解脲脲原体克隆复合体2亚群新毒力因子鉴定及致病作用研究

Ureaplasma urealyticum can cause a variety of genitourinary tract diseases and seriously affect human reproductive health, and its pathogenicity was closely related to serotypes and virulence factors. However, the molecular mechanisms of inducing host cell injury, inflammation and infection of this species have not yet been clearly elucidated. According to our previously developed eMLST scheme, U.urealyticum clonal complex sub-group 2 has been proved to be associated with high pathogenicity. In this study, we will apply the third generation high-throughput sequencing technology PacBio RS II to obtain the genome sequence of this sub-group to reveal the genetic background of virulence factors (i.e. adhesion factor). Moreover, we will clarify the correlation between cell invasion and its genetic backgrounds in cellular level using mutant library technology by virulence factor mutant screening system. We will also discuss the further role of virulence factors in pathogenesis by animal model of genital tract infection. The aim of this study was to provide new clues to investigate the pathogenic mechanisms and also establish a novel clinical diagnostic technology to differentiate colonization from infection based on virulence factor identification technology.

解脲脲原体（U. urealyticum）可引起泌尿生殖道多种感染性疾病，严重影响人类生殖健康，其致病性与血清型和毒力因子密切相关，但其导致宿主细胞损伤、炎症及感染等确切分子机制尚未阐明。本课题组应用最新建立的eMLST分型技术，已证明解脲脲原体克隆复合体2亚群与临床致病高关联。本研究拟采用PacBio RS II第三代高通量测序技术，揭示该亚群基因组中以粘附因子为代表的重要毒力因子的遗传背景。在此基础上，通过构建基于突变体库的毒力因子突变株筛选体系，比较高毒力株与毒力因子突变株对细胞粘附能力与诱导细胞凋亡能力的差异，从细胞水平阐明其对细胞侵袭能力与基因组背景之间的关联。并通过构建小鼠生殖道感染模型，从动物水平验证毒力因子在其致病机制中的作用。为解脲脲原体致病机制研究提供新线索，也为建立基于毒力因子检测的致病性解脲脲原体临床诊断新技术奠定基础，极可能为区分解脲脲原体定植与感染提供科学依据。

解脲脲原体可引起多种泌尿生殖道感染性疾病，严重影响人类生殖健康。本研究对解脲脲原体克隆复合体2亚群临床菌株基因组进行Oxford Nanopore测序，并分析了不同血清型解脲脲原体菌株携带的毒力因子多带抗原MBA的基因周围结构。同时，本研究还阐明解脲脲原体不同标准菌株与临床菌株脂结合膜蛋白（LAMPs）的致病机制。研究表明，MBA的变异具有灵活性和复杂性，串联重复序列数目的变化并不只是发生在MBA的C端，还可见于其他下游包含串联重复序列的基因。倒置后的基因片段不仅可与其近段基因发生融合，还可与插入的其他基因发生融合。解脲脲原体标准菌株UUR8及临床菌株LAMPs均能明显抑制U937细胞增殖，而标准菌株UPA3 LAMPs只能轻微抑制细胞增殖。流式细胞术结果表明所有解脲脲原体LAMPs均可使U937细胞阻滞在G1期。本研究还发现，使用标准菌株UUR8或临床菌株LAMPs处理细胞后，细胞周期阻滞与p53、p21的mRNA和蛋白水平升高有关，同时伴随CDK2，CDK4，CDK6和cyclin E1水平的降低；而使用标准菌株UPA3 LAMPs处理后，只有cyclin E1发生了下调。标准菌株UUR8和临床菌株的LAMPs可通过p53非依赖的方式增加p21表达，从而诱导细胞发生G1期阻滞来抑制U937细胞的增殖，而标准菌株UPA3 LAMPs可轻微诱导细胞周期发生阻滞。本研究可为探索解脲脲原体感染的致病机制提供新的线索和研究手段，也为建立基于毒力因子检测的致病性解脲脲原体临床诊断新技术提供科学依据。