LiaSR双组分信号转导系统调控变异链球菌耐酸和生物膜形成能力的机制研究  

Role and related mechanisms of LiaSR two-component system in acid tolerance and biofilm formation of Streptococcus mutans

作　　者：黄珊 杜景云 李艺君 吴敏婧 陈帅[1] 江山[1] 黄晓晶[1] Huang Shan;Du Jingyun;Li Yijun;Wu Minjing;Chen Shuai;Jiang Shan;Huang Xiaojing(Department of Endodontics,School and Hospital of Stomatology,Fujian Medical University&Fujian Key Laboratory of Oral Diseases&Fujian Provincial Engineering Research Center of Oral Biomaterial&Stomatological Key Laboratory of Fujian College and University&Institute of Stomatology,Fujian Medical University&Research Center of Oral Tissue Engineering,Fujian Medical University,Fuzhou 350002,China)

机构地区：[1]福建医科大学口腔医学院·附属口腔医院牙体牙髓科、福建省口腔疾病研究重点实验室、福建省口腔生物材料工程技术研究中心、福建省高校口腔医学重点实验室、福建医科大学口腔医学研究院、福建医科大学口腔组织工程研究中心,福州350002

出　　处：《中华口腔医学杂志》2024年第1期54-63,共10页Chinese Journal of Stomatology

基　　金：国家自然科学基金(81600861);闽江学者科研启动基金(2018-KQMJ-02)。

摘　　要：目的探究LiaSR双组分信号转导系统对变异链球菌(Sm)593号(Sm 593)临床株耐酸和生物膜形成的影响及相关机制。方法以Sm 593及其liaS和liaR基因敲除株为研究菌株,采用全自动生长曲线测定仪检测并绘制pH=5.5环境中各菌株的生长曲线;用菌落计数法检测细菌的适应性耐酸能力;使用Laurdan探针、氢-钾腺苷三磷酸(ATP)酶试剂盒、质子通透性检测实验和实时荧光定量PCR(RT-qPCR)探究LiaSR双组分信号转导系统介导的耐酸机制。体外构建各菌株的生物膜,通过结晶紫染色法、菌落计数法、SYTOX探针检测法和蒽酮-硫酸法探究各菌株的生物膜总量、活菌数、胞外DNA(eDNA)和胞外多糖含量;采用RT-qPCR法检测胞外多糖合成相关基因的表达情况。结果pH=5.5环境中,liaS和liaR基因敲除株的生长受到显著抑制(P<0.05)。适应性耐酸曲线显示敲除株与野生株相比,固有耐酸和适应性耐酸能力均显著下降(P<0.05)。对三菌株无预酸化处理20和40 min以及预酸化处理20和40 min后,liaS基因敲除株生存率[(8.98±2.00)%、(0.18±0.07)%、(14.88±8.64)%、(0.82±0.91)%]和liaR基因敲除株生存率[(0.38±0.19)%、(0.34±0.18)%、(7.89±2.02)%、(1.52±0.37)%]均分别显著低于野生株[(32.49±9.75)%、(1.27±0.32)%、(62.76±29.06)%、(8.02±1.25)%](均P<0.05)。此外,liaS和liaR敲除株膜流动性(0.18±0.04和0.18±0.05)均显著低于野生株(0.08±0.05)(均P<0.01);不饱和脂肪酸合成基因fabM表达水平(0.52±0.11和0.57±0.05)与野生株(1.04±0.30)相比均显著降低约1/2(均P<0.001);H+-ATP酶活性(917.06±59.53和469.53±47.65)与野生株(127.00±50.71)相比显著升高7.22和3.70倍(均P<0.001),且编码H+-ATP酶基因atpD的表达水平(3.39±0.21和1.94±0.17)也较野生株(1.00±0.15)显著升高3.39和1.94倍(均P<0.01)。liaR基因敲除株的终末pH值(4.76±0.01)显著低于野生株(4.90±0.00),liaS基因敲除株的终末pH值(5.19±0.01)显著高于野生株(均P<0.001)。liaSObjective To investigate the role and related mechanisms of the LiaSR two-component system in acid tolerance and biofilm formation abilities of Streptococcus mutans(Sm)593.Methods The growth curves of various Sm strains in pH=5.5 brian heart infusion(BHI)medium were analyzed.And colony forming unit(CFU)was also performed to evaluate the acid tolerance of Sm.Laurdan probe,H+-K+adenosine triphosphate(ATP)ase activity analysis kit,proton permeability assay and real-time fluorescence quantitative PCR(RT-qPCR)were conducted to detect the acid tolerant mechanisms of LiaSR two-component system in Sm.Crystal violet staining,CFU,SYTOX probe and anthrone-sulfuric method were used to analyze the properties and structures of the Sm biofilms.RT-qPCR was conducted to detect the expression levels of underlying regulated genes.Results The growth of mutants in acidic BHI were inhibited(P<0.05).The acid tolerance of mutants significantly decreased compared to the wild-type strain(P<0.05).In mutants,the activity of H+-ATPase(917.06±59.53 and 469.53±47.65)were elevated by 7.22-folds and 3.70-folds compared to the wild-type strain(127.00±50.71)(P<0.001,P<0.001)and the encoded gene atpD(3.39±0.21 and 1.94±0.17)were also elevated by 3.39-folds and 1.94-folds compared to the wild-type strain(1.00±0.15)(P<0.001,P=0.001).The Laurdan generalized polarization of mutants(0.18±0.04 and 0.18±0.05)increased significantly compared to the wild-type strain(0.08±0.05)(P=0.006,P=0.003)and the expression levels of fabM gene were decreased in mutants(0.52±0.11 and 0.57±0.05)by 1/2(P=0.014,P=0.022).In liaR deletion mutant,the reduced terminal pH(4.76±0.01)can also be observed(P<0.001).The total amount of the biofilms of three Sm didn't show significant differences(P>0.05).But the number of viable bacteria of mutants′biofilms were decreased[Sm 593:(12.00±2.80)×107 CFU/ml;SmΔliaS:(2.95±1.13)×107 CFU/ml;SmΔliaR:(7.25±1.60)×107 CFU/ml](P=0.001,P=0.024).The extracellular DNA were increased by 18.00-folds and 6.50-folds in mutants′bio

关 键 词：龋齿 链球菌 变异 生物膜 LiaSR双组分信号转导系统 耐酸 

分 类 号：R78[医药卫生—口腔医学]