Western blot (WB) analysis of toxin-coregulated pilus protein A (TcpA). Whole-cell lysates of classical V. cholerae O395 (wild-type [wt] TcpA and TcpA deleted [ΔtcpA]) were electrophoresed through 10% polyacrylamide gel and detected by anti-classical TcpA 11 to 18 peptide tertiary sera. The corresponding preimmune (PI) serum was used as the negative control. Some anti-TcpA peptide sera were more reactive in Western blotting (TcpA 11, TcpA 14, and TcpA 18), while others were less so (TcpA 13, TcpA 15, and TcpA 17), and two were negative (TcpA 12 and TcpA 16).

Rabbit anti-TcpA 11 to 18 sera specific for sequences from classical V. cholerae O1 toxin-coregulated pilus protein A (TcpA). Rabbits were immunized with synthetic TcpA polypeptides coupled to keyhole limpet hemocyanin (KLH). The immunization protocol employed multiple routes and multiple injections. Serum samples were collected on days 0 (preimmune), 28 (primary), 42 (secondary), 70 (tertiary), and 87 (quaternary). Primary rabbit serum was serially diluted in twofold increments, starting at 1:1,000. Enzyme-linked immunosorbent assay results are reported as optical densities measured at 450 nm.

Transmission immunoelectron microscopic analysis of anti-TcpA (toxin-coregulated pilus protein A)-specific antibodies binding to classical V. cholerae O1 TCP. Bacteria were incubated with various TcpA peptide-specific sera on 4% Formvar-coated, 300-mesh nickel support grids, washed to remove unbound antibody, and fixed in 2.0% paraformaldehyde-0.5% glutaraldehyde buffer. Bound antibody was detected with 10.0-nm gold particle-conjugated goat anti-rabbit IgG. Grids were negatively stained with 0.5% phosphotungstic acid (pH 6.5) and viewed with a transmission electron microscope at 25,000× magnification (inset). The inset shows the relationship of TCP and bacteria. The pilus shown in the individual panels corresponds to a magnified view of the pilus in the inset.

Western blot and immunoelectron microscopic analyses of V. cholerae expressing wild-type TcpA (toxin-coregulated pilus protein A), TcpA-deleted strains, and various pilin mutant strains that express TcpA with an Ala substitution in a specific interactive residue (8) (A) Anti-TcpA 15 tertiary serum (upper panel) diluted 1:12,500 was used to probe the membranes and compared to mouse monoclonal antibody 169.1 (lower panel), which is specific for TcpA 5 and known to provide protection in the infant mouse assay (15). (B) The effect of the D175A alteration on binding of anti-TcpA 15 serum to TCP was tested. The D175A change resulted in the complete loss of binding of TcpA 15 serum. An isotype control monoclonal antibody was negative for binding to TCP.

Soluble toxin-coregulated pilus protein A (TcpA) can interact with some anti-TcpA peptide antisera. (Top) A pulldown analysis which assessed the ability of anti-classical TcpA peptide serum to interact with TcpA monomers was performed. Purified, biotin-labeled TcpA was incubated overnight at 4°C with anti-TcpA peptide prebleed or tertiary serum, and then 20 μl of streptavidin-Sepharose (in PBS-1% nonfat milk) was added for 2 h at room temperature, followed by washing, electrophoresis, and subsequent Western blot analysis with horseradish peroxidase-labeled goat anti-rabbit IgG, antibody. (Bottom) Purified TcpA lysate was incubated overnight at 4°C with His-Select nickel affinity beads and washed, and anti-TcpA peptide preimmune (PI) or tertiary serum was added and incubated overnight at 4°C. Samples were washed before pellets were resuspended and analyzed in a Western blot as described for the top panel. rIg, rabbit IgG control.

Classical, toxin-coregulated pilus protein A (TcpA) peptides that were synthesized for this study and their locations on the classical TcpA monomer. TcpA peptides 11 to 18 were synthesized with a C residue at the C termini and conjugated by a disulfide bond to keyhole limpet hemocyanin (KLH) for immunization of rabbits as described in Materials and Methods. Colored peptides are correlated with the colored amino acids in the space-filling model of TcpA and define the locations of the primary amino acid sequences of the TcpA peptides on the folded TcpA monomer. RasMol version 2.7.2.1 molecular visualization software was used to display and manipulate the Brookhaven Protein Data Bank (available through the National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov]) TcpA file 1OQV (3).

Survival of infant mice following oral challenge with virulent classical V. cholerae O1 bacteria treated with anti-TcpA (toxin-coregulated pilus protein A) peptide serum. Groups of 10 4- to 5-day-old CD-1 mice were orally gavaged with 4.4 × 10⁶ CFU of classical V. cholerae O1 Ogawa bacteria (representing 4.4 50% lethal doses) which was mixed with tertiary antisera (A and B) or preimmune antisera (C); some pups were not infected (C). Mice were kept at 30°C and monitored for death every 4 h starting 24 h after oral challenge. The protection data for anti-TcpA 15 serum are representative of seven independent challenge assays. In six independent experiments, protective efficacy ranged between 70 and 100% (mean, 91.4%) for anti-TcpA 15 serum.

Computer-generated space-filling model of TcpA (toxin-coregulated pilus protein A) 15 and its location on TCP. The RasMol version 2.7.2.1 software was used to display and manipulate the Brookhaven Protein Data Bank files 1OQV (TcpA, panel A) and 1OR9 (TCP model, panels B to D) (3). (A) TcpA 15 comprises amino acids 170 to 183. TcpA 15 contains four (K172, D175, H181, and E183) of the six interactive amino acids (K172, D175, H181, E183, K184, and K187) previously identified as being important for TCP function. The TcpA residues (N173, L174, L176 [cyan], I179, H181, and V182) that do not vary between biotypes or colonizing-competent environmental isolates of V. cholerae are colored red. Eight of the 14 residues that comprised TcpA 15, A170, S171, K172, D175 (yellow amino acid), T177, N178, T180, and E183 (orange amino acids), vary between naturally occurring, functional TcpA alleles. Residue L176 is involved with Y51 in a hydrogen bond interaction. (B) The primary amino acid sequence of TcpA peptides (11 to 18) used to immunize rabbits are shown in the one-letter code and color coded for the individual TcpA peptides which are displayed on the TCP fiber model. Lysine residues are underlined. A TCP fiber model has been proposed (3). TcpA 15 is red and is partially exposed, nested between other TcpA peptides, all of which are displayed in a repeating pattern on the surface of the TCP fiber. (C) Residues 30 to 199 of selected chains (C, B, E, H, G. J, M, and L) of the TCP model were restricted (displayed without N termini) with the RasMol program and displayed. This TCP representation shows that D175 (yellow) is exposed and available for antibody binding. There is a recessed “pocket,” 30 by 20 Å, where TcpA 15 is located within the quaternary structure of TCP. Orange residues represent amino acids that can vary in TcpA 15, while red residues are conserved. Leucine 176 is cyan, and Y51 is blue. Residues 50 to 70 (pink, except 51) represent the sequence area in the α/β loop that bind the D region on an adjacent monomer. (D) Enlarged area showing the face of TcpA on adjacent monomers that are proposed to interact. The color scheme for the amino acids is as above.