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

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)。liaS和liaR基因敲除株与野生株相比,Sm 593生物膜总量未发生明显变化,生物膜活菌数均显著少于野生株(均P<0.05),且eDNA含量均显著高于野生株(均P<0.01)。liaS基因敲除株生物膜水溶性多糖含量与野生株相比显著增加1.88倍(P=0.003)。liaS和liaR基因敲除株胞外多糖合成相关基因gtfD表达水平较野生株显著升高47.43和16.90倍(均P<0.05),liaS基因敲除株中gtfC基因比野生株显著高表达1.65倍(P=0.014)。结论LiaSR双组分信号转导系统通过调控膜不饱和脂肪酸合成和胞膜流动性,参与Sm 593的耐酸过程;LiaR反应调节蛋白还可参与调节膜质子通透性,增强Sm 593的耐酸能力;此双组分信号转导系统可负向调控生物膜胞外基质的产生。

Objective 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′biofilms(128.73±15.65 and 46.38±5.52)compared to the wild-type strain(7.16±3.62)(P<0.001,P=0.003).Water-soluble exopolysaccharides could be found up-regulated in liaS deletion mutant[(138.73±10.12)μg/ml](P=0.003)along with the expression level of gtfC gene(1.65±0.39)(P=0.014).The expression level of gtfD were elevated by 47.43-folds and 16.90-folds in mutants(P<0.001,P=0.010).Conclusions The LiaSR two-component system can promote the expression of fabM gene and increase the fluidity of Sm which contributes to acid tolerance.The LiaR can also decrease the proton permeability and restrict the entrance of H+.The LiaSR two-component system can negatively regulate the production of the extracellular matrix in Sm biofilm.