pORF5质粒蛋白拮抗LL37抗菌肽增强沙眼衣原体感染的初步研究

pORF5 plasmid protein promotes Chlamydia trachomatis infection through inhibition of the antibacterial peptide LL37： a preliminary study

目的 探讨pORF5质粒蛋白能否通过抑制LL37抗菌肽增强沙眼衣原体（Chlamydia trachomatis,Ct）感染,并初步探讨其分子机制.方法 表达并纯化GST-pORF5融合蛋白,融合蛋白经蛋白酶酶切制备不含GST标签的pORF5蛋白;pORF5蛋白和LL37单独或共孵育衣原体后再感染HeLa细胞,间接免疫荧光技术计数衣原体包涵体形成数量;荧光定量PCR检测TNF-α、LL37基因表达水平.结果 单独Ct感染组衣原体包涵体形成单位（IFU）为3.8×105/ml,LL37处理组IFU为2.0 × 105/ml,pORF5蛋白处理组IFU为3.0×105/ml,pORF5蛋白和LL37共处理组IFU为3.1×10/ml.pORF5蛋白处理组、LL37与pORF5蛋白共处理组和单独Ct感染组之间比较,差异无统计学意义（P＞0.05）,但明显高于LL37单独处理组（P＜0.05）.pORF5蛋白和LL37共同处理组TNF-α表达水平明显高于LL37单独处理组（P＜ 0.01）;pORF5蛋白处理组较Ct处理组LL37基因转录水平下降了19％.结论 pORF5质粒蛋白能拮抗LL37抗菌肽增强Ct感染,其机制可能与上调肿瘤坏死因子α、下调LL37表达相关.

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 downregulation of LL37 expression in Ct-infected cells.