四型分泌系统：介导肺炎链球菌血清型替换的新机制研究

Streptococcus pneumoniae is a major etiological agent for community acquired pneumonia. Although prophylactic approaches are applied, serotype replacement could bring a remarkable challenge to the effectiveness of certain serotype of vaccine. .The recombination of capsule polysaccharide synthesis gene cluster (cps) could alter the serotype, and contribute to a rise in the prevalence rate of non-vaccine serotypes. Amount of studies have shown that S. pneumoniae could uptake small DNA fragment through transformation and subsequently integrate it into genome by recombination. However, the mechanism of long DNA segments recombination remains largely unknown. .Our previous researches have showed: i) type IV secretion system (T4SS) is widely distributed in S. pneumoniae and could mediate the horizontal gene transfer (HGT) of long DNA segments. ii) T4SS has been identified in the newly emerging serotypes, and iii) its binding site, oriT, has been identified within or in the flanking region of the cps locus of lots of prevalent serotypes. Therefore, we proposed that the HGT mediated by T4SS may be an important mechanism for long segment transfer and recombination of cps. . In this project, firstly, we aimed to study the association of T4SS and related elements with long segments cps recombination via the analysis of S. pneumoniae genomes from public database. Secondly, we try to examine whether T4SS could mediate the horizontal transfer of long segments cps between model strains using the conjugation experiments and determine its transfer rates. Finally, we will evaluate the risk of serotype replacement in clinical strains. Using this framework, we wanted to find out the effect of vaccination in the pandemic of non-vaccine serotypes and predict the trends of serotype replacement in clinic. The molecular elements identified in our studies could be a potential target for the development of new vaccines.

肺炎链球菌是社区获得性肺炎的首位细菌性病原，接种疫苗能有效预防该菌感染，但血清型替换常导致现有疫苗失效。血清型基因决定簇（cps）重组是引起血清型替换的主要原因之一，其中cps短片段重组可由自然转化介导，但长片段重组机制尚不明确。我们前期发现：该菌中广泛存在介导长片段基因转移的四型分泌系统（T4SS）；新出现及分离增加的血清型菌株携带T4SS编码基因，其cps及两侧序列存在T4SS相关转移起始位点。由此推测：T4SS介导cps基因水平转移是引起大片段cps重组的重要机制之一。本研究拟通过分析已公布的基因组数据，明确长片段cps重组与T4SS及相关元件的关联性；采用模式菌接合转移实验，证实T4SS介导长片段cps转移的能力；分析并验证临床流行株中T4SS与长片段cps重组及血清型替换的关系。本研究力求阐明肺炎链球菌血清型替换的分子机制，为预测流行血清型替换趋势，及新疫苗开发提供科学依据。

肺炎链球菌（SP）是一种重要的人类致病菌，可引发多种侵袭性肺炎球菌病（IPD）。由于肺炎链球菌耐药日益严重，WHO将疫苗接种作为预防和应对肺炎链球菌感染性疾病最有效、最经济的措施。但是，SP的血清型决定基因cps存在基因水平传播可能使SP发生疫苗逃逸，导致IPD发生率显著回升。SP基因组上的四型分泌系统（T4SS）相关基因可能和cps基因水平转移相关。为了明确上述问题，我们首先鉴定了数据库中3969株SP的多位点序列型别、血清型和T4SS。明确了全球范围内疫苗覆盖血清型的SP占比为48.5%。并且ST型和血清型不存在特定的对应关系，提示在SP中血清型转化普遍发生。更为重要的是T4SS阳性菌株占比为10.8%，而T4SS阳性菌株绝大多数在在疫苗覆盖的血清型菌株中发现（82.9%），提示肺疫苗覆盖血清型的在全球范围内传播可能与T4SS介导的基因水平转移相关。另外，我们在临床上收集了166株SP菌株并对上述菌株进行了全基因组测序。结果表明，与全球数据相同，我国临床流行菌株的序列型别和血清型不存在特定的对应关系，且cps基因存在水平转移的特征。上述结果提示，疫苗接种依旧是全球范围内应对SP感染的主要措施，但是血清型转化而导致疫苗逃逸是目前全球SP防控面临的重要挑战。我们针对革兰氏阳性细菌的基因组进行了一系列的后续研究：1. 发现了一种染色体编码(CE)-T4SS，能够以不精确的方式介导大规模的全基因组基因转移。2. 发现Fsr密度感应系统与粪肠球菌血液感染显著相关，这也为探索粪肠球菌侵袭性的研究提供了重要的信息。3. 发现外膜蛋白：OprD/Occ与鲍曼不动杆菌ST2/KL22克隆的毒力密切相关，这为后期多耐药高毒力细菌的检测提供了新的靶点。