沙眼衣原体质粒编码蛋白pgp5导致输卵管病变的作用机制研究

Chlamydia trachomatis (C.t) genitourinary tract infection is one of the important causes of the oviduct infertility, abortion, ectopic pregnancy in vomen. Our previous work has demonstrated for the first time that chlamydial plasmid-encoded protein pgp5 can inhibit chromosome gene expression by using the newest chlamydial transformation system （J Bacteriol 2014）,and the infection of chlamydia muridarum with pgp5 mutation reduced in significantly less hydrosalpinx in mice, However, how pgp5 inhibits the expression of these genes and the mechanism that pgp5 plays an important role in the process of the chlamydial pathogenicity are still kept unknown. Based on our findings, bioinformatics, molecular cloning technique, , electrophoretic mobility shift assay(EMSA), transcription activity assays will be used to find the biological characteristics of pgp5 and the molecular mechanism of gene expression regulation of pgp5, and then genetic recombination, antisense nucleic acid technology, chlamydial transformation system, quantitive RT-PCR technology will be used to construct new Chlamydia muridarum strains with single pgp5 regulation gene mutation, these new mutant Chlamydia will be injected into mice to find which gene regulated by pgp5 plays the decisive role in the process of chlamydial pathogenicity in fallopian tube caused by pgp5 and the molecular mechanism that the pgp5 regulated gene results in hydrosalpinx in mice. The completion of this project can reveal the molecular mechanism that pgp5 results in hydrosalpinx in mice, moreover this work can provides a basis for clinical treatment of chlamydial infection and the reduction of the incidence of oviduct infertility caused by Chlamydia.

沙眼衣原体（C.t）泌尿生殖道感染是女性输卵管性不孕、流产、异位妊娠的重要原因之一。申请者曾通过最新的衣原体转化技术首次发现质粒编码蛋白Pgp5能够抑制染色体基因的表达（J Bacteriol 2014），动物实验结果显示无pgp5的突变衣原体在小鼠输卵管中致病性明显减低，但pgp5如何抑制染色体基因的表达及其在衣原体致病过程中发挥重要作用的机理尚不明确。本项目拟在此基础上，通过生物信息学、分子克隆表达、凝胶迁移滞后实验、体外转录活性测定等试图发现pgp5的生物学特性及其调控染色体基因表达的分子机制；利用基因重组、反义核酸、衣原体转化、定量PCR等技术构建新的单一pgp5调控基因突变的衣原体，感染小鼠后探索这些pgp5调控基因中对致病性具有决定作用的基因及其致病机制。本项目的完成试图揭示pgp5导致衣原体输卵管致病的分子机制，为治疗C.t的感染、降低C.t所致输卵管性不孕的发生率提供新思路

沙眼衣原体泌尿生殖道感染是女性输卵管性不孕、流产、异位妊娠的重要原因之一。前期研究发现质粒编码蛋白Pgp5能够抑制染色体基因的表达，动物实验显示无pgp5基因的突变衣原体在小鼠输卵管中致病性明显减低，但pgp5如何抑制染色体基因的表达及其在衣原体致病过程中发挥作用的机理尚不明确。本项目在此基础上，用pgp5突变衣原体和对照衣原体分别感染小鼠，发现输卵管病变的发生率下降至25%-38%，较野生型衣原体导致的80%致病率有了明显降低，证实了pgp5是衣原体致病的重要毒力因子。构建了pgp5全长和不同长度基因片段的原核表达载体，表达纯化蛋白，免疫家兔产生pgp5的多克隆抗体，进行Pgp5生物学特性的鉴定，定量PCR检测可见在衣原体生长发育周期中pgp5的mRNA在早期含量高且随时间而减少，Pgp5的蛋白水平也在感染后12小时即可检测到。应用凝胶迁移滞后实验（EMSA）检测Pgp5蛋白与调控基因启动子区域的结合，发现Pgp5不能和所调控基因的启动子区域结合。课题组获得了调控基因TC0181(编码蛋白GlgA)氨基酸突变的衣原体株，动物实验发现GlgA氨基酸突变后，衣原体导致小鼠的输卵管病变率明显降低，提示GlgA可能是Pgp5调控基因中最具有重要作用的基因。本项目pgp5是衣原体致病的重要毒力因子，揭示了pgp5导致衣原体输卵管致病的分子机制，为降低女性感染患者输卵管性不孕的发病率、治疗沙眼衣原体感染提供新的思路。.课题组按计划基本完成相应内容，获得了重要的实验资料和结果，在《Front. Cell. Infect. Microbiol》、《Pathog Dis》、《Arch Microbiol》、《Acta Biochim Pol》和《中华传染病杂志》等杂志发表论文8篇，其中SCI论文4篇，培养硕士研究生4名，参加第一届全国衣原体学术研讨会和中华医学会第四届皮肤科实验研究学术会议等学术交流活动。