Abstract: Ma Kangkang, Li Zhongyu*, Su Shengmei, Cao Wenjuan, Dai Wenting, Yang Xiaoyu, He Hongmei, Zhong Guangming. *Institute of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, Hunan, China Corresponding author: Li Zhongyu, Email: nhlzhy1023@126.com 【Abstract】 Objective To investigate whether pORF5 plasmid protein promotes Chlamydia trachomatis （Ct） infection through inhibition of the antibacterial peptide LL37, and to explore its potential molecular mechanism. Methods After expression and purification, the glutathione S-transferase （GST）-pORF5 fusion protein was digested with proteases to obtain pORF5 protein without GST tag. Some Hela cells were classified into four groups: Ct group infected with unpretreated Ct, pORF5 group infected with Ct that had been pretreated with pORF5 of 30 μg/ml for 2 hours, LL37 group infected with Ct that had been pretreated with LL37 of 20 μg/ml for 2 hours, combination group infected with Ct that had been pretreated with pORF5 of 30 μg/ml and LL37 of 20 μg/ml for 2 hours. After additional culture, indirect immunofluorescence assay was performed to count the number of Ct inclusion-forming units （IFUs）, and real-time, fluorescence-based quantitative PCR and enzyme-linked immunosorbent assay （ELISA） to detect the protein and mRNA expressions of tumor necrosis factor-α （TNF-α） respectively. Some Hela cells were divided into three groups: blank control group remaining untreated, Ct group infected with unpretreated Ct, pORF5 group infected with Ct that had been pretreated with pORF5 of 30 μg/ml for 2 hours. After 6 hours of additional culture, real-time, fluorescence-based quantitative PCR and ELISA were conducted to measure the mRNA and protein expressions of LL37, respectively. Results The concentration of IFUs was significantly higher in the Ct group, pORF5 group and combination group than in the LL37 group （3.8 × 105/ml, 3.0 × 105/ml and 3.1 × 105/ml vs. 2.0 × 105/ml, all P < 0.05）, with no significant differences between the Ct group, pORF5 group and combination group （P > 0.05）. The expressions of TNF-α mRNA and protein were significantly increased in the combination group compared with the LL37 group. The mRNA expression of LL37 was reduced by 19% in the pORF5 group compared with the Ct group. Conclusions pORF5 plasmid protein could promote Ct infection by counteracting the antibacterial peptide LL37, which may be associated with the up-regulation of TNF-α expression and down-regulation of LL37 expression in Ct-infected cells.

Key words: Chlamydia trachomatis, pORF5 plasmid protein, LL37, Antimicrobial peptides