伴放线聚集杆菌通过微调过氧化氢促进副血链球菌生物膜形成及毒力的机制研究

Aggregatibacter actinomycetemcomitans (Aa) is a well-known pathogen of periodontitis, especially localized aggressive periodontitis(LAP）. It has been reported that Aa and Streptococcus parasanguinis (Sp) are co-localized and are associated with the development of LAP. Recent studies have found that Aa and Sp can form thick biofilms together which are regulated by fine-tuned H2O2. The poxL mutant, which lost the ability to produce H2O2, is deficient in dual-species biofilm formation. Comparing the level of gene transcripts between poxL mutant and wild type Sp when they both co-cultured with Aa, two surface protein genes were found to be down-regulated in poxL mutant, one encodes the amylase binding protein AbpA and the other encodes a C5a peptidase homologous protein SPAF_0194. According to the literature, both the proteins might play important roles in the colonization and invasion of Sp in the host. The aim of this study was to elucidate the functions of the two proteins in dual-species biofilm formation, host colonization and invasion through constructing mutants of the two genes, in vitro biofilm model, expression and purification of the proteins, fluorescence quantitative PCR and experimental animal models et al.To explore whether Aa has the the keystone pathogen-like effect of up-regulating the virulence of the commensal bacteria and work with them synergistically to cause periodontitis. The results of this study will help to elucidate the mechanisms of the co-infection of Aa and Sp in the development of periodontitis. It also can provide new ideas and therapeutic targets for the prevention and treatment of periodontitis.

伴放线聚集杆菌(Aa)是牙周炎公认的致病菌，近年研究显示Aa与副血链球菌(Sp)共同检出并与局限型侵袭性牙周炎发生发展相关。申请人研究发现，Aa和Sp可形成增厚的双菌种生物膜，并受微调的H2O2调控。敲除Sp产H2O2基因poxL则无双菌种生物膜形成。比较poxL突变株与野生株在与Aa共培养时基因转录组水平，发现两个表面蛋白基因明显下调，它们分别编码淀粉酶结合蛋白AbpA和C5a肽酶同源蛋白SPAF_0194。结合文献报道，推测两者在Sp定植和入侵宿主中有重要作用。本课题拟通过相应基因突变株的构建、体外生物膜培养、蛋白质纯化和功能检测、实验动物模型等，阐明两者在Aa和Sp生物膜形成、定植和入侵宿主中的作用和机制，探究Aa是否具有上调共生菌Sp毒力并与之协同作用从而导致牙周炎的关键致病菌样作用，成果有助于阐明Aa和Sp在牙周炎发生发展中的致病机制，为防治牙周炎提供新思路和治疗靶点。

伴放线聚集杆菌(Aggregatibacter actinomycetemcomitans，A.a)是牙周炎公认的致病菌，近年研究显示A.a与副血链球菌(Streptococcus parasanguinis，S.p)共同检出并与局限型侵袭性牙周炎发生发展相关。本研究期望通过对A.a和S.p的双菌种生物膜的形成和调节机制进行深入研究，阐明两者可能的相互作用机制，为防治牙周炎提供新思路和治疗靶点。.在研究中，我们通过体外细菌生物膜培养、基因敲除、蛋白质表达纯化，免疫印迹等技术研究了淀粉酶结合蛋白AbpA在混合生物膜中的作用，研究发现，在A.a和S.p共培养的生物膜中，AbpA的表达上调，敲除abpA基因使混合生物膜形成明显减少，但对于S.p单菌种生物膜却影响不大，提示AbpA并不调节S.p的生物膜形成，而是介导了A.a和S.p两者之间的相互作用，在混合生物膜形成中发挥重要作用。此外，通过构建重组abpA表达质粒，我们成功表达并纯化了rAbpA，发现rAbpA可以促进S.p在唾液包被孔板表面的生物膜形成，对ΔabpA无明显作用，推测游离状态的AbpA主要通过与锚定状态AbpA相互作用来调节生物膜形成。本研究首次发现唾液淀粉酶结合蛋白AbpA除介导链球菌黏附到唾液淀粉酶和获得性膜从而促进定植之外的另一种重要功能——即参与到链球菌与A.a之间的相互作用和混合生物膜的形成。.此外，通过采集牙周炎患者临床样本，我们分析了 A.a、S.p在牙周病患者唾液、龈沟液中检出与相关炎症消退因子、与牙周炎治疗预后之间的关系，发现A.a检出与否是预测牙周基础治疗后患者预后的重要指标之一，A.a阳性与进一步附着丧失相关。我们还发现A.a检出阳性位点的龈沟液中促炎消退因子LXA4显著降低，进一步揭示了A.a可能在牙周炎的致病过程中发挥了关键致病菌作用及潜在机制，为最终阐明牙周炎的发病机制及牙周炎的防治提供了新的理论依据。