【作者】 聂杰； Daniel R·Utter； 李永亮； 王晓燕； 何雪松； Jeffrey McLean； Batbileg Bor；

【Author】 Jie Nie;Daniel R.Utter;Yongliang Li;Xiaoyan Wang;Xuesong He;Jeffrey McLean;Batbileg Bor;Department of Cariology and Endodontology,Peking University School of Stomatology;Department of Organismic and Evolutionary Biology,Harvard University;The Forsyth Institute;Department of Periodontics,University of Washington;

【机构】 北京大学口腔医学院牙体牙髓科； Department of Organismic and Evolutionary Biology,Harvard University； The Forsyth Institute； Department of Periodontics,University of Washington；

【摘要】 目的:解糖微小寄生菌（Saccharibacteria）专性寄生于宿主细菌表面,形成独特的"细菌-细菌"寄生关系,并可能在牙周炎等口腔疾病中发挥重要作用。作为近年才得以分离培养的新微生物,较少的可培养菌株数量限制了对其寄生行为的深入理解。本研究通过平行诱饵技术分离解糖微小寄生菌,从株水平比较细菌间差异对其寄生行为的影响,并通过测序技术在基因水平上初步探讨菌株间差异对寄生行为的影响。材料和方法:收集志愿者的非刺激性唾液样本（n=16）置于放线菌选择性培养基中厌氧培养,将分离鉴定得到的不同放线菌保存于96孔板中建立宿主库。收集牙周炎患者的非刺激性唾液（n=16）,离心后使用0.45μm滤网过滤得到含解糖微小寄生菌的菌液。将过滤后的菌液分别加入放线菌宿主库孔板中,微需氧条件下培养得到稳定的解糖微小寄生菌-宿主共培养物,达到分离解糖微小寄生菌的目的。观察不同解糖微小寄生菌的生物学特性及其与不同宿主放线菌（n=15）的结合能力,并选取同一菌种不同菌株的解糖微小寄生菌与放线菌宿主进行"生长-抑制"试验,分析菌株间差异对其寄生行为的影响。之后进行全基因组测序并分析不同菌株的基因特点。结果:通过分离鉴定获得76种不同的放线菌种纳入宿主库。采用平行诱饵技术新发现14株解糖微小寄生菌,其中10株属于HMT-352菌种。HMT-352菌种的10个菌株与宿主的结合能力各不相同,"生长-抑制"试验表明各菌株与放线菌的寄生行为也各不相同。测序分析结果提示编码未知蛋白的GC₀0000518基因可能在解糖微小寄生菌的寄生行为中发挥关键作用。结论:通过建立放线菌宿主库,使用平行诱饵技术可以高效的分离口腔解糖微小寄生菌。不同菌株表现出不同的宿主结合能力和寄生生长特性,在菌株水平比较解糖微小寄生菌的表型和基因差异更有助于我们进一步探寻寄生行为的内在机制。更多还原

【Abstract】 Objective:Saccharibacteria,which are obligate episymbionts growing on the surface of host bacteria forming a unique "bacteria-bacteria" parasitic relationship,may play an important role in oral disease,such as periodontitis.As a new microorganism that has only been isolated and cultured in recent years,the limited number of culturable strains also impede the in-depth understanding of its biological characteristics.The current study isolated Saccharibacteria using the parallel baiting method and compared the differences in its parasitic behavior at the strain level,and further explored its underlying mechanism at the gene level by sequencing.Materials and methods:Non-stimulating saliva samples（n=16） from volunteers were collected and placed in Actinomyces-selective medium,cultivating under anaerobic condition.The isolated Actinomyces strains were identified and then placed in 96-well plates to establish the host bank.Non stimulating saliva（n=16） from patients with periodontitis was collected,centrifuged and filtered with 0.45μm filter to isolate Saccharibacteria.The filtered Saccharibacteria were added to the host bank plate,then cultured under microaerobic conditions,and stable Saccharibacteria-host basibionts were obtained to isolated Saccharibacteria monoculture.The biological characteristics,including host range and growth dynamics during the establishment of their episymbiotic relationship with host bacteria,of the isolated Saccharibacteria strains with different hosts（n=15） were observed.Ten Saccharibacteria strains from the same species were selected for "growth-crash" experiments with host Actinomyces strains to compare the differences on their parasitic behavior at strain level.Then the genetic characteristics of different strains are analyzed by whole genome sequencing.Results:76 different Actinomyces were isolated and identified to establish the host bank.A total of 14 Saccharibacteria strains were isolated,of which 10 belong to the Saccharibacteria species HMT-352.The HMT-352 strains exhibited a diverse host range and varied growth dynamics with different hosts.Growth-crash assays showed that the parasitic behavior of the 10 strains with the Actinomyces hosts also differed.Furthermore,genomic analyses showed that the GC₀0000518 gene,which encodes an unknown protein,may play a key role in the parasitic ability of Saccharibacteria.Conclusion:By establishing a host bank of Actinomyces,the parallel bait method could isolate oral Saccharibacteria more efficiently.Different strains from HMT-352exhibited different host range and varied growth dynamics.Comparing the phenotypic and genetic differences of Saccharibacteria at the strain level could be more helpful for us to further explore the underlying mechanism of its parasitic behavior.更多还原