Genome-wide study of Pseudomonas aeruginosa outer membrane protein immunogenicity using self-assembling protein microarrays

Abstract

Pseudomonas aeruginosa is responsible for potentially life-threatening infections in individuals with compromised defense mechanisms and those with cystic fibrosis. P. aeruginosa infection is notable for the appearance of a humoral response to some known antigens, such as flagellin C, elastase, alkaline protease, and others. Although a number of immunogenic proteins are known, no effective vaccine has been approved yet. Here, we report a comprehensive study of all 262 outer membrane and exported P. aeruginosa PAO1 proteins by a modified protein microarray methodology called the nucleic acid-programmable protein array. From this study, it was possible to identify 12 proteins that trigger an adaptive immune response in cystic fibrosis and acutely infected patients, providing valuable information about which bacterial proteins are actually recognized by the immune system in vivo during the natural course of infection. The differential detections of these proteins in patients and controls proved to be statistically significant (P<0.01). The study provides a list of potential candidates for the improvement of serological diagnostics and the development of vaccines.

FIG. 1.

NAPPA quality control. (a) Expression clones encoding the target proteins fused to a C-terminal GST tag were printed along with a polyclonal anti-GST antibody in duplicate on the array surface. DNA capture was confirmed by PicoGreen (PG) staining (DNA), in situ protein expression and capture were assessed by GST detection using a monoclonal antibody (protein), and after incubation with serum, the response was measured with a horseradish peroxidase-linked anti-human IgG secondary antibody (serum). (b) DNA-versus-protein scatter plot showing background negative control spots (blue) and expression clone spots (brown) on a logarithmic plot. All clone spots showed protein and DNA levels at least three standard deviations higher than the mean background level (red lines). (c) Scatter plot comparing protein signal intensities (anti-GST antibody) and serum signal intensities (anti-human IgG antibody) for data from panel a. Red features correspond to visually confirmed hits. Duplicate signals for one example of the hits are indicated by the black arrows on both the serum panel and the graph.

FIG. 2.

Serum screening. (a) A representative group of serum slides for controls and CF and non-CF (NCF) patients. (b and c) Reproducibility scatter plots of the serum responses for independently processed samples (samples A and B) from patients CF-1 and NCF-1, as seen in panel a. All detected spots are represented in green, and the visually confirmed hits are highlighted with red circles. The R² values for CF-1 and NCF-1 were 0.85 and 0.80, respectively.

FIG. 3.

Microarray analysis. (a) Z-scores were computed from the averages of all four features for each antigen (duplicate features from duplicate slides). Here, the Z-scores of the 48 visually confirmed positives were used to create a heat map. (b) A map displaying the visually confirmed hits from panel a. The order of antigens in the heat map and the order in the chart are the same.

FIG. 4.

Western blot confirmation. Proteins of interest were generated by IVTT and analyzed by Western blotting using patient serum as the primary antibody. The numbers above the lanes correspond to antigens tested, and their NAPPA results for that specific patient are represented by a plus or minus sign. NAPPA images for some hits that were negative by Western blotting are shown. Proteins tested are numbered as follows: 1, PA1080; 2, PA0044; 3, PA2373; 4, PA2130; 5, PA2760; 6, no DNA control; 7, PA3841; 8, PA0973; 9, PA0807; 10, PA2685; 11, PA4179; 12, PA1082; 13, PA1302; 14, PA1094; 15, PA4837. Molecular masses are given to the left of the gels.