Identification of proteins interacting with pORF5 in the pathogenesis of C. trachomatis.

OBJECTIVE

This study is to identify and investigate the proteins interacting with pORF5 implicated in the pathogenesis of C. trachomatis.

METHODS

The isobaric tags for relative and absolute quantitation (iTRAQ) approach combined with nano liquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) analysis was applied to identify and quantify the differentially expressed proteins in the pORF5-transfected HeLa (pORF5-HeLa) cells and the control vector-transfected HeLa (vector-HeLa) cells. Quantitative real-time PCR (qRT-PCR) and Western blot analysis were performed to detect the mRNA and protein expression levels.

RESULTS

Totally 3355 proteins were quantified by employing biological replicates, 314 of which were differentially expressed between the pORF5-HeLa and vector-HeLa cells. Nine differentially expressed proteins (HIST1H1C, HBA1, PARK7, HMGB1, HMGB2, CLIC1, KRT7, SFN, and CDKN2A) were subjected to qRT-PCR, and two over-expressed proteins (HMGB1 and PRAK7) were subjected to the Western blot analysis, to validate the proteomic results. The results from the qRT-PCR and Western blot analysis were consistent with the findings from the proteomic analysis. Moreover, pORF5 could inhibit the TNF-α-induced apoptosis in HeLa cells. Through siRNA-mediated functional screening, the high-mobility group box 1 (HMGB1) was shown to be relevant to the inhibition of the apoptotic response in the host cells.

CONCLUSION

Identification of key proteins interacting with pORF5 could contribute to the understanding and further exploration of the function of pORF5 in the pathogenic mechanisms of C. trachomatis.