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

Yan Zou; Wenting Dai; Wenbo Lei; Shengmei Su; Qiulin Huang; Zhou Zhou; Chaoqun Chen; Zhongyu Li

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

Am J Transl Res. 2018 Jun 15;10(6):1633-1647. eCollection 2018.

Authors

Yan Zou¹, Wenting Dai¹, Wenbo Lei¹, Shengmei Su¹, Qiulin Huang², Zhou Zhou¹, Chaoqun Chen¹, Zhongyu Li^{1

3}

Affiliations

¹ Pathogenic Biology Institute, School of Medicine, University of South China Hengyang 421001, Hunan, China.
² Department of General Surgery, The First Affiliated Hospital of University of South China Hengyang 421001, Hunan, China.
³ Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study Hengyang 421001, Hunan, China.

PMID: 30018706
PMCID: PMC6038076

Abstract

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.

Keywords: Chlamydia trachomatis (C. trachomatis); HMGB1; isobaric tags for relative and absolute quantitation (iTRAQ); pORF5; quantitative proteomic analysis.