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The exposure of blood to bioincompatible materials used for dialysis triggers leukocyte activation and protein adsorption. We describe a single-step, postmanufacturing method for surface modification to create biomaterials used in medical devices and dialysis with altered surface characteristics. Peptides derived from the receptor-binding domain of the type IV pilin of Pseudomonas aeruginosawere synthesized using land d-amino acids to generate l-K122-4, enantiomer d-K122-4, and d-retroinverso RI-K122-4 peptides. l-K122-4, d-K122-4, and RI-K122-4 peptides, but not control peptides, bound durably to the surfaces of materials used in medical devices and dialysis including silicone and polysulfone. d-K122-4 enantiomeric peptides were protease resistant on polysulfone and could remain bound to the surface for up to 28 days. To demonstrate that K122-4 peptides could be used to modify material surfaces, d-K122-4 peptide was conjugated to polyethylene glycol (d-K122-4-PEG) and applied to polysulfone. When compared with untreated material, d-K122-4-PEG reduced the surface adsorption of albumin or immunoglobulin G to polysulfone. In coincubation experiments, although uncoated polysulfone induced pro-interleukin-1β cytokine expression in leukocytes, cellular activation was prevented when leukocytes were incubated with d-K122-4-PEG–modified polysulfone. These data demonstrate the proof of principle that K122-4 peptides can be applied to modify the surface characteristics of materials used for dialysis.

Importance of the field:CF airway mucus can be infected by opportunistic microorganisms, notably Pseudomonas aeruginosa. Once organisms are established as biofilms, even the most potent antibiotics have little effect on their viability, especially during late-stage chronic infections. Better understanding of the mechanisms used by P. aeruginosato circumvent host defenses and therapeutic intervention strategies is critical for advancing novel treatment strategies.Areas covered in this review:Inflammatory injury in CF lung, role of neutrophils in pathogenesis, P. aeruginosabiofilms, mucoidy and its relationship with poor airway oxygenation, mechanisms by which P. aeruginosabiofilms in the CF airway can be killed.What the reader will gain:An understanding of the processes that P. aeruginosaundergoes during CF airway disease and clues to better treat such infections in future.Take home message:The course of CF airway disease is a process involving host and microbial factors that often dictate frequency of pulmonary exacerbations, thus affecting the overall course. In the past decade significant discoveries have been made regarding the pathogenic processes used by P. aeruginosato bypass the immune system. Many new and exciting features of P. aeruginosanow illuminate weaknesses in the organism that may render it susceptible to inexpensive compounds that force its own destruction.

Type IV pilin monomers assemble to form fibers called pili that are required for a variety of bacterial functions. Pilin monomers oligomerize due to the interaction of part of their hydrophobic N-terminal α-helix. Engineering of a truncated pilin fromPseudomonas aeruginosastrain K122-4, where the first 28 residues are removed from the N terminus, yields a soluble, monomeric protein. This truncated pilin is shown to bind to its receptor and to decrease morbidity and mortality in mice upon administration 15 min before challenge with a heterologous strain of Pseudomonas. The structure of this truncated pilin reveals an α-helix at the N terminus that lies across a 4-stranded antiparallel β-sheet. A model for a pilus is proposed that takes into account both electrostatic and hydrophobic interactions of pilin subunits as well as previously published x-ray fiber diffraction data. Our model indicates that DNA or RNA cannot pass through the center of the pilus, however, the possibility exists for small organic molecules to pass through indicating a potential mechanism for signal transduction.

Fibers of pilin monomers (pili) form the dominant adhesin of Pseudomonas aeruginosa,and they play an important role in infections by this opportunistic bacterial pathogen. Blocking adhesion is therefore a target for vaccine development. The receptor-binding site is located in a C-terminal disulphide-bonded loop of each pilin monomer, but functional binding sites are displayed only at the tip of the pilus. A factor complicating vaccination is that different bacterial strains produce distinct, and sometimes highly divergent, pilin variants. It is surprising that all strains still appear to bind a common receptor, asialo-GM1. Here, we present the 1.63 Å crystal structure of pilin from P. aeruginosastrain PAK. The structure shows that the proposed receptor-binding site is formed by two β-turns that create a surface dominated by main-chain atoms. Receptor specificity could therefore be maintained, whilst allowing side-chain variation, if the main-chain conformation is conserved. The location of the binding site relative to the proposed packing of the pilus fiber raises new issues and suggests that the current fiber model may have to be reconsidered. Finally, the structure of the C-terminal disulphide-bonded loop will provide the template for the structure-based design of a consensus sequence vaccine.

A representative combinatorial peptide library technique is proposed to simplify and reduce both synthesis and screening efforts. A multiple column peptide synthesizer was used to perform parallel synthesis of the peptide libraries. The peptide libraries were synthesized with a set of 10 selected amino acids representing the basic physicochemical properties associated with naturally occurring amino acid side-chains. Three series of two-position defined hexapeptide libraries were made and used to map the epitope of a monoclonal antibody, which was raised against the receptor binding domain of Pseudomonas aeruginosa strain K pilin. The capability of the peptide libraries to map and represent other epitope sequences was addressed. The native epitope sequence was mapped successfully together with sequences that were not previously recognized. These results illustrated the feasibility of using the representative peptide library.Copyright 1994, 1999 Academic Press

The four synthetic peptide antigens, PAK 128-144, PAO 128-144, KB7 128-144 and P1 126-148, correspond in amino acid sequence to the C-terminal receptor binding regions of four strains (PAK, PAO, KB7, P1) of Pseudomonas aeruginosa pilin. The NMR solution structures of the trans forms of the peptides show conserved β-turns which have been implicated in antibody and receptor recognition. The interactions between these peptides and a cross-reactive monoclonal antibody, PAK-13, have been studied using two-dimensional1H NMR spectroscopy in order to map the antigenic determinants recognized by the antibody. Residues for which spectral changes were observed upon antibody binding differed from peptide to peptide but were mostly confined to one or both of the turn regions and to the hydrophobic pockets. Conformational changes in the β-turns and hydrophobic pockets of these peptides upon antibody binding were also monitored by examination of the pattern of nuclear Overhauser effects (NOEs) versus transferred nuclear Overhauser effects (TRNOEs) for the free versus the bound peptides. Although TRNOEs developed strongly between side chain resonances in the hydrophobic pockets of the peptides, no additional backbone TRNOEs were observed in the presence of antibody, suggesting no major conformational changes in the secondary structures of the peptides upon binding. This implies a flexible antibody combining site, a feature which is discussed with respect to cross-reactivity, strain specificity, and the design of a synthetic peptide vaccine effective against a broad spectrum of P. aeruginosa strains. The binding of the PAK peptide to a disaccharide receptor analog, (βGalNAc(1-4)βGal), was also studied using 1H NMR in order to map the “adhesintope” recognized by the receptor. Spectral changes observed in the peptide spectrum with the binding of receptor were similar to those seen for the binding of antibody, suggesting that the epitope recognized by the antibody is structurally coincident with the adhesintope recognized by the receptor. The relevancy of this result is discussed with respect to immunogenicity versus pathogenicity, and the proper design of a vaccine which could prevent the mutational escape of the pathogen away from the host’s defence systems.

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