Undressing of Waddlia chondrophila to enrich its outer membrane proteins to develop a new species-specific ELISA

Abstract

Waddlia chondrophila , an obligate intracellular bacterium of the Chlamydiales order, is considered as an agent of bovine abortion and a likely cause of miscarriage in humans. Its role in respiratory diseases was questioned after the detection of its DNA in clinical samples taken from patients suffering from pneumonia or bronchiolitis. To better define the role of Waddlia in both miscarriage and pneumonia, a tool allowing large-scale serological investigations of Waddlia seropositivity is needed. Therefore, enriched outer membrane proteins of W. chondrophila were used as antigens to develop a specific ELISA. After thorough analytical optimization, the ELISA was validated by comparison with micro-immunofluorescence and it showed a sensitivity above 85% with 100% specificity. The ELISA was subsequently applied to human sera to specify the role of W. chondrophila in pneumonia. Overall, 3.6% of children showed antibody reactivity against W. chondrophila but no significant difference was observed between children with and without pneumonia. Proteomic analyses were then performed using mass spectrometry, highlighting members of the outer membrane protein family as the dominant proteins. The major Waddlia putative immunogenic proteins were identified by immunoblot using positive and negative human sera. The new ELISA represents an efficient tool with high throughput applications. Although no association with pneumonia and Waddlia seropositivity was observed, this ELISA could be used to specify the role of W. chondrophila in miscarriage and in other diseases.

Conserved features and major differences in the outer membrane protein composition of chlamydiae

“…This is a performance comparable to or better than achieved in other outer membrane proteome studies (Chung et al ., ; Thein et al ., ; Cao et al ., ; Gesslbauer et al ., ). The same enrichment protocol was much less efficient for Parachlamydia and Waddlia and consistent with a recent study on immunogenic proteins of Waddlia (Lienard et al ., ). In outer membrane fractions of these organisms, non‐OMPs such as ribosomal proteins, elongation factors and heat‐shock proteins were detected in high amounts.…”

Simkania negevensis, an insight into the biology and clinical importance of a novel member of the Chlamydiales order

“…Such recommendations should also be applied to detect a Simkania infection in order to enable inter-laboratories comparison of obtained results. Alternatively, an ELISA assay developed in our laboratory to detect W. chondrophila (Lienard et al, 2014) could be adapted for Simkania infections. Such ELISA might be a good alternative, as the results are not biased by subjective analysis.…”

Eukaryotic Cell Permeabilisation to Identify New Putative Chlamydial Type III Secretion System Effectors Secreted within Host Cell Cytoplasm

“…Twenty-one C. trachomatis and 28 W. chondrophila proteins were identified in this analysis. Among them are abundant proteins such as chaperones, ribosomal proteins or elongation and initiation factors that are repeatedly retrieved in W. chondrophila mass spectrometry analyses [36,38,39] and thus likely represent contaminant abundant proteins. The remaining 13 C. trachomatis and 19 W. chondrophila putative secreted proteins include the Chlamydial Protease-like Activity Factor (CPAF), that was retrieved in screenings with both bacteria, and the Chlamydiaceae specific CADD (Chlamydia protein Associating with Death Domains).…”

“…All bacterial proteins identified in the PFO-treated sample and absent in mock-treated control as well as those enriched more than 1.5 times in the PFO-treated sample versus the mock control are presented in Supplementary Table S1. This list includes several proteins, such as chaperones (DnaK, GroL, GroS), ribosomal proteins (50S ribosomal proteins L7/L12, L9 and L10, 30S ribosomal protein S6) or elongation and initiation factors (Elongation factors Tu, Ts and G, Translation initiation factor IF-1) that are repeatedly retrieved in mass spectrometry analyses performed on different fractions containing W. chondrophila proteins [36,38,39]. They are probably very abundant proteins and likely represent technical contaminants.…”