Animal protection and structural studies of a consensus sequence vaccine targeting the receptor binding domain of the type IV pilus of Pseudomonas aeruginosa

Abstract

One of the main obstacles in the development of a vaccine against Pseudomonas aeruginosa is the requirement that it is protective against a wide range of virulent strains. We have developed a synthetic-peptide consensus-sequence vaccine (Cs1) that targets the host receptor-binding domain (RBD) of the type IV pilus of P. aeruginosa. Here, we show that this vaccine provides increased protection against challenge by the four piliated strains that we have examined (PAK, PAO, KB7 and P1) in the A.BY/SnJ mouse model of acute P. aeruginosa infection. To further characterize the consensus sequence, we engineered Cs1 into the PAK monomeric pilin protein and determined the crystal structure of the chimeric Cs1 pilin to 1.35 A resolution. The substitutions (T130K and E135P) used to create Cs1 do not disrupt the conserved backbone conformation of the pilin RBD. In fact, based on the Cs1 pilin structure, we hypothesize that the E135P substitution bolsters the conserved backbone conformation and may partially explain the immunological activity of Cs1. Structural analysis of Cs1, PAK and K122-4 pilins reveal substitutions of non-conserved residues in the RBD are compensated for by complementary changes in the rest of the pilin monomer. Thus, the interactions between the RBD and the rest of the pilin can either be mediated by polar interactions of a hydrogen bond network in some strains or by hydrophobic interactions in others. Both configurations maintain a conserved backbone conformation of the RBD. Thus, the backbone conformation is critical in our consensus-sequence vaccine design and that cross-reactivity of the antibody response may be modulated by the composition of exposed side-chains on the surface of the RBD. This structure will guide our future vaccine design by focusing our investigation on the four variable residue positions that are exposed on the RBD surface.

Figure 1. Amino acid sequences of the Cs1 consensus sequence and the receptor-binding domains from six strains of P. aeruginosa

Alignment of amino acid sequences of the receptor-binding domain from P. aeruginosa strains PAK, PAO, KB7, K122-4, CD4 and P1. Boxed residues show identical positions. Shaded residues indicate positions with conservative substitutions.

Figure 2. Protection of A.BY/SnJ mice from challenge by different strains of P. aeruginosa after active immunization by pilin peptide-tetanus toxoid conjugates

(a) Challenge by PAK strain bacteria. (b) Challenge by PAO strain bacteria. (c) Challenge by KB7 bacteria. (d) Challenge by P1 bacteria. (e) Challenge by PAK bacteria. (f) Challenge by PAO bacteria. (g) Challenge by P1 bacteria. For panels (e)–(g) the open squares denote Cs1-TT and filled triangles denote the scrambled Cs1 peptide-TT (Scr-TT). The strain used for challenge is indicated in the top left of each panel. TT indicates animals immunized with tetanus toxoid alone and Adjuvax indicates animals that received adjuvant alone. A scrambled Cs1 peptide conjugated to tetanus toxoid was used as a control to demonstrate dramatic protection of the peptide beyond the adjuvant effect of tetanus toxoid alone (unpublished results).

Figure 3

Representative sigmaA-weighted 2 mFo-Fc electron density map of Cs1 pilin, contoured at 2 σ, showing the quality of X-ray diffraction data for Cs1 pilin.

Figure 4. Ribbon diagrams showing similarities among Cs1, PAK and K122-4 monomeric pilin structures

(a) Cs1 pilin. The α-helix spanning residues 25–51 and the four strands of the major β-sheet are labeled. The receptor binding domain is boxed. Residue 29 is the start of the monomeric pilin construct. Residues 1–28 were removed to prevent polymerization. C denotes the C-terminal of the pilin. (b) Overlay of Cs1 and PAK pilin. (c) Overlay of Cs1 and K122-4 pilin. The superimposed region of Cs1 and K122-4 includes the major β-sheet and the RBD is shown (as discussed in the text). In (b) and (c), the regions highlighted in red are the regions of the two proteins that deviate by 2 Å or less (and used for r.m.s.d. calculations).

Figure 5. Stereo images of stick representations showing the similarities in backbone conformation of the entire receptor binding domain (128–143) from the crystallographically determined structures of the three pilin proteins

The view has been rotated by 180° about the horizontal and 90° about the vertical axes of the page relative to the orientation in Figure 4. (a) Cs1 pilin protein (PDB ID, 2PY0). (b) Overlay of the RBD (128–143) of Cs1, PAK (PDB ID, 1DZO) and K122-4 (PDB ID, 1QVE) pilins, showing only main-chain atoms. (c) Overlay of the RBD (128–143) of Cs1, PAK, and K122-4 pilins, showing main-chain and side-chain atoms. The r.m.s.d. between residues 128–143 truncated and full-length forms of PAK pilin is 0.17 Å for main-chain atoms, so only the truncated pilin structure has been shown. In each diagram, the backbone of Cs1 pilin is shown in light blue, PAK pilin in light green and K122-4 in orange.

Figure 6. Stick representations showing the differences in backbone conformation of the RBD region in each of the three pilin proteins

(a) Cs1, PAK and K122-4 pilin are superimposed by the backbone atoms of residues 128–132. The differences in position of the first β-turn (residues 134–137) are shown when the RBD regions are superimposed in this way. Hydrogen bonds are shown by red dashes. Stick and ball models are used to show the side-chains of residues 132 and 114, which form hydrogen bonds with main-chain atoms to terminate the β-strand of the RBD. (b) Cs1, PAK and K122-4 pilin are superimposed by the backbone atoms of residues 134–137. The differences in position of the second β-turn (residues 139–142) are shown when the RBD regions are superimposed in this way. In both panels, the backbone carbon atoms of Cs1 pilin are shown in light blue, PAK pilin in light green and K122-4 in orange.

Figure 7. Stick diagrams showing the interactions of buried side-chains of the RBD

(a) Cs1 pilin, showing the hydrogen bonding of the side-chain of Gln133 with Ser131 and Asn111. The side-chain of Ile138 is shown as a space-filling model. The same interactions shown in (a) are observed in the PAK pilin structure. (b) K122-pilin, showing the side-chain of Ala133. The side-chains of Leu138, Leu33 and Leu111 (equivalent to Leu108 in the K122-4 sequence alignment) are shown as space-filling models to demonstrate the hydrophobic interactions on the buried face of the RBD of K122-4 pilin. Both panels show the conserved hydrogen bond of the side-chain of Ser131 with the backbone nitrogen of residue 33.

Figure 8. Molecular surfaces showing the positions of variable and conserved residues on the solvent accessible surface of the Cs1 pilin receptor binding domain

(a) The contribution of conserved position side-chains are shown in red, green and blue. Red indicates an acidic residue side-chain (Asp134). Green indicates a hydrophobic residue side-chain (Phe137). Blue indicates a basic residue side-chain (Lys140). Yellow indicates the disulfide bond. The surface contributions from backbone atoms are highlighted in orange. Light grey highlights variable position side-chains (Lys130, Asp132, Pro135, Gln136, Ile138 and Ser143). (b) The contributions of residues in the RBD region to the molecular surface are shown in blue. The side-chains of variable positions Asp132 and Gln136 are shown in red. Lys128 and Ser143, which are outside of the disulfide loop, are also shown in red, as their identities vary among strains. The contributions of the side-chains to the molecular surface of the consensus sequence substitution sites 130 and 135 are shown in orange. The contribution of Ile138 is shown in white because although exposed on the RBD surface, 83% of the surface area of Ile138 is buried and we do not expect this side-chain to be significant in antibody recognition. The side-chain of variable position Gln133 is buried, and therefore is not visible on the molecular surface. In both panels, dark gray denotes region of the molecular surface from residues not in the RBD region. The solvent accessible surface was calculated using a 1.4 Å probe.