变形链球菌LuxS调控功能的甲基代谢机制研究

Methyl-metabolism contributes to the LuxS regulation of Streptococcus mutans

目的 研究甲基代谢通路在变形链球菌LuxS相关调控功能中的作用,为探讨变形链球菌LuxS调控机制及临床生物膜防龋策略的制定提供依据.方法 以构建的变形链球菌代谢SmUA159野生株（WT）及LuxS缺陷株（KO）、甲基代谢恢复株（KO-S）、空质粒对照株（KO-P）为研究模型,实时PCR观察生物膜及耐酸相关基因转录水平;甲基噻唑基四唑（methyl thiazolyl tetrazolium,MTT）比色法和共聚焦显微镜分别观察生物膜数量和结构;以不同pH环境下变形链球菌生物膜量的变化为指标,观察不同生物膜状态下各菌株的耐酸能力.结果 实时PCR结果发现,4种被检测基因smu.44、smu.46、smu.238及gtfD在野生株WT的表达水平分别为1.289±0.051、1.694±0.140、1.565±0.107及1.667±0.196,在缺陷株KO中的表达水平分别为1.001±0.045、1.007±0.151、1.000±0.021及1.012±0.196,4种基因在KO中的表达均显著下调（P＜0.05）;4种基因在KO-S中的表达水平分别为4.662±0.091、5.019±0.258、3.462± 0.029及3.071 ±0.136,均显著高于KO及WT中的表达水平（P＜0.05）.4种菌株形成的生物膜数量（A值）分别为WT：1.592±0.213、KO：0.939±0.029、KO-S：2.177±0.226、KO-P：1.020±0.093,KO及KO-P的生物膜数量均显著小于WT （P＜0.05）,KO-S的生物膜数量显著高于其他3种菌株（P＜0.05）.WT生物膜结构均匀致密,KO、KO-S的生物膜可见大量较大团块及宽而长的沟裂,KO-S生物膜中大团块消失,代之以致密小颗粒,沟裂数量变少且较窄而短.耐酸实验显示,pH=4.3时KO及KO-P的生物膜减少量大于WT及KO-S.结论 与野生株相比,变形链球菌LuxS缺陷株生物膜形成及耐酸能力在基因及生理水平均存在异常;甲基代谢循环恢复后,被检测基因变化恢复,生物膜数量完全恢复,生物结构部分恢复,耐酸能力得到恢复;甲基代谢循环通路在LuxS对变形链球菌生物膜及耐酸能力的调控中发挥重要作用.

Objective To investigate the predominant contribution of methyl-metabolism pathway to the regulation of LuxS of Strecptococcus mutans.Methods The differences in biofilm formation and aciduricity of Strecptococcus mutans among the methyl-metabolism-complementation strain（KO-S）,the parental wide-type strain（WT） and the luxS null strain（KO） were observed by real-time PCR for monitoring the transcriptional level of genes related to biofilm formation（smu.238,gtfD） and aciduricity（smu.44,smu.46） of the studied strains,methyl thiazolyl tetrazolium（MTT） for quantifying the biofilm of the exhibited strains and confocal laser scanning microscopy for estimating the structure of the biofilm.Results The transcriptional level of smu.44,smu.46,smu.238,gtfD in WT were 1.289±0.051,1.694±0.140,1.565±0.107,1.667±0.196 respectively; in KO were 1.001 ±0.045,1.007±0.151,1.000±0.021,1.012±0.196 respectively,downregulated compared with WT（P〈0.05）; in KO-S were 4.662 ± 0.091,5.019 ± 0.258,3.462 ± 0.029,3.071 ±0.136 respectively,upregulated compared both with KO and with WT（P〈0.05）.The quantity of biofilms formed by the studied strains were WT（1.592± 0.213）,KO（0.939± 0.029）,KO-S（2.177 ± 0.226）,KO-P（1.020±0.093）,respectively,representing a less quantity by KO and KO-P than WT（P〈0.05） and a more quantity by KO-S than other three stains（P〈0.05）.According to the observation of biofilms texture by confocal laser scanning microscopy,the WT biofilm was condensed and even.In contrast,fissures and gaps were found scattered in biofilms of KO,KO-P while lessened in that of KO-S,in which high-density bacterial aggregates were observed.The acid assay indicated a smaller biofilm decrease by WT and KO-S than that by KO and KO-P（P〈0.05）.Conclusions The methyl-metabolism pathway contributes to LuxS regulation on biofilm formation and auiduricity of Strecptococcus mutans.