(a) Side view of the toxin-coregulated pilus (TCP) model depicted as a ribbon model.(b) Close-up views of the stabilizing salt bridges between Arg26

Fig. S10 .
Fig. S10.Zoomed-in view of the linker between TcpB domain 1 and domain 2 (green).In this case, the two Gly residues (Gly231 and Gly239) and one Ser residue (Ser240) are depicted as a stick model.

Fig. S11 .
Fig. S11.Protein sequence alignments among secreted proteins in T4bP.A sequence alignment of the 20 N-terminal amino acids of Vibrio cholerae TcpF and putative secreted proteins from Vibrio mimicus, Photobacterium kishitanii, Enterovibrio coralii, and Allivibrio fischeri in Clade 1. Middle panel: A sequence alignment of the 20 N-terminal amino acids of Escherichia coli CofJ, Escherichia coli LngJ, predicted secreted proteins from Escherichia albertii, Escherichia lignolyticus, and Salmonella enterica in Clade 2. Lower panel: A sequence alignment of the 20 N-terminal amino acids among Citrobacter rodentium CfcJ, predicted secreted proteins from Enterobacter mori, Enterobacter cloacae, Klebsiella sp., and Citrobacter werkmanii in Clade 3. The details of the clades and accession number of amino acid sequences are shown in Fig. 4d.The mature N-terminal sequences of secreted proteins generated from N-terminal signal sequence cleavage were predicted by SignalP-6.0(https://services.healthtech.dtu.dk/service.php?SignalP).The black underlines indicate the position of T4bP-SS of TcpF or CofJ.The black dashed underline indicates the predicted position of T4bP-SS in CfcJ.The figures were generated with ESPript (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).

Fig. S12 .
Fig. S12.Comparison of the diameter of the toxin-coregulated pilus (TCP) model and TcpF trimer.Left panel: Superposition of a TcpA filament model shown as a blue ribbon model with the electron density map of TCP (ID: EMD-1955).The diameter of this filament model is approximately 90 Å.Right panel: model of the top view of the TcpF trimer.The maximum diameter of the TcpF trimer is approximately 110 Å.

Fig. S13 .
Fig. S13.Structural model of the TcpA filament-TcpB monomer complex.Left panel: Side view of the TcpA filament-TcpB monomer complex depicted as a ribbon model, constructed by superimposing the TcpB monomer model (magenta) onto a TcpA molecule (green) at the bottom of the TcpA filament model (gray) with Coot (51) and PyMOL.The α/β loop of TcpB, which has a disulfide bond (Cys85-Cys107) and is longer than that of TcpA, collides with the D-region of TcpA (right panel).

Fig. S14 .
Fig. S14.Location of residues important for V. cholerae colonization.Top view (left) and side view (right) of the TcpF trimer with the surface model.Glu251, Glu252, and Tyr292 of TcpF, which are important for V. cholerae colonization, are depicted in red.