Use of phage display to identify potential Pseudomonas aeruginosa gene products relevant to early cystic fibrosis airway infections

Abstract

Pseudomonas aeruginosa airway infections are a major cause of morbidity and mortality in patients with cystic fibrosis. Treatment of established infections is difficult, even with microbiologically active agents. Thus, prevention of infection is an important goal of management. Isolates from cystic fibrosis patients appear to originate from the environment but adapt to the milieu of the airway of the cystic fibrosis patient and evolve toward a common phenotype. Identification of the antigens expressed early in infection may lead to novel targets for vaccine development. Immunogenic peptides were identified in a J404 random nonapeptide phage display library with serum from cystic fibrosis patients obtained within the first year of P. aeruginosa infection. One hundred sixty-five reactive clones were verified by plaque lift assays, and their inserts were sequenced. The sequenced nonapeptides were compared with the published sequence of strain PAO1, identifying homologies to 76 genes encoding outer membrane and secreted proteins. The majority of these were proteins involved in small-molecule transport, membrane structural proteins, and secreted factors. An in silico analysis was performed that suggested that the occurrence of multiple matches to predominantly outer membrane and secreted proteins was not attributable to random chance. Finally, gene expression array data from early isolates of P. aeruginosa from cystic fibrosis patients was compared with the results from phage display analysis. Eleven outer membrane and secreted proteins were common between the two data sets. These included genes involved in iron acquisition, antibiotic efflux, fimbrial biogenesis, and pyocin synthesis. These results demonstrate the feasibility and validity of this novel approach and suggest potential targets for future development.

FIG. 1.

Scheme for panning and evaluation of potential targets. Details are given in the text. The number of phage particles panned/recovered in each component is noted beneath the component. The total number of genes encoding proteins in each cytoplasmic location is noted beneath the location. IM, inner membrane; OM, outer membrane; assoc., associated.

FIG. 2.

Distribution of functional classes of Pseudomonas genes that were sequenced from the J404 phage display library. Proteins of all 27 known functional classes were hit: (A) hypothetical, unknown, and unclassified, (B) membrane proteins, (C) small-molecule transport, (D) putative enzymes, (E) transcriptional regulators, (F) two-component regulatory systems, (G) amino acid biosynthesis, (H) energy metabolism, (I) adaptation and protection, (J) carbon compound catabolism, (K) translation, posttranslational modification, and degradation, (L) DNA replication, recombination, modification, and repair, (M) biosynthesis of cofactors and prosthetic groups, (N) cell wall, lipopolysaccharide, and capsule, (O) secreted factors, (P) protein secretion and export apparatus, (Q) motility and attachment, (R) central internal metabolism, (S) transcription and RNA processing, (T) nucleotide biosynthesis and metabolism, (U) fatty acid and phospholipid metabolism, (V) chemotaxis, (W) related to phages, transposons, and plasmids, (X) chaperones and heat shock proteins, (Y) cell division, (Z) antibiotic resistance/susceptibility, and (AA) quinolone signal response.

FIG. 3.

Distribution of BLAST hits per gene for the P. aeruginosa (Pa) genome compared with the average over 100 random genomes. The variance of the random genome distribution for each number of BLAST hits per gene is indicated by ±2 standard deviation error bars. A selected set of 76 outer membrane and secreted proteins (Pa OM/sec) and 390 cytoplasmic proteins (Pa cyt) are also shown.

FIG. 4.

Distribution of functional classes overlapping the microarray data. The transcriptional profiles of four isolates from cystic fibrosis patients were compared to that of PAO1. All cystic fibrosis-activated genes were used for comparison to the phage display data. Overlaps were found in 23 of 26 functional classes: (A) hypothetical, unknown, and unclassified, (B) membrane proteins, (C) small-molecule transport, (D) putative enzymes, (E) transcriptional regulators, (F) two-component regulatory systems, (G) amino acid biosynthesis, (H) energy metabolism, (I) adaptation and protection, (J) carbon compound catabolism, (K) translation, posttranslational modification, and degradation, (L) DNA replication, recombination, modification, and repair, (M) biosynthesis of cofactors and prosthetic groups, (N) cell wall, lipopolysaccharide, and capsule, (O) secreted factors, (P) protein secretion and export apparatus, (Q) motility and attachment, (R) central internal metabolism, (S) transcription and RNA processing, (T) nucleotide biosynthesis and metabolism, (U) fatty acid and phospholipid metabolism, (X) chaperones and heat shock proteins, and (Z) antibiotic resistance and susceptibility.