Proteomic identification of OprL as a seromarker for initial diagnosis of Pseudomonas aeruginosa infection of patients with cystic fibrosis

Abstract

Identification of new immunogenic antigens that diagnose initial Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF) alone or as an adjunct to microbiology is needed. In the present study, a proteomic analysis was performed to obtain a global assessment of the host immune response during the initial P. aeruginosa infection of patients with CF. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to identify outer membrane protein L (OprL), a non-type III secretion system (TTSS) protein, as an early immunogenic protein during the initial P. aeruginosa infection of patients with CF. Longitudinal Western blot analysis of sera from 12 of 14 patients with CF detected antibodies to OprL during the initial P. aeruginosa infection. In addition, also detected were antibodies to ExoS, ExoU, or ExoS and ExoU, the latter indicating sequential P. aeruginosa infections during initial infections. Detection of serum reactivity to OprL, along with proteins of the TTSS, and in conjunction with microbiology may diagnose initial P. aeruginosa infections in patients with CF.

FIG. 1.

Immunoreactivity of sera from a patient with CF. P. aeruginosa Sup (15 μg of protein) was subjected to 2D SDS-PAGE with a pH 3 to 10 isoelectric focusing (IEF) strip, followed by 12% SDS-PAGE. Proteins were transferred to PVDF membranes, blocked, and subjected to Western blotting with serum from a patient with CF. Blots were washed, incubated with a secondary antibody (goat anti-human IgG-HRP), washed, incubated in Super Signal West Pico, and exposed to X-ray film. An image of the X-ray is shown. Blots were also probed for ExoS. Proteins 10 and 17, which migrated differently than known TTSS proteins (arrows) were subjected to MALDI-TOF MS. The serum sample used to generate this figure was chosen based upon the presence of antibodies to numerous P. aeruginosa proteins which could be resolved into ∼20 distinct immunoreactive dots.

FIG. 2.

Longitudinal reactivity of serum to Oprl and TTSS proteins. Proteins ExoS (S), ExoU (U), PcrV (V), PopB (B), and OprL (L) and P. aeruginosa Sup (Ly) (1.0 μg of protein) were separated by 12% SDS-PAGE. Proteins were transferred to PVDF membranes, blocked, and subjected to Western blotting with serum from three sequential blood samples drawn from patient 14 (referenced in Table 1). Blots were washed and incubated with a secondary antibody (goat anti-human IgG-HRP), washed, and incubated in Super Signal. The blot was exposed to X-ray film. The upper to lower panels represent early to late times of collection; images are of identically timed exposures to X-ray film. The migration of protein markers is shown to the left of the film.

FIG. 3.

ELISA of OprL and PopB in serum from a patient with CF. An ELISA was performed with OprL or PopB (50 to 200 ng). Plates were blocked and incubated with the indicated diluted serum from patient 5 (1/500 to 1/16,000, expressed as a reciprocal number) or without a primary antibody. Wells were washed and incubated with 100 μl of a secondary antibody (goat anti-human at a 1:10,000 final dilution) and then washed and developed with 3,3′,5,5′-tetramethylbenzidine. Absorbance was read at 450 nm (OD 450).