Antigen‐antibody interactions: Elucidation of the epitope and strain‐specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K

Abstract

The C-terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strainspecific monoclonal antibody PK99H, which blocks pilus-mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134-140 (Asp-Glu-Gln-PheIle-Pro-Lys) by using a single alanine replacement analysis on the 17-residue synthetic peptide corresponding to the PAK C-terminal sequence 128-144. Indeed, a 7-residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17-residue peptide. This epitope was found to contain two critical residues (PheI3' and LysI4O) and one nonessential residue ( G l r~'~~) .Interestingly, the peptide, Phe-Ile-Pro-Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strainspecificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122-4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain-specificity of PK99H for strains PAO, K122-4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain-specificity of PK99H. Similarly, cross-reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence.

Immunization with aPseudomonas aeruginosaElastase Peptide Reduces Severity of Experimental Lung Infections Due toP. aeruginosaorBurkholderia cepacia

“…Each peptide contained alpha-Nbenzoylbenzoyl ornithine as the N-terminal group for the purposes of photocross-linking and the quantitation of peptide : protein ratios. The synthesis, purification, and characterization of the peptides were performed as described elsewhere [24].…”

Interaction of the receptor binding domains of Pseudomonas aeruginosa pili strains PAK, PAO, KB7 and P1 to a cross-reactive antibody and receptor analog: implications for synthetic vaccine design

“…Synthesis of P. aeruginosa pilin peptides P. aeruginosa strain PAK pilin residues 128 to 144, Ac-KCTSDQDEQFIPKGCSK-amide, PAO pilin residues 128 to 144, Ac-ACKSTQDPMFTPKGCDN-amide, KB7 pilin residues 128 to 144, Ac-SCATTVDAKFRPNGCTDamide, and P1 pilin residues 126 to 148, Ac-NCKITKTP-TAWKPNYAPANCPKS-amide were synthesized and puri®ed by solid phase peptide synthesis and puri®ed by reversed-phase HPLC as previously reported (Wong et al, 1992(Wong et al, , 1995. All peptides were studied in their oxidized forms with the disul®de bridge between residues 129 and 142 in the PAK, PAO and KB7 peptides and between residues 127 and 145 in the P1 peptide (Figure 1).…”

“…A strain-speci®c monoclonal antibody, PK99H, directed against the PAK pilus was found to block pilus-mediated adherence to buccal and tracheal epithelial cells (Doig et al, 1990) and provided passive immunity against a homologous strain in challenge studies in mice (Sheth et al, 1995). The PK99H epitope was located in the sequence 134 to 140 (Asp-Glu-Gln-Phe-Ile-Pro-Lys) in PAK, and was found to contain three critical residues, Phe137, Ile138 and Lys140, two of which are located in the b-turns (Wong et al, 1992). A second monoclonal antibody, PAK-13, raised against the PAK pilus is cross-reactive with all four pilin peptides and their native pili and recognizes the same epitope in the PAK peptide as PK99H (Sheth et al, 1995).…”

Animal Protection and Structural Studies of a Consensus Sequence Vaccine Targeting the Receptor Binding Domain of the Type IV Pilus of Pseudomonas aeruginosa

“…36 Specific methods for SPPS, purification and characterization of the peptides have been described. 37 Peptides were synthesized with a photo-reactive group, benzoyl benzoic acid, attached to the N terminus. [38][39][40] These peptides were conjugated to the carrier protein tetanus toxoid (TT).…”