Immunoproteomic Identification and Serological Responses to Novel Chlamydia pneumoniae Antigens That Are Associated with Persistent C. pneumoniae Infections

Abstract

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.

Biomarker Candidates of Chlamydophila pneumoniae Proteins and Protein Fragments Identified by Affinity-Proteomics Using FTICR-MS and LC-MS/MS

“…Sera from 39 human donors were tested for antibodies against C. pneumoniae, C. trachomatis, and C. psittaci using the MIF assay (Savyon Diagnostics, St. Ashdod, Israel) [8]. One serum tested positive for C. trachomatis and was excluded from the study.…”

“…Chlamydia samples were purified using the 2D Clean-up Kit (Amersham Biosciences, Uppsala, Sweden) and applied overnight on 17 cm IPG strips (pH range 3-10 NL) using a passive in-gel rehydration method, as described [8]. Following separation, proteins were visualized with sensitive colloidal Coomassie staining [9], and gels were scanned with a Bio-Rad (Laboratories GmbH, München, Germany) GS-710 imaging densitometer.…”

“…One serum tested positive for C. trachomatis and was excluded from the study. C. pneumoniae TW-183 were cultured as previously described [8].…”

“…Immunoblotting was performed as previously described [8], and electronic image files matched with the 2DE maps of the Coomassie-stained gels. For the analysis of the immunoblots, the maximum intensity of each spot was determined using the AIDA software package (Raytest/Fuji, Straubenhardt, Germany) [8].…”

“…In a previous study, an affinity-proteomics approach combined with FTICR-MS with high specificity and reproducibility has been developed and applied for the identification of C. pneumoniae antigenic structures relevant for serodiagnosis [8]. The combination of 2-DE immunoproteomics with FTICR-MS and LC-MS/MS is shown here to be highly successful in identifying several hitherto unknown antigens and, particularly, the identification of several neo-antigenic protein fragments.…”