c-di-GMP信号通路在变形链球菌致龋过程中作用的初步研究
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摘要
龋病是一种最为常见的、严重危害人类口腔健康的口腔感染性疾病之一。变形链球菌( Streptococcus mutans, S. mutans)是人类龋病的主要致病菌,其致龋毒力因子主要表现在对牙面的粘附和利用碳水化合物产生多糖以及产酸、耐酸。含有GGDEF结构域的蛋白是近年来发现的一种新型蛋白,其主要功能是参与单磷酸鸟苷环二聚体(bis-(3’-5’)-cyclic dimeric guanosine monophosphate, c-di-GMP)的合成。c-di-GMP已被证实是原核细胞信号转导中一类新的第二信使,它的作用涉及生物膜的形成、毒力因子的表达、细胞间的通讯、激发免疫系统的应答等多个方面,是细菌生存和代谢的关键性调节因子之一,但其在变形链球菌中的研究尚属空白。本研究主要是针对外源性c-di-GMP对变形链球菌致龋过程作用及其机制的初步研究,为防龋探索一条新的途径。
第一部分:外源性c-di-GMP对变形链球菌致龋过程影响的初步研究
对生物膜影响的研究:在96孔细胞培养板上形成变形链球菌生物膜,不同浓度的外源性c-di-GMP作用于生物膜48 h,将生物膜进行染色,乙醇/丙酮混合液显色,使用酶标仪测定光密度值,观测生物膜形成量的改变,等质量的生理盐水作为阴性对照。同时在离体牙的新鲜釉质片上形成变形链球菌生物膜,200μM的c-di-GMP与等量的生理盐水分别作用于生物膜48 h,扫描电镜观察结构的改变。结果c-di-GMP处理组与生理盐水组相比,生物膜形成能力明显下降,细菌排列无明显规律,细胞外基质减少,提示200μM的c-di-GMP可有效抑制变形链球菌生物膜形成能力。
对产酸、耐酸影响的研究:外源性c-di-GMP与生理盐水分别加入至含变形链球菌UA159菌液的不同pH的BHI培养基中,精密数显酸度计测终末pH值,以pH的差值(△pH)表示产酸结果,比较两组△pH,分析c-di-GMP对变形链球菌UA159产酸能力的影响;在pH=5的培养液中孵育2 h,以孵育前后细菌存活数量的比值评价细菌耐酸能力。结果: c-di-GMP处理组与生理盐水组相比,产酸、耐酸能力明显下降,提示c-di-GMP可以抑制变形链球菌的产酸、耐酸能力。
对胞外多糖形成能力影响的研究:将变形链球菌UA159厌氧培养,外源性c-di-GMP与生理盐水分别加入至变形链球菌培养液中培养24 h,用等同培养基体积的蒸馏水洗2次,将3次上清液合并作为水溶性胞外多糖,水洗后的菌胞中加入等体积0.5 mol/L NaOH洗3次,合并上清液,作为水不溶性胞外多糖。上述两部分上清液各取1.5ml,加入3倍体积无水乙醇,4℃过夜,离心后弃水相,将沉淀分别在等体积的蒸馏水及0.1mol/L NaOH中溶解,用蒽酮法分别测定水溶性及水不溶性葡聚糖的含量,在OD620比色,以葡萄糖标准液绘制标准曲线,根据标准曲线计算UA159合成的水溶性及水不溶性葡聚糖的含量,对两组进行比较。结果发现干预组生成水不溶性糖的量明显减少。提示外源性c-di-GMP对变形链球菌合成胞外多糖的能力有一定的影响。
对唾液包被的HA表面黏附的影响:选取荧光标记物BCECF/AM标记变形链球菌标准菌株UA159,用唾液包被的羟基磷灰石(SHA)模拟口腔中的牙面情况,外源性c-di-GMP与生理盐水分别与荧光标记的UA159菌液,SHA共同孵育30分钟,于荧光酶标仪上以激发波长485 nm、发射波长535 nm测定各孔,扣除阴性对照的结果作为测定的荧光值。按照公式计算黏附率,比较两组的区别。结果: c-di-GMP处理组与生理盐水组相比,粘附能力明显下降。综述上述各结果,提示外源性c-di-GMP对变形链球菌的致龋特性均有一定程度的抑制能力,因此可以单独也可以与其它的抗龋制剂联合应用控制龋病,有望成为一种新型防龋方法。
第二部分:外源性c-di-GMP防龋的动物实验研究
选用20 d龄的SD大鼠感染致龋菌,并饲以致龋饲料2000 #诱龋成功后,分别用适量外源性c-di-GMP、氟化钠水溶液、0.9%的氯化钠给大鼠施药,用唾液取样细菌培养记录大鼠口腔内变形链球菌数量、龋齿记分观察外源性c-di-GMP在动物口内对致龋菌的生长繁殖以及对龋病发生、发展的影响,并以氟化钠为阳性对照。结果:实验中大鼠食物、水消耗,各组间未见明显差异,保证了各组大鼠所受致龋攻击性相同。药物处理期间,大鼠口腔黏膜未见红斑、肿胀等异常表现。提示外源性c-di-GMP没有明显毒性,同时,发现外源性c-di-GMP处理组大鼠咬合面龋及光滑面龋齿计分显著低于阴性对照组,但是大鼠口腔内变形链球菌定植数量未见明显差异,提示了c-di-GMP作为未来新型防龋途径的可行性。
第三部分:变形链球菌gcp基因失活菌株的构建、鉴定及致龋特性研究
以前期研究中pMD19T-gcp为模板, PCR扩增873bp的gcp基因内部序列,连接T载体后将该内部片段定向插入自杀载体pVA8912,酶切鉴定,发现PCR产物及插入片段大小与预期值相符,表明成功构建了打靶载体pVA8912/gcp;将鉴定正确的质粒转化变形链球菌UA159株,挑取阳性克隆,提取基因组DNA,用PCR结合酶切鉴定发现gcp基因失活株基因组中gcp基因内部成功插入了打靶载体片段。结果表明成功构建了变形链球菌gcp基因失活株,为该基因功能的研究奠定了基础。
厌氧培养变形链球菌gcp基因失活株,在不同时间点测定OD600值,绘制生长曲线,发现gcp基因失活株和野生菌的生长曲线无显著性差异,表明gcp的基因失活对变形链球菌的生长无显著影响。
将gcp失活菌和野生菌菌悬液转入无菌的96孔酶标板中厌氧培养48h,黏附菌使用结晶紫溶液染色,乙醇/丙酮混合液显色,测量OD575值,以定量反映生物膜形成量。结果发现gcp失活菌生物膜形成量显著低于野生菌,表明gcp可能与变形链球菌生物膜形成能力有关。
荧光标记gcp失活菌和野生菌,与唾液包被的羟基磷灰石粉末共同孵育后,测定羟基磷灰石沉淀的荧光值,比较粘附率的差异,发现gcp失活菌的黏附率(45.33%±6.35%)低于野生菌(64.05%±6.73%),差异有显著性(P <0.05);说明gcp可能对变形链球菌与唾液包被的羟基磷灰石的黏附有关,对这种黏附有一定的抑制作用。
总之,本研究证明变形链球菌内部存在c-di-GMP信号通路,该通路介导变形链球菌的生物膜形成及在离体牙釉质表面的黏附,外源性c-di-GMP具有降低变形链球菌生物膜的形成,产酸、耐酸、体外黏附等特性。上述结果为揭示变形链球菌黏附致龋的分子机理以及建立新的防龋途径提供了实验依据。
Dental caries is a common oral infectious disease that affect people’s health. One of the dorminant pathogenic microorganisms cause human dental caries is Streptococcus mutans ( S. mutans), which attaches on the surface of the teeth, produce polysaccharide and acid, and survive acid environment. With the contribution of GGDEF, S. mutans generates c-di-GMP, which has been reported mediating biofilm formation and virulent factors expression in Staphlococcus Aureus, Vibrio Cholerae, Pseudomonus Aurugenosa, and it also stimulated innate immunity which limited the infection of the bacteriae mentioned above. It is one of key regulatory factors inbacterium exist and metabolism,however, the research in S. mutans is still unknown. In this study, we focus on the effect of exogenous c-di-GMP on S. mutans induced dental caries and its mechanism,which might theoretically indicate a new way in caries prevention.
Section I:exogenous c-di-GMP’s effect on dental caries caused by S Mutans
Biofilm Formation:48 hours after exposed to exogenous c-di-GMP with different dosage, biofilm formed in 96 well plates by S Mutans were stained and dissolved by ethanol/acetone, the OD measured by spectrophotometry presents the amount of biofilm in each well. 200μM c-di-GMP and the saline with the same volume incubated with S Mutans on the fresh tooth slices, the biofilm on the slices were investigated with scanning electronic microscope. Result shown that c-di-GMP decreased the biofilm formation and the bacteriae disorganized on the tooth slices with less extracellular matrix, which implies that 200uM c-di-GMP inhibit S Mutans’s capability to form biofilm.
Acid generation and acid resistance: Exogenous c-di-GMP and saline added into variable pH bacteria medium with UA159 S Mutans strain, the change of pH values (△pH)at the end of culture were compared;after cultured in pH=5 medium for 2 hours, the number of live bacteriea presents the ability of acid resistance. Results: after treat with c-di-GMP, S. Mutans’s capability of acid generation and acid resistance decreased.
Extracellular polysaccharide formation: S. Mutans strain UA159 cultured in anaerobic environment 24 hour after exogenous c-di-GMP and saline added into culture system, S. Mutans were broken by distilled water (x2 times) and the supernatant (supernatant I) of 3 centrifugations were collected and combined as water-soluble extracellular polysaccharide. The pellets were added with same volume 0.5 mol/L NaOH, centrifuged, collected supernatant, and repeated 3 times. Combine the supernatant (supernatant II) as water-insoluble polysaccharide. 1.5ml from supernatant I and II (respectively) plus 3 volumes of ethanol, 4℃overnight, collect and centrifuge and discard the upper water phase. Pellets dissolved in distilled water and 0.1mol/L NaOH with same volume. Anthrone method measured the content of glucan in the water-soluble and–insoluble supernatant,OD620,the standard curve was made with glucose and calculate the water-soluble and–insoluble glucan generation. With the result shown that exogenous c-di-GMP treatment significantly decreased the formation of water-insoluble glucan.
Attached to the surface of HA coated with saliva: fluorescence- BCECF/AM labeled UA159 strain put on SHA coated with saliva mimic oral teeth surface, exogenous c-di-GMP and saline co-cultured with UA159 for 30 minutes, fluorescence measured presents the bacteria attached on SHA (485nm→535nm). Results: c-di-GMP treatment decreased the bacteria attachment.
Token together, exogenous c-di-GMP inhibits S Mutans’capacity to form dental caries. So it is possible that exogenous c-di-GMP could be used as anti-caries agent, either alone or with other methods.
Section II: Exogenous c-di-GMP Prevent Caries Formation in Animals Dental Caries were made on the teeth of 20 day age SD rat infected with S Mutans, and fed with caries inducing food 2000 #. Exogenous c-di-GMP, NaF solution and 0.9% NaCl applied to the teeth of the rats, the bacteria number and scores of dental caries were recorded. Results shown that the food and water consume are same among different treatments, which enabled same number of dental caries on the teeth. No red plague, swelling appeared during the treatment, which implies that exogenous c-di-GMP has no toxic effect in the oral cavity. Exogenous c-di-GMP treatment group downgrade the scores of dental caries on occlusal surface and smooth surface, but no difference has been found on the number of bacterial plagues, implies that c-di-GMP could be used as anti-caries agent.
Section III: S Mutans with gcp gene inactivation, strain construction, identification and its caries inducing characters
Take pMD19T-gcp as template, PCR amplify gcp sequence (873bp) , after connected to T vector, the gene frame inserted into suicide vector pVA8912. Enzymatic analysis found the product of PCR product had the same size as the inserted piece, which implies the construct of pVA8912/gcp is successful. Thereby pVA8912/gcp transformed into UA159 and positive clone selected. The selected clone was then verified by PCR and enzymatic analysis on the extracted genomic DNA of the positive clone, with the result shown that the construction of S. Mutans with gcp gene inactivation strain was successful.
S. Mutans with gcp gene inactivation strain was culture in anaerobic environment, the growth curve based on values of OD 600 presents the bacterial growth condition. Results shown that no difference between S. Mutans with gcp gene inactivation strain and wildtype bacteria was found, which implies that inactivation of gcp gene did not change the growth of S Mutans.
S. Mutans with gcp gene inactivation strain and wildtype bacteria were plated into 96 well plates and cultured in anaerobic environment for 48 hours, the bacteria attached were stained with crystal violet, ethanol/acetone dissolve the precipitated stain and the spectrophotometric values of OD575 represent the amount of biofilm formation, with the results shown that S. Mutans with gcp gene inactivation strain formed much less biofilm as compared with wildtype. This implies that gcp gene has relationship with S. Mutans’capacity to form biofilm.
Fluorescence labeled S. Mutans with gcp gene inactivation strain and wildtype bacteria were put on SHA coated with saliva,the fluorescence on SHA represents the number of bacteria attached. S. Mutans with gcp gene inactivation strain (45.33%±6.35%)attached less (P <0.05)than wildtype bacteria(64.05%±6.73%), which implies that gcp gene could inhibit the SHA surface attachment by S Mutans. Conclusion
In our study, c-di-GMP signaling pathway has been found existed in S. Mutans and mediated its capacity of the biofilm formation and surface attachment. Exogenous c-di-GMP reduced S. Mutans’ability of biofilm formation, acid generation, acid resistance and surface attachment ex vivo. In brief, our research provided molecular mechanism for explanation how S Mutans’cause dental caries and experimental evidences for developing new anti-caries method.
第一部分:外源性c-di-GMP对变形链球菌致龋过程影响的初步研究
对生物膜影响的研究:在96孔细胞培养板上形成变形链球菌生物膜,不同浓度的外源性c-di-GMP作用于生物膜48 h,将生物膜进行染色,乙醇/丙酮混合液显色,使用酶标仪测定光密度值,观测生物膜形成量的改变,等质量的生理盐水作为阴性对照。同时在离体牙的新鲜釉质片上形成变形链球菌生物膜,200μM的c-di-GMP与等量的生理盐水分别作用于生物膜48 h,扫描电镜观察结构的改变。结果c-di-GMP处理组与生理盐水组相比,生物膜形成能力明显下降,细菌排列无明显规律,细胞外基质减少,提示200μM的c-di-GMP可有效抑制变形链球菌生物膜形成能力。
对产酸、耐酸影响的研究:外源性c-di-GMP与生理盐水分别加入至含变形链球菌UA159菌液的不同pH的BHI培养基中,精密数显酸度计测终末pH值,以pH的差值(△pH)表示产酸结果,比较两组△pH,分析c-di-GMP对变形链球菌UA159产酸能力的影响;在pH=5的培养液中孵育2 h,以孵育前后细菌存活数量的比值评价细菌耐酸能力。结果: c-di-GMP处理组与生理盐水组相比,产酸、耐酸能力明显下降,提示c-di-GMP可以抑制变形链球菌的产酸、耐酸能力。
对胞外多糖形成能力影响的研究:将变形链球菌UA159厌氧培养,外源性c-di-GMP与生理盐水分别加入至变形链球菌培养液中培养24 h,用等同培养基体积的蒸馏水洗2次,将3次上清液合并作为水溶性胞外多糖,水洗后的菌胞中加入等体积0.5 mol/L NaOH洗3次,合并上清液,作为水不溶性胞外多糖。上述两部分上清液各取1.5ml,加入3倍体积无水乙醇,4℃过夜,离心后弃水相,将沉淀分别在等体积的蒸馏水及0.1mol/L NaOH中溶解,用蒽酮法分别测定水溶性及水不溶性葡聚糖的含量,在OD620比色,以葡萄糖标准液绘制标准曲线,根据标准曲线计算UA159合成的水溶性及水不溶性葡聚糖的含量,对两组进行比较。结果发现干预组生成水不溶性糖的量明显减少。提示外源性c-di-GMP对变形链球菌合成胞外多糖的能力有一定的影响。
对唾液包被的HA表面黏附的影响:选取荧光标记物BCECF/AM标记变形链球菌标准菌株UA159,用唾液包被的羟基磷灰石(SHA)模拟口腔中的牙面情况,外源性c-di-GMP与生理盐水分别与荧光标记的UA159菌液,SHA共同孵育30分钟,于荧光酶标仪上以激发波长485 nm、发射波长535 nm测定各孔,扣除阴性对照的结果作为测定的荧光值。按照公式计算黏附率,比较两组的区别。结果: c-di-GMP处理组与生理盐水组相比,粘附能力明显下降。综述上述各结果,提示外源性c-di-GMP对变形链球菌的致龋特性均有一定程度的抑制能力,因此可以单独也可以与其它的抗龋制剂联合应用控制龋病,有望成为一种新型防龋方法。
第二部分:外源性c-di-GMP防龋的动物实验研究
选用20 d龄的SD大鼠感染致龋菌,并饲以致龋饲料2000 #诱龋成功后,分别用适量外源性c-di-GMP、氟化钠水溶液、0.9%的氯化钠给大鼠施药,用唾液取样细菌培养记录大鼠口腔内变形链球菌数量、龋齿记分观察外源性c-di-GMP在动物口内对致龋菌的生长繁殖以及对龋病发生、发展的影响,并以氟化钠为阳性对照。结果:实验中大鼠食物、水消耗,各组间未见明显差异,保证了各组大鼠所受致龋攻击性相同。药物处理期间,大鼠口腔黏膜未见红斑、肿胀等异常表现。提示外源性c-di-GMP没有明显毒性,同时,发现外源性c-di-GMP处理组大鼠咬合面龋及光滑面龋齿计分显著低于阴性对照组,但是大鼠口腔内变形链球菌定植数量未见明显差异,提示了c-di-GMP作为未来新型防龋途径的可行性。
第三部分:变形链球菌gcp基因失活菌株的构建、鉴定及致龋特性研究
以前期研究中pMD19T-gcp为模板, PCR扩增873bp的gcp基因内部序列,连接T载体后将该内部片段定向插入自杀载体pVA8912,酶切鉴定,发现PCR产物及插入片段大小与预期值相符,表明成功构建了打靶载体pVA8912/gcp;将鉴定正确的质粒转化变形链球菌UA159株,挑取阳性克隆,提取基因组DNA,用PCR结合酶切鉴定发现gcp基因失活株基因组中gcp基因内部成功插入了打靶载体片段。结果表明成功构建了变形链球菌gcp基因失活株,为该基因功能的研究奠定了基础。
厌氧培养变形链球菌gcp基因失活株,在不同时间点测定OD600值,绘制生长曲线,发现gcp基因失活株和野生菌的生长曲线无显著性差异,表明gcp的基因失活对变形链球菌的生长无显著影响。
将gcp失活菌和野生菌菌悬液转入无菌的96孔酶标板中厌氧培养48h,黏附菌使用结晶紫溶液染色,乙醇/丙酮混合液显色,测量OD575值,以定量反映生物膜形成量。结果发现gcp失活菌生物膜形成量显著低于野生菌,表明gcp可能与变形链球菌生物膜形成能力有关。
荧光标记gcp失活菌和野生菌,与唾液包被的羟基磷灰石粉末共同孵育后,测定羟基磷灰石沉淀的荧光值,比较粘附率的差异,发现gcp失活菌的黏附率(45.33%±6.35%)低于野生菌(64.05%±6.73%),差异有显著性(P <0.05);说明gcp可能对变形链球菌与唾液包被的羟基磷灰石的黏附有关,对这种黏附有一定的抑制作用。
总之,本研究证明变形链球菌内部存在c-di-GMP信号通路,该通路介导变形链球菌的生物膜形成及在离体牙釉质表面的黏附,外源性c-di-GMP具有降低变形链球菌生物膜的形成,产酸、耐酸、体外黏附等特性。上述结果为揭示变形链球菌黏附致龋的分子机理以及建立新的防龋途径提供了实验依据。
Dental caries is a common oral infectious disease that affect people’s health. One of the dorminant pathogenic microorganisms cause human dental caries is Streptococcus mutans ( S. mutans), which attaches on the surface of the teeth, produce polysaccharide and acid, and survive acid environment. With the contribution of GGDEF, S. mutans generates c-di-GMP, which has been reported mediating biofilm formation and virulent factors expression in Staphlococcus Aureus, Vibrio Cholerae, Pseudomonus Aurugenosa, and it also stimulated innate immunity which limited the infection of the bacteriae mentioned above. It is one of key regulatory factors inbacterium exist and metabolism,however, the research in S. mutans is still unknown. In this study, we focus on the effect of exogenous c-di-GMP on S. mutans induced dental caries and its mechanism,which might theoretically indicate a new way in caries prevention.
Section I:exogenous c-di-GMP’s effect on dental caries caused by S Mutans
Biofilm Formation:48 hours after exposed to exogenous c-di-GMP with different dosage, biofilm formed in 96 well plates by S Mutans were stained and dissolved by ethanol/acetone, the OD measured by spectrophotometry presents the amount of biofilm in each well. 200μM c-di-GMP and the saline with the same volume incubated with S Mutans on the fresh tooth slices, the biofilm on the slices were investigated with scanning electronic microscope. Result shown that c-di-GMP decreased the biofilm formation and the bacteriae disorganized on the tooth slices with less extracellular matrix, which implies that 200uM c-di-GMP inhibit S Mutans’s capability to form biofilm.
Acid generation and acid resistance: Exogenous c-di-GMP and saline added into variable pH bacteria medium with UA159 S Mutans strain, the change of pH values (△pH)at the end of culture were compared;after cultured in pH=5 medium for 2 hours, the number of live bacteriea presents the ability of acid resistance. Results: after treat with c-di-GMP, S. Mutans’s capability of acid generation and acid resistance decreased.
Extracellular polysaccharide formation: S. Mutans strain UA159 cultured in anaerobic environment 24 hour after exogenous c-di-GMP and saline added into culture system, S. Mutans were broken by distilled water (x2 times) and the supernatant (supernatant I) of 3 centrifugations were collected and combined as water-soluble extracellular polysaccharide. The pellets were added with same volume 0.5 mol/L NaOH, centrifuged, collected supernatant, and repeated 3 times. Combine the supernatant (supernatant II) as water-insoluble polysaccharide. 1.5ml from supernatant I and II (respectively) plus 3 volumes of ethanol, 4℃overnight, collect and centrifuge and discard the upper water phase. Pellets dissolved in distilled water and 0.1mol/L NaOH with same volume. Anthrone method measured the content of glucan in the water-soluble and–insoluble supernatant,OD620,the standard curve was made with glucose and calculate the water-soluble and–insoluble glucan generation. With the result shown that exogenous c-di-GMP treatment significantly decreased the formation of water-insoluble glucan.
Attached to the surface of HA coated with saliva: fluorescence- BCECF/AM labeled UA159 strain put on SHA coated with saliva mimic oral teeth surface, exogenous c-di-GMP and saline co-cultured with UA159 for 30 minutes, fluorescence measured presents the bacteria attached on SHA (485nm→535nm). Results: c-di-GMP treatment decreased the bacteria attachment.
Token together, exogenous c-di-GMP inhibits S Mutans’capacity to form dental caries. So it is possible that exogenous c-di-GMP could be used as anti-caries agent, either alone or with other methods.
Section II: Exogenous c-di-GMP Prevent Caries Formation in Animals Dental Caries were made on the teeth of 20 day age SD rat infected with S Mutans, and fed with caries inducing food 2000 #. Exogenous c-di-GMP, NaF solution and 0.9% NaCl applied to the teeth of the rats, the bacteria number and scores of dental caries were recorded. Results shown that the food and water consume are same among different treatments, which enabled same number of dental caries on the teeth. No red plague, swelling appeared during the treatment, which implies that exogenous c-di-GMP has no toxic effect in the oral cavity. Exogenous c-di-GMP treatment group downgrade the scores of dental caries on occlusal surface and smooth surface, but no difference has been found on the number of bacterial plagues, implies that c-di-GMP could be used as anti-caries agent.
Section III: S Mutans with gcp gene inactivation, strain construction, identification and its caries inducing characters
Take pMD19T-gcp as template, PCR amplify gcp sequence (873bp) , after connected to T vector, the gene frame inserted into suicide vector pVA8912. Enzymatic analysis found the product of PCR product had the same size as the inserted piece, which implies the construct of pVA8912/gcp is successful. Thereby pVA8912/gcp transformed into UA159 and positive clone selected. The selected clone was then verified by PCR and enzymatic analysis on the extracted genomic DNA of the positive clone, with the result shown that the construction of S. Mutans with gcp gene inactivation strain was successful.
S. Mutans with gcp gene inactivation strain was culture in anaerobic environment, the growth curve based on values of OD 600 presents the bacterial growth condition. Results shown that no difference between S. Mutans with gcp gene inactivation strain and wildtype bacteria was found, which implies that inactivation of gcp gene did not change the growth of S Mutans.
S. Mutans with gcp gene inactivation strain and wildtype bacteria were plated into 96 well plates and cultured in anaerobic environment for 48 hours, the bacteria attached were stained with crystal violet, ethanol/acetone dissolve the precipitated stain and the spectrophotometric values of OD575 represent the amount of biofilm formation, with the results shown that S. Mutans with gcp gene inactivation strain formed much less biofilm as compared with wildtype. This implies that gcp gene has relationship with S. Mutans’capacity to form biofilm.
Fluorescence labeled S. Mutans with gcp gene inactivation strain and wildtype bacteria were put on SHA coated with saliva,the fluorescence on SHA represents the number of bacteria attached. S. Mutans with gcp gene inactivation strain (45.33%±6.35%)attached less (P <0.05)than wildtype bacteria(64.05%±6.73%), which implies that gcp gene could inhibit the SHA surface attachment by S Mutans. Conclusion
In our study, c-di-GMP signaling pathway has been found existed in S. Mutans and mediated its capacity of the biofilm formation and surface attachment. Exogenous c-di-GMP reduced S. Mutans’ability of biofilm formation, acid generation, acid resistance and surface attachment ex vivo. In brief, our research provided molecular mechanism for explanation how S Mutans’cause dental caries and experimental evidences for developing new anti-caries method.
引文
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