基因工程改性变异链球菌替代疗法防龋研究
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摘要
龋齿在大多数国家是主要的口腔问题。根据第三次全国口腔健康调查的结果,我国5岁儿童乳牙龋病的患病率为66.0%,12岁儿童恒牙龋病的患病率为28.9%,35-44岁中年人龋病患病率为88.1%，65-74岁老年人龋病患病率为98.4%(全国牙病防治指导组,2008)。虽然包括食品加氟等预防龋齿的有效方式己在临床中被应用(Hinman, Sterritt et al.1996; Ellwood, Blinkhorn et al.1998),但是对于如何对抗引起感染的的病原体微生物却缺少合适的治疗方法(Gu, Lux et al.2002; Luoma, Murtomaa et al.2009)。这是因为口腔是一个拥有丰富多样微生物群落的复杂的生态系统,对它采取任何干扰措施,都会造成一系列无法预期的连锁反应(Brook1999).因此为了消除口腔病原菌而提出的细菌替代疗法成为研究的新思路。
变异链球菌(Streptococcus mutans, S. mutans)被认为是与人类口腔龋齿最密切相关的主要病原体。其在代谢过程中产生的酸性物质,因造成牙面的矿物质溶解,成为其主要致龋毒力因子(Loesche1986)。此外,变异链球菌对牙面的蔗糖依赖性黏附能力帮助细菌不会在咀嚼黏附或唾液冲刷的的过程中被冲洗掉。本次实验中,我们尝试加强低产酸效应菌株的蔗糖黏附能力以增强其与更具致龋性的亲代菌株之间的竞争力,理论上这将最终导致效应菌株在同一个小生境中抢先占据相应的位点(Hillman, Brooks et al.2000)。
在本研究中,我们构建出gcrR缺陷的变异链球菌效应株MS-gcrR-def并与野生型UA159比较其形态变化,黏附和产酸能力的差异。根据一系列体外实验和动物致龋实验的结果,说明缺失了整个gcrR基因的开放阅读框(open reading frame, ORF)的变异株MS-gcrR-def展示出较低的产酸和较强的定植能力,以及较低的致龋能力。
本研究分以下三个部分进行：
第一部分变异链球菌gcrR缺陷株MS-gcrR-def的构建
首先根据Genebank中S. mutans的gcrR基因序列及其上下游同源基因,用Primer5.0设计克隆引物gcrR-For和gcrR-Rev,将其PCR产物插入质粒pMD18-T,形成新的重组质粒pMD-G。根据重组质粒pMD-G的序列在保留gcrR基因翻译起始密码子上游720bp和终止密码子下游705bp同源区的基础上,利用反向PCR方法,为了完全去除gcrR的开放阅读框架,设计反向PCR引物gcrR-rp-For和gcrR-rp-Rev。为了在后续实验中保证与卡那霉素抗性基因的定向连接,在下游引物gcrR-rp-For和上游引物gcrR-rp-Rev的5’端分别加入Clal和Xhol酶切位点。此外以质粒pEGFP-N1为模板,设计克隆引物KanR-For和KanR-Rev,获得卡那抗性片段(KanR,795bp),为了便于在接下来的实验中插入,在上下游引物的5’端插入Clal和Xhol。将反向PCR获得的包含质粒pMD18-T和gcrR上下游的线性片段及通过PCR获得的卡那霉素抗性片段,经Clal和Xho1酶切后实用T4连接酶连接得到重组质粒命名为pMD-GK(4915bp)。以上引物序列送至上海生工合成。为获得相应的PCR产物,在含有Ex Taq DNA聚合酶和Buffer的50μ1混合物中加入相应引物,并放置入PCR仪(ABI),运行以下程序：95℃预变性3min,然后30个循环的95℃变性30s并在合适的温度下退火30s,72℃延伸30s,循环结束后72℃延伸3min。
为确认所构建的质粒pMD-GK序列正确,以其为模板,用gcrR-For和gcrR-Rev进行PCR后做1%琼脂糖凝胶电泳鉴定,见约2.1kb处有一特异条带。以Cla I和Xho I酶切重组质粒pMD-GK后经1%琼脂糖凝胶电泳鉴定,放出长约795的卡那抗性片段。以BamHI和Sal I酶切重组质粒后经1%琼脂糖凝胶电泳鉴定,放出长2.2kb的片段。将其送至上海生工测序,得到gcrR上下游片段和卡那抗性片段都是正确的。由于pMD18是在大肠杆菌中复制的质粒,在链球菌中通常不能复制,并且同时拥有抗性基因的标记,是-种较为方便取得的自杀载体,可用于同源重组(Ortiz-Martin, Macho et al.2006)。同源重组是将外源基因定位导入受体细胞染色体上的方法,因为在该座位有与导入基因同源的序列,通过单一或双交换,新基因片段可替换有缺陷的基因片段,达到修改基因的目的(Burr, Burr et al.1988).
由于变异链球菌是转化率很低的革兰氏阳性菌,本身的转化效率很低且菌株间转化能力有较大的差异(边专and樊明文1996),所以本实验用感受态刺激肽(Competence stimulating peptide, CSP)诱导变异链球菌UA159,使之较容易摄取外源性DNA,根据本实验室先前的研究(Lau, Sung et al.2002),其转化效率提高了20倍以上。加入感受态刺激肽,如文献所描述的方法将质粒pMD-GK转化入变链UA159,并涂于含1mg/ml卡那霉素的TH平板上(Li, Lau et al.2001)。37℃厌氧过夜培养并从中选择转化株。载体pMD-GK进入细菌内,获得了随机插入变异链球菌基因组的机会。同时由于位于卡那抗性片段两侧的gcrR上下游同源臂的存在,可与染色体上的互补序列结合,并在复制过程中诱导发生染色体上的gcrR基因与质粒上的卡那抗性基因互换。由于抗性基因的标记,最终能在含卡那霉素的琼脂平板上生长的即为成功发生同源重组的转化株。通过微量反应糖管和菌落形态的的观察,可以确认转化子是变异链球菌。提取变异株的染色体DNA并将其作为模板,加入引物P1/P2和RT-gcrRF/RT-gcrRR。扩增应获得包含gcrR上游及KanR内部的拼接片段并应同时缺失gcrR基因片段,通过凝胶电泳观察并回收前者用以测序。根据电泳和测序的结果证实变异株成功敲除了gcrR基因并插入卡那霉素抗性片段。此变异株被命名为MS-gcrR-def。
第二部分MS-gcrR-def生化特性和形态方面的研究
对比变异链球菌UA159和gcrR缺陷株MS-gcrR-def的黏附,产酸和致龋能力的改变,探讨MS-gcrR-def是否适合作为龋病替代治疗的效应菌株。为了证明其gcrR缺陷对变异链球菌黏附相关基因的影响,在相同的含2%蔗糖的BHI培养基中分别培养变异链球菌UA159和变异株MS-gcrR-def。8000g离心后收集菌体,分别提取其总RNA,加入终浓度为10μg/ml的溶菌酶和20μ g/ml的变溶菌素37℃孵育1h。然后加入Trzol混匀并加入异丙醇沉淀。用70%乙醇清洗沉淀RNA,用无核酸酶水溶解保存。取5μ1RNA溶液加入适量DuRed,2%凝胶电泳观察RNA是否完整并测量其浓度。通过逆转录的方法获得其cDNA,简单来说,提取变链UA159和变异株MS-gcrR-def的总RNA,并按照说明书使用cDNA第一链合成试剂盒(TaKaRa)获得cDNA。Real time PCR,即荧光定量PCR技术,逆转录获得的cDNA,与相应引物一起加入SYBR green预混试剂系统(TaKaRa)。放入7500荧光定量PCR系统(ABI)进行扩增。其循环条件如下：95-C预变性2min,循环40次95℃5s,60℃60s。为了量化gtfD和gbpC基因,以DNA回旋酶亚基A (gyrA)为内参,以确保参与比较的DNA量是一致的。检测出参与表达的gbpC(葡聚糖结合蛋白C)基因与gtfD(葡萄糖基转移酶D)基因的结果说明在变异株MS-gcrR-def中gtfD相对于野生型或补偿株表达增加(MS-gcrR-def平均RQ=3.8497,UA159平均RQ=0.719,补偿株MS-gcrR-def-com平均RQ=1.4487)。同时gbpC的表达在变异株中也大幅增加(MS-gcrR-def平均RQ=19.9497for, UA159平均RQ=0.8317, MS-gcrR-def-com平均RQ=1.0913)。其中变异株gtfD的表达量是野生株的5倍,gbpC的表达量则高达是24倍。
对于体外黏附试验,在24孔板中,加入含2%蔗糖的BHI培养基后,1：20接种过夜培养并调整为相同OD值(0D470=1.0)的变链UA159,变异株MS-gcrR-def和补偿株MS-gcrR-def-com菌液,分别培养2h,4h,6h,一式三份,37℃厌氧培养。在指定时间用酶标仪测试其在470nm处的吸光度。去掉浮游细菌和培养基,用蒸馏水清洗三次,室温或温箱干燥后用0.1%结晶紫染色15min。然后再用蒸馏水清洗三次,室温或温箱干燥后,加入30%乙酸溶液,将生物膜上的结晶紫漂洗释放,测定其在562nm处的吸光度。结果表明变异株的黏附能力远远强于野生株,尤其是在早期,这一差异的更显著。
野生型和变异株菌液经革兰氏染色,使用光学显微镜观察发现,在含2%蔗糖的BHI培养基中,变异株表现出更多的细菌间的黏附和聚集成团的现象,该结果在早期定植中显得尤其重要。
将培养了2h,4h,6h,12h,24h的生物膜样本先用PBS漂洗清除游离细菌,用分子探针SYT09和碘化丙啶染色5min。染色样本在共聚焦显微镜(ZEISS LSM510META)下观察其红绿荧光并用软件Image pro-plus6.0分析。图像表明,变异株能在成膜早期(2h-4h)抢先在Si02上定植并形成生物膜。
将变异株与野生型同时1：20接种到含4mlBHI培养基的六孔板中,37℃厌氧培养4h,6h,8h,11h’和24h。用蒸馏水清洗除去游离细菌后,收集附着细菌,提取其总DNA。通过荧光定量技术,分析其中变异株所占比例,结果表明,在同时培养的混合生物膜内变异株达到90%以上。结果说明,在变异株与野生型UA159同时存在的时候,变异株能抢先定植固体表面,并生物膜中限制野生株的生长。
为了进一步探讨变异株能否作为合适的替代疗法效应菌,我们同时检测了其产酸能力。在体外产酸试验中,MS-gcrR-def, MS-gcrR-def-com和野生型UA159以1：100的比例接种到含2%蔗糖的BHI培养基中,并在培养前测量其pH值(7.3)。经过16h的培养,pH值再次被分别测量。变异株的终末pH为4.11-4.13,其△pH为3.17-3.19,小于野生型和补偿株的的△pH3.23-3.25,且此差异有统计学意义。此外在生长曲线的观察中我们发现在生长稳定期,变异株的总量少于UA159,说明培养基中累积的酸性物质,对变异株的抑制更为明显。以上结果表明,MS-gcrR-def的黏附性与UA159相比显著增强,产酸能力减少。
第三部分MS-gcrR-def在SD大鼠中的致龋能力
在喂食三天抗生素后,12只大鼠分为两组。用棉拭子在牙面上接种浓度为2X109CFU/ml的UA159和MS-gcrR-def的菌液。连续三天,都用新鲜菌液接种。接种后,用棉拭子收集牙面的细菌并在MSB板上培养,以确认定植成功。继续向MS-gcrR-def组的大鼠提供含1%菌液的蒸馏水,并一直用致龋饲料2000##喂养。接种过后40天,处死大鼠并取出上下颌骨行龋齿记分。结果显示MS-gcrR-def的致龋能力较野生株降低。
Dental caries are major oral health problem in most countries, affecting60-90%of schoolchildren and the vast majority of adults. According to the third national survey on oral health epidemiology in China,66.0%and28.9%of children at age5and12were affected by dental caries, meanwhile88.1%and98.4%of adults at35-44and65-74. Although fluoride and other preventive methods have led to decline in dental caries, little strategy puts effort on the actual pathogen for infection because the oral cavity is a complex ecosystem in which a rich and diverse micro biota has evolved. Methods such as probiotic approach (i.e. whole bacteria replacement therapy) are being investigated in order to eliminate pathogenic members of oral cavity.
Mutans Streptococi is recognized as a main pathogen of dental caries in humans. In the procession of caries, the paramount virulence factor is the ability to produce acid.The sucrose-dependent adherence of S.mutans to teeth surface helps the bacteria not to be washed away with chewing or the flow of saliva. Interestingly, the rationale of the sucrose-dependent adherence could be used for the low acid-producing strains to enhance their adherence ability, so as to them to occupy the same ecological niche in plaque like their more cariogenic progenitor does.
In the present study, we generated a gcrR-deficient S. mutans mutant and compared the morphological characteristics and the ability of adhesion and acid-production between the mutant and wild-type strains. It demonstrated that the mutant strain that is deficient in the WHOLE ORF of gcrR gene showed both lower acid production and stronger colonization potential in comparison with the wild-type strain.
PART1Construction of an S.mutans gcrR-knockout mutant
First of all, according to the gcrR gene sequence and its upstream and downstream homologous genes from the Genebank, the primers were designed. The resulting amplicon (DNA of S.mutans UA159as templates and gcrR-For/gcrR-Rev as primers) was a2118bp fragment including gcrR upstream (720bp) and gcrR downstream (705bp), which was then purified with a PCR Purification Kit (Qiagen, Netherlands), and ligated to the plasmid pMD18-T simple, according to the manufactures'instructions. The resultant plasmid pMD-G was used as a template in another round of amplification with the gcrR-rp-For/gcrR-rp-Rev primers set to generate gcrR-deficient ligation. The amplification product was then ligated to795bp of Kanamycin Resistance cassette (from pEGFP-N1vector) in the presence of T4DNA ligase (New England Biolabs, USA) to generate a new plasmid pMD-GK.
Primers were designed by using Primer premier5.0software (Premier, Canada) and synthesized by Sangon Biotech Co.,Ltd (Shanghai, China). PCR amplicons were generated in a50μl final reaction volume with ExTaq mixture (Takara, Japan) using Primers gcrR-For/gcrR-Rev, gcrR-rp-For/gcrR-rp-Rev, and KanR-For/KanR-Rev. The reaction mixtures were placed in an automated thermal (Applied Biosystems, USA) that was programmed to run one cycle of denaturation at95℃for2min followed by40cycles of denaturation at95℃for5sec, primers annealing at60℃for30sec.
To PMD-GK as a template for PCR with gcrR-For gcrR-Rev do a1%agarose gel electrophoresis, and approximately2.1kb at a specific band. Cleave the recombinant to Cla I and Xho I the plasmid pMD-GK by1%agarose gel electrophoresis, release the Kana resistant fragment of about795.1%agarose gel electrophoresis to the BamH I and Sal I cleave the recombinant plasmid after release2.2kb fragment. Will be sent to Shanghai Sangon sequencing, get downstream fragment and gcrR the Kana resistant fragment are100%correct. Since pMD18plasmid replication in E. coli, Streptococcus usually cannot be copied, and also has a resistance gene marker, is a more convenient to obtain the suicide vector can be used for homologous recombination. Homologous recombination is the positioning of the exogenous gene import receptor on the cell chromosome, since the sequence homology with the introduced gene, by a single or double-swap new gene fragment can replace a defective gene fragment in the seat, to modify the purpose of the gene.
S.mutans UA159cells were transformed with the final plasmid pMD-GK in the presence of competence-stimulating peptide, as described elsewhere. Transformed colonies were selected on TH-agar plated with1mg/ml Kanamycin after overnight incubation at37℃with5%CO2.
Kanamycin-resistant transformants were screened for deletion of the whole gcrR gene and replacement with Kanamycin-resistant cassette as a result of double-crossover recombination. Chromosomal DNA was isolated from selected transformants and from the UA159progenitor and used as a template for PCR amplification with primers P1/P2and RT-gcrRF/RT-gcrRR. The resulting amplicons (the gcrR upstream-KanR cassette junction and the part of gcrR gene) were analyzed by agarose gel electrophoresis and their sizes were compared with those predicted following a successful allelic exchange event. Nucleotide sequencing of the purified amplicons with the same primers confirmed the knockout of gcrR and insertion of the Kanamycin resistant cassette. The resulting mutant which contains the gcrR upstream-KanR cassette junction and lacks the gcrR gene was designated as MS-gcrR-def.
PART2Biochemical and morphology study of the MS-gcrR-def
S.mutans wild-type and the mutant MS-gcrR-def was grown in BHI supplemented with2%sucrose at37℃under5%CO2to mid-exponential-phase culture (OD470=0.5), then harvested by centrifugation at8000g and digested by N-acetylmuramidase (mutanolysinl; Sigma-Aldrich, USA) at20μg/ml and lysozyme (Sigma-Aldrich, USA) at10μg/ml at37℃for60min before homogenized in Trizol reagent (Invitrogen, USA). Following isopropanol precipitation, the RNA pellets were washed in70%ethanol and finally resuspended in nuclease-free water. RNA integrity was assessed by DuRed staining of the23S and16S rRNA subunits on a2%agarose gel, and quantified spectrophotometrically at A260for concentration and at A260/280for purity.
RT-PCR was conducted to characterize the transcription of S.mutans gcrR gene. Briefly,1μg of DNase-treated total RNA isolated from S.mutans wild-type or the mutant MS-gcrR-def was reverse-transcripted to cDNA by using a first-strand cDNA synthesis kit (TaKaRa) in accordance with the recommendation of the supplier.
Single-strand cDNA obtained in the above experiment, was amplified for gtfD and gbpC in a TaKaRa SYBR Premix system. Amplification was performed in a7500real-time PCR system (Applied Biosystems, USA) according to the following thermal cycling protocol:95℃for2min for initial denaturation, followed by40cycles of three steps consisting of94℃for5s, the optimal temperature (Table2) for60s, and72℃for60s. Quantification of gtfD and gbpC cDNA was normalized to the amount of cDNA derived from a DNA gyrase, subunit A protein (gyrA) control, the expression of which does not vary under the experimental conditions.
The results of real-time RT-PCR revealed gtfD expression was increased in the MS-gcrR-def (mean RQ=3.8497for MS-gcrR-def,0.719for UA159and1.4487for MS-gcrR-def-com; n=3) relative to that in the wild type or compensated mutant. Likewise, gbpC expression was increased in MS-gcrR-def compared to that in the wild type or compensated mutants (mean RQ=19.9497for MS-gcrR-def,0.8317for UA159and1.0913 for MS-gcrR-def-com; n=3). Approximately, there were a fivefold and twenty-fourfold increase in gtfD and gbpC expression in the MS-gcrR-def gcrR knockout mutant relative to the wild type and compensated mutant.
S.mutans wild-type and mutant strains were monitored for adherence to the walls of24well cell culture plates following2h,4h and6h-growths in BHI supplemented with2%sucrose. Briefly, overnight cultures of S.mutans UA159, the mutants MS-gcrR-def and MS-gcrR-def-com were inoculated as described above into BHI with control water and then standardized respectively to with1.0OD units (OD470) with fresh physiological saline. The three resulting bacteria suspensions were diluted1:20with fresh BHI with2%sucrose respectively and distributed into2.0ml fresh medium in the wells of sterile24-well cell culture plates in triplicate. The plate were kept at37℃with5%CO2for2h,4h and6h in triplicate. Bacterial growth was measured at470nm using a microplate reader. Then the media was discarded and the plates were rinsed with distilled water for3times and dried in room temperature.0.1%Crystal violet dye was added into the plates and kept for15min. Then the plates were rinsed with distilled water for3times and dried in room temperature or incubator. Thirty percent of acetic acid was added into plate to dissolve crystal violet. Biofilm formation was measured at562nm using a microplate reader.
According to the result of adhesion test, the adhesion ability of gcrR deficient mutants was much stronger than that of the wild type. Furthermore, the difference in the early stage was greater than in the late stage.
The two bacteria suspensions were diluted1:20with fresh BHI respectively or together and distributed4.0ml aliquots into the wells of sterile6-well cell culture plates to obtain two-bacterial biofilm cells at37℃for4h,6h,8h,11h and24h. In the6-well plates, each well was gently rinsed with distilled water to remove loose bacteria, and firmly attached cells were scraped from the substrate by pipetting and rinsed with distilled water to make sure all cells were collected. Total DNA was extracted from the same numbers of cells of the control and test groups.
According to the result of Real time PCR, the mutant possessed an apparent advantage over wild type. The gcrR defective-bacteria accounted for more than90%in all adherent bacteria. Interestingly, the initial advantage continued to expand over time.
In vitro acid production, the initial pH of the culture when mixed with the cell suspensions of S.mutans, MS-gcrR-def or MS-gcrR-def-com was measured at a constant pH of7.3and then the fall in pH over a16h period were measured again by pH meter to obtain the ApH of these cultures.
the final pH of the above two cultures were significantly lower than those values for the culture of MS-gcrR-def which were in the range of4.11-4.13after16h of incubation at37℃.The MS-gcrR-def had a small average ApH of between3.17-3.19which was substantially smaller than the average ApH of the wild-type and MS-gcrR-def-com which ranged between3.23-3.25.There were significant differences between UA159and MS-gcrR-def (p<0.05). There were significant differences between MS-gcrR-def-com and MS-gcrR-def (p<0.05).
After incubation at37℃with5%CO2, S.mutans UA159, MS-gcrR-def were harvested at mid-exponential phage (OD470=0.5) and the culture densities were standardized to1.0OD units with fresh physiological saline. The resulting cells suspensions were diluted by1:20into fresh BHI and distributed2.0ml aliquots into the wells of24-well plates in triplicate. The cells were incubated for24h at37℃and5%CO2in and OD470measurements were obtained with gentle agitation preceding each reading in a Microplate Reader. Doubling times from each of three independent experiments were determined for cells in exponential phase and at points corresponding.
We observed equal survivor-ship for all S.mutans strain following24h of growth. MS-gcrR-def ultimately attained a lower final resting culture density than wild-type. Taken together, these findings support the involvement of GcrR in the ability of S.mutans to withstand acid stress and indicate that defects in acid tolerance arise from gcrR deficiency.
The cells suspensions were smeared on glass slides stained with Gram's iodine and observed under an optical microscope. The results of the wild type and gcrR-deficient mutant stain were obviously Gram positive. Interestingly, the mutants were more easily clump together than the wild type and the process occurred rapidly. It contributed to bacterial colonization in early stage.
The biofilm samples were gently rinsed with PBS three times to remove unattached cells, dried for5min, and then stained with molecular Probes SYTO9and propdium iodide (Invitrogen, USA). The stained samples were then examined by CLSM (ZEISS LSM510META; Carl Zeiss, Germany). The red and green fluorescence intensity was quantified respectively by Image pro-plus6.0(Media Cybernetics, USA).
According to the figures, at the early stage (at2h and4h) MS-gcrR-def formed biofilms more quickly than UA159and the biofilms were thicker relative to the wild type.
It demonstrated that MS-gcrR-def showed both lower acid production and stronger colonization potential in comparison with the wild-type strain.
Part3Assessment of the cariogenic potential of MS-gcrR-def in SD rats.
After feeding with appropriate antibiotic for3days, two groups of six rats each were challenged with oral swabs saturated with2X109CFU/ml of S.mutans UA159and MS-gcrR-def per ml. Over the next3day, the rats were challenged repeatedly with fresh swabs of the appropriate strain, and on the next day postchallenge, swab samples were collected and plated onto MSB agar plated to confirm colonization. The rats of MS-gcrR-def group were provided water supplemented with1%the appropriate strain and maintained on caries-promoting diet2000#. Forty days postchallenge, the rats were sacrificed, and their maxillary and mandibles were removed and subsequently processed for caries scores.MS-gcrR-def was significantly less cariogenic than the UA159wild-type progenitor in germfree rats.
变异链球菌(Streptococcus mutans, S. mutans)被认为是与人类口腔龋齿最密切相关的主要病原体。其在代谢过程中产生的酸性物质,因造成牙面的矿物质溶解,成为其主要致龋毒力因子(Loesche1986)。此外,变异链球菌对牙面的蔗糖依赖性黏附能力帮助细菌不会在咀嚼黏附或唾液冲刷的的过程中被冲洗掉。本次实验中,我们尝试加强低产酸效应菌株的蔗糖黏附能力以增强其与更具致龋性的亲代菌株之间的竞争力,理论上这将最终导致效应菌株在同一个小生境中抢先占据相应的位点(Hillman, Brooks et al.2000)。
在本研究中,我们构建出gcrR缺陷的变异链球菌效应株MS-gcrR-def并与野生型UA159比较其形态变化,黏附和产酸能力的差异。根据一系列体外实验和动物致龋实验的结果,说明缺失了整个gcrR基因的开放阅读框(open reading frame, ORF)的变异株MS-gcrR-def展示出较低的产酸和较强的定植能力,以及较低的致龋能力。
本研究分以下三个部分进行：
第一部分变异链球菌gcrR缺陷株MS-gcrR-def的构建
首先根据Genebank中S. mutans的gcrR基因序列及其上下游同源基因,用Primer5.0设计克隆引物gcrR-For和gcrR-Rev,将其PCR产物插入质粒pMD18-T,形成新的重组质粒pMD-G。根据重组质粒pMD-G的序列在保留gcrR基因翻译起始密码子上游720bp和终止密码子下游705bp同源区的基础上,利用反向PCR方法,为了完全去除gcrR的开放阅读框架,设计反向PCR引物gcrR-rp-For和gcrR-rp-Rev。为了在后续实验中保证与卡那霉素抗性基因的定向连接,在下游引物gcrR-rp-For和上游引物gcrR-rp-Rev的5’端分别加入Clal和Xhol酶切位点。此外以质粒pEGFP-N1为模板,设计克隆引物KanR-For和KanR-Rev,获得卡那抗性片段(KanR,795bp),为了便于在接下来的实验中插入,在上下游引物的5’端插入Clal和Xhol。将反向PCR获得的包含质粒pMD18-T和gcrR上下游的线性片段及通过PCR获得的卡那霉素抗性片段,经Clal和Xho1酶切后实用T4连接酶连接得到重组质粒命名为pMD-GK(4915bp)。以上引物序列送至上海生工合成。为获得相应的PCR产物,在含有Ex Taq DNA聚合酶和Buffer的50μ1混合物中加入相应引物,并放置入PCR仪(ABI),运行以下程序：95℃预变性3min,然后30个循环的95℃变性30s并在合适的温度下退火30s,72℃延伸30s,循环结束后72℃延伸3min。
为确认所构建的质粒pMD-GK序列正确,以其为模板,用gcrR-For和gcrR-Rev进行PCR后做1%琼脂糖凝胶电泳鉴定,见约2.1kb处有一特异条带。以Cla I和Xho I酶切重组质粒pMD-GK后经1%琼脂糖凝胶电泳鉴定,放出长约795的卡那抗性片段。以BamHI和Sal I酶切重组质粒后经1%琼脂糖凝胶电泳鉴定,放出长2.2kb的片段。将其送至上海生工测序,得到gcrR上下游片段和卡那抗性片段都是正确的。由于pMD18是在大肠杆菌中复制的质粒,在链球菌中通常不能复制,并且同时拥有抗性基因的标记,是-种较为方便取得的自杀载体,可用于同源重组(Ortiz-Martin, Macho et al.2006)。同源重组是将外源基因定位导入受体细胞染色体上的方法,因为在该座位有与导入基因同源的序列,通过单一或双交换,新基因片段可替换有缺陷的基因片段,达到修改基因的目的(Burr, Burr et al.1988).
由于变异链球菌是转化率很低的革兰氏阳性菌,本身的转化效率很低且菌株间转化能力有较大的差异(边专and樊明文1996),所以本实验用感受态刺激肽(Competence stimulating peptide, CSP)诱导变异链球菌UA159,使之较容易摄取外源性DNA,根据本实验室先前的研究(Lau, Sung et al.2002),其转化效率提高了20倍以上。加入感受态刺激肽,如文献所描述的方法将质粒pMD-GK转化入变链UA159,并涂于含1mg/ml卡那霉素的TH平板上(Li, Lau et al.2001)。37℃厌氧过夜培养并从中选择转化株。载体pMD-GK进入细菌内,获得了随机插入变异链球菌基因组的机会。同时由于位于卡那抗性片段两侧的gcrR上下游同源臂的存在,可与染色体上的互补序列结合,并在复制过程中诱导发生染色体上的gcrR基因与质粒上的卡那抗性基因互换。由于抗性基因的标记,最终能在含卡那霉素的琼脂平板上生长的即为成功发生同源重组的转化株。通过微量反应糖管和菌落形态的的观察,可以确认转化子是变异链球菌。提取变异株的染色体DNA并将其作为模板,加入引物P1/P2和RT-gcrRF/RT-gcrRR。扩增应获得包含gcrR上游及KanR内部的拼接片段并应同时缺失gcrR基因片段,通过凝胶电泳观察并回收前者用以测序。根据电泳和测序的结果证实变异株成功敲除了gcrR基因并插入卡那霉素抗性片段。此变异株被命名为MS-gcrR-def。
第二部分MS-gcrR-def生化特性和形态方面的研究
对比变异链球菌UA159和gcrR缺陷株MS-gcrR-def的黏附,产酸和致龋能力的改变,探讨MS-gcrR-def是否适合作为龋病替代治疗的效应菌株。为了证明其gcrR缺陷对变异链球菌黏附相关基因的影响,在相同的含2%蔗糖的BHI培养基中分别培养变异链球菌UA159和变异株MS-gcrR-def。8000g离心后收集菌体,分别提取其总RNA,加入终浓度为10μg/ml的溶菌酶和20μ g/ml的变溶菌素37℃孵育1h。然后加入Trzol混匀并加入异丙醇沉淀。用70%乙醇清洗沉淀RNA,用无核酸酶水溶解保存。取5μ1RNA溶液加入适量DuRed,2%凝胶电泳观察RNA是否完整并测量其浓度。通过逆转录的方法获得其cDNA,简单来说,提取变链UA159和变异株MS-gcrR-def的总RNA,并按照说明书使用cDNA第一链合成试剂盒(TaKaRa)获得cDNA。Real time PCR,即荧光定量PCR技术,逆转录获得的cDNA,与相应引物一起加入SYBR green预混试剂系统(TaKaRa)。放入7500荧光定量PCR系统(ABI)进行扩增。其循环条件如下：95-C预变性2min,循环40次95℃5s,60℃60s。为了量化gtfD和gbpC基因,以DNA回旋酶亚基A (gyrA)为内参,以确保参与比较的DNA量是一致的。检测出参与表达的gbpC(葡聚糖结合蛋白C)基因与gtfD(葡萄糖基转移酶D)基因的结果说明在变异株MS-gcrR-def中gtfD相对于野生型或补偿株表达增加(MS-gcrR-def平均RQ=3.8497,UA159平均RQ=0.719,补偿株MS-gcrR-def-com平均RQ=1.4487)。同时gbpC的表达在变异株中也大幅增加(MS-gcrR-def平均RQ=19.9497for, UA159平均RQ=0.8317, MS-gcrR-def-com平均RQ=1.0913)。其中变异株gtfD的表达量是野生株的5倍,gbpC的表达量则高达是24倍。
对于体外黏附试验,在24孔板中,加入含2%蔗糖的BHI培养基后,1：20接种过夜培养并调整为相同OD值(0D470=1.0)的变链UA159,变异株MS-gcrR-def和补偿株MS-gcrR-def-com菌液,分别培养2h,4h,6h,一式三份,37℃厌氧培养。在指定时间用酶标仪测试其在470nm处的吸光度。去掉浮游细菌和培养基,用蒸馏水清洗三次,室温或温箱干燥后用0.1%结晶紫染色15min。然后再用蒸馏水清洗三次,室温或温箱干燥后,加入30%乙酸溶液,将生物膜上的结晶紫漂洗释放,测定其在562nm处的吸光度。结果表明变异株的黏附能力远远强于野生株,尤其是在早期,这一差异的更显著。
野生型和变异株菌液经革兰氏染色,使用光学显微镜观察发现,在含2%蔗糖的BHI培养基中,变异株表现出更多的细菌间的黏附和聚集成团的现象,该结果在早期定植中显得尤其重要。
将培养了2h,4h,6h,12h,24h的生物膜样本先用PBS漂洗清除游离细菌,用分子探针SYT09和碘化丙啶染色5min。染色样本在共聚焦显微镜(ZEISS LSM510META)下观察其红绿荧光并用软件Image pro-plus6.0分析。图像表明,变异株能在成膜早期(2h-4h)抢先在Si02上定植并形成生物膜。
将变异株与野生型同时1：20接种到含4mlBHI培养基的六孔板中,37℃厌氧培养4h,6h,8h,11h’和24h。用蒸馏水清洗除去游离细菌后,收集附着细菌,提取其总DNA。通过荧光定量技术,分析其中变异株所占比例,结果表明,在同时培养的混合生物膜内变异株达到90%以上。结果说明,在变异株与野生型UA159同时存在的时候,变异株能抢先定植固体表面,并生物膜中限制野生株的生长。
为了进一步探讨变异株能否作为合适的替代疗法效应菌,我们同时检测了其产酸能力。在体外产酸试验中,MS-gcrR-def, MS-gcrR-def-com和野生型UA159以1：100的比例接种到含2%蔗糖的BHI培养基中,并在培养前测量其pH值(7.3)。经过16h的培养,pH值再次被分别测量。变异株的终末pH为4.11-4.13,其△pH为3.17-3.19,小于野生型和补偿株的的△pH3.23-3.25,且此差异有统计学意义。此外在生长曲线的观察中我们发现在生长稳定期,变异株的总量少于UA159,说明培养基中累积的酸性物质,对变异株的抑制更为明显。以上结果表明,MS-gcrR-def的黏附性与UA159相比显著增强,产酸能力减少。
第三部分MS-gcrR-def在SD大鼠中的致龋能力
在喂食三天抗生素后,12只大鼠分为两组。用棉拭子在牙面上接种浓度为2X109CFU/ml的UA159和MS-gcrR-def的菌液。连续三天,都用新鲜菌液接种。接种后,用棉拭子收集牙面的细菌并在MSB板上培养,以确认定植成功。继续向MS-gcrR-def组的大鼠提供含1%菌液的蒸馏水,并一直用致龋饲料2000##喂养。接种过后40天,处死大鼠并取出上下颌骨行龋齿记分。结果显示MS-gcrR-def的致龋能力较野生株降低。
Dental caries are major oral health problem in most countries, affecting60-90%of schoolchildren and the vast majority of adults. According to the third national survey on oral health epidemiology in China,66.0%and28.9%of children at age5and12were affected by dental caries, meanwhile88.1%and98.4%of adults at35-44and65-74. Although fluoride and other preventive methods have led to decline in dental caries, little strategy puts effort on the actual pathogen for infection because the oral cavity is a complex ecosystem in which a rich and diverse micro biota has evolved. Methods such as probiotic approach (i.e. whole bacteria replacement therapy) are being investigated in order to eliminate pathogenic members of oral cavity.
Mutans Streptococi is recognized as a main pathogen of dental caries in humans. In the procession of caries, the paramount virulence factor is the ability to produce acid.The sucrose-dependent adherence of S.mutans to teeth surface helps the bacteria not to be washed away with chewing or the flow of saliva. Interestingly, the rationale of the sucrose-dependent adherence could be used for the low acid-producing strains to enhance their adherence ability, so as to them to occupy the same ecological niche in plaque like their more cariogenic progenitor does.
In the present study, we generated a gcrR-deficient S. mutans mutant and compared the morphological characteristics and the ability of adhesion and acid-production between the mutant and wild-type strains. It demonstrated that the mutant strain that is deficient in the WHOLE ORF of gcrR gene showed both lower acid production and stronger colonization potential in comparison with the wild-type strain.
PART1Construction of an S.mutans gcrR-knockout mutant
First of all, according to the gcrR gene sequence and its upstream and downstream homologous genes from the Genebank, the primers were designed. The resulting amplicon (DNA of S.mutans UA159as templates and gcrR-For/gcrR-Rev as primers) was a2118bp fragment including gcrR upstream (720bp) and gcrR downstream (705bp), which was then purified with a PCR Purification Kit (Qiagen, Netherlands), and ligated to the plasmid pMD18-T simple, according to the manufactures'instructions. The resultant plasmid pMD-G was used as a template in another round of amplification with the gcrR-rp-For/gcrR-rp-Rev primers set to generate gcrR-deficient ligation. The amplification product was then ligated to795bp of Kanamycin Resistance cassette (from pEGFP-N1vector) in the presence of T4DNA ligase (New England Biolabs, USA) to generate a new plasmid pMD-GK.
Primers were designed by using Primer premier5.0software (Premier, Canada) and synthesized by Sangon Biotech Co.,Ltd (Shanghai, China). PCR amplicons were generated in a50μl final reaction volume with ExTaq mixture (Takara, Japan) using Primers gcrR-For/gcrR-Rev, gcrR-rp-For/gcrR-rp-Rev, and KanR-For/KanR-Rev. The reaction mixtures were placed in an automated thermal (Applied Biosystems, USA) that was programmed to run one cycle of denaturation at95℃for2min followed by40cycles of denaturation at95℃for5sec, primers annealing at60℃for30sec.
To PMD-GK as a template for PCR with gcrR-For gcrR-Rev do a1%agarose gel electrophoresis, and approximately2.1kb at a specific band. Cleave the recombinant to Cla I and Xho I the plasmid pMD-GK by1%agarose gel electrophoresis, release the Kana resistant fragment of about795.1%agarose gel electrophoresis to the BamH I and Sal I cleave the recombinant plasmid after release2.2kb fragment. Will be sent to Shanghai Sangon sequencing, get downstream fragment and gcrR the Kana resistant fragment are100%correct. Since pMD18plasmid replication in E. coli, Streptococcus usually cannot be copied, and also has a resistance gene marker, is a more convenient to obtain the suicide vector can be used for homologous recombination. Homologous recombination is the positioning of the exogenous gene import receptor on the cell chromosome, since the sequence homology with the introduced gene, by a single or double-swap new gene fragment can replace a defective gene fragment in the seat, to modify the purpose of the gene.
S.mutans UA159cells were transformed with the final plasmid pMD-GK in the presence of competence-stimulating peptide, as described elsewhere. Transformed colonies were selected on TH-agar plated with1mg/ml Kanamycin after overnight incubation at37℃with5%CO2.
Kanamycin-resistant transformants were screened for deletion of the whole gcrR gene and replacement with Kanamycin-resistant cassette as a result of double-crossover recombination. Chromosomal DNA was isolated from selected transformants and from the UA159progenitor and used as a template for PCR amplification with primers P1/P2and RT-gcrRF/RT-gcrRR. The resulting amplicons (the gcrR upstream-KanR cassette junction and the part of gcrR gene) were analyzed by agarose gel electrophoresis and their sizes were compared with those predicted following a successful allelic exchange event. Nucleotide sequencing of the purified amplicons with the same primers confirmed the knockout of gcrR and insertion of the Kanamycin resistant cassette. The resulting mutant which contains the gcrR upstream-KanR cassette junction and lacks the gcrR gene was designated as MS-gcrR-def.
PART2Biochemical and morphology study of the MS-gcrR-def
S.mutans wild-type and the mutant MS-gcrR-def was grown in BHI supplemented with2%sucrose at37℃under5%CO2to mid-exponential-phase culture (OD470=0.5), then harvested by centrifugation at8000g and digested by N-acetylmuramidase (mutanolysinl; Sigma-Aldrich, USA) at20μg/ml and lysozyme (Sigma-Aldrich, USA) at10μg/ml at37℃for60min before homogenized in Trizol reagent (Invitrogen, USA). Following isopropanol precipitation, the RNA pellets were washed in70%ethanol and finally resuspended in nuclease-free water. RNA integrity was assessed by DuRed staining of the23S and16S rRNA subunits on a2%agarose gel, and quantified spectrophotometrically at A260for concentration and at A260/280for purity.
RT-PCR was conducted to characterize the transcription of S.mutans gcrR gene. Briefly,1μg of DNase-treated total RNA isolated from S.mutans wild-type or the mutant MS-gcrR-def was reverse-transcripted to cDNA by using a first-strand cDNA synthesis kit (TaKaRa) in accordance with the recommendation of the supplier.
Single-strand cDNA obtained in the above experiment, was amplified for gtfD and gbpC in a TaKaRa SYBR Premix system. Amplification was performed in a7500real-time PCR system (Applied Biosystems, USA) according to the following thermal cycling protocol:95℃for2min for initial denaturation, followed by40cycles of three steps consisting of94℃for5s, the optimal temperature (Table2) for60s, and72℃for60s. Quantification of gtfD and gbpC cDNA was normalized to the amount of cDNA derived from a DNA gyrase, subunit A protein (gyrA) control, the expression of which does not vary under the experimental conditions.
The results of real-time RT-PCR revealed gtfD expression was increased in the MS-gcrR-def (mean RQ=3.8497for MS-gcrR-def,0.719for UA159and1.4487for MS-gcrR-def-com; n=3) relative to that in the wild type or compensated mutant. Likewise, gbpC expression was increased in MS-gcrR-def compared to that in the wild type or compensated mutants (mean RQ=19.9497for MS-gcrR-def,0.8317for UA159and1.0913 for MS-gcrR-def-com; n=3). Approximately, there were a fivefold and twenty-fourfold increase in gtfD and gbpC expression in the MS-gcrR-def gcrR knockout mutant relative to the wild type and compensated mutant.
S.mutans wild-type and mutant strains were monitored for adherence to the walls of24well cell culture plates following2h,4h and6h-growths in BHI supplemented with2%sucrose. Briefly, overnight cultures of S.mutans UA159, the mutants MS-gcrR-def and MS-gcrR-def-com were inoculated as described above into BHI with control water and then standardized respectively to with1.0OD units (OD470) with fresh physiological saline. The three resulting bacteria suspensions were diluted1:20with fresh BHI with2%sucrose respectively and distributed into2.0ml fresh medium in the wells of sterile24-well cell culture plates in triplicate. The plate were kept at37℃with5%CO2for2h,4h and6h in triplicate. Bacterial growth was measured at470nm using a microplate reader. Then the media was discarded and the plates were rinsed with distilled water for3times and dried in room temperature.0.1%Crystal violet dye was added into the plates and kept for15min. Then the plates were rinsed with distilled water for3times and dried in room temperature or incubator. Thirty percent of acetic acid was added into plate to dissolve crystal violet. Biofilm formation was measured at562nm using a microplate reader.
According to the result of adhesion test, the adhesion ability of gcrR deficient mutants was much stronger than that of the wild type. Furthermore, the difference in the early stage was greater than in the late stage.
The two bacteria suspensions were diluted1:20with fresh BHI respectively or together and distributed4.0ml aliquots into the wells of sterile6-well cell culture plates to obtain two-bacterial biofilm cells at37℃for4h,6h,8h,11h and24h. In the6-well plates, each well was gently rinsed with distilled water to remove loose bacteria, and firmly attached cells were scraped from the substrate by pipetting and rinsed with distilled water to make sure all cells were collected. Total DNA was extracted from the same numbers of cells of the control and test groups.
According to the result of Real time PCR, the mutant possessed an apparent advantage over wild type. The gcrR defective-bacteria accounted for more than90%in all adherent bacteria. Interestingly, the initial advantage continued to expand over time.
In vitro acid production, the initial pH of the culture when mixed with the cell suspensions of S.mutans, MS-gcrR-def or MS-gcrR-def-com was measured at a constant pH of7.3and then the fall in pH over a16h period were measured again by pH meter to obtain the ApH of these cultures.
the final pH of the above two cultures were significantly lower than those values for the culture of MS-gcrR-def which were in the range of4.11-4.13after16h of incubation at37℃.The MS-gcrR-def had a small average ApH of between3.17-3.19which was substantially smaller than the average ApH of the wild-type and MS-gcrR-def-com which ranged between3.23-3.25.There were significant differences between UA159and MS-gcrR-def (p<0.05). There were significant differences between MS-gcrR-def-com and MS-gcrR-def (p<0.05).
After incubation at37℃with5%CO2, S.mutans UA159, MS-gcrR-def were harvested at mid-exponential phage (OD470=0.5) and the culture densities were standardized to1.0OD units with fresh physiological saline. The resulting cells suspensions were diluted by1:20into fresh BHI and distributed2.0ml aliquots into the wells of24-well plates in triplicate. The cells were incubated for24h at37℃and5%CO2in and OD470measurements were obtained with gentle agitation preceding each reading in a Microplate Reader. Doubling times from each of three independent experiments were determined for cells in exponential phase and at points corresponding.
We observed equal survivor-ship for all S.mutans strain following24h of growth. MS-gcrR-def ultimately attained a lower final resting culture density than wild-type. Taken together, these findings support the involvement of GcrR in the ability of S.mutans to withstand acid stress and indicate that defects in acid tolerance arise from gcrR deficiency.
The cells suspensions were smeared on glass slides stained with Gram's iodine and observed under an optical microscope. The results of the wild type and gcrR-deficient mutant stain were obviously Gram positive. Interestingly, the mutants were more easily clump together than the wild type and the process occurred rapidly. It contributed to bacterial colonization in early stage.
The biofilm samples were gently rinsed with PBS three times to remove unattached cells, dried for5min, and then stained with molecular Probes SYTO9and propdium iodide (Invitrogen, USA). The stained samples were then examined by CLSM (ZEISS LSM510META; Carl Zeiss, Germany). The red and green fluorescence intensity was quantified respectively by Image pro-plus6.0(Media Cybernetics, USA).
According to the figures, at the early stage (at2h and4h) MS-gcrR-def formed biofilms more quickly than UA159and the biofilms were thicker relative to the wild type.
It demonstrated that MS-gcrR-def showed both lower acid production and stronger colonization potential in comparison with the wild-type strain.
Part3Assessment of the cariogenic potential of MS-gcrR-def in SD rats.
After feeding with appropriate antibiotic for3days, two groups of six rats each were challenged with oral swabs saturated with2X109CFU/ml of S.mutans UA159and MS-gcrR-def per ml. Over the next3day, the rats were challenged repeatedly with fresh swabs of the appropriate strain, and on the next day postchallenge, swab samples were collected and plated onto MSB agar plated to confirm colonization. The rats of MS-gcrR-def group were provided water supplemented with1%the appropriate strain and maintained on caries-promoting diet2000#. Forty days postchallenge, the rats were sacrificed, and their maxillary and mandibles were removed and subsequently processed for caries scores.MS-gcrR-def was significantly less cariogenic than the UA159wild-type progenitor in germfree rats.
引文
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2 Abrams G. D., Bauer H., Sprinz H. (1962). " Influence of the normal flora on mucosal morphology and cellular renewal in the ileum. " A comparison of germ-free and conventional mice, DTIC Document.
3 Alvarez-Olmos M.I., Oberhelman R.A. (2001). " Probiotic agents and infectious diseases:a modern perspective on a traditional therapy." Clinical infectious diseases 32(11):1567-1576.
4 Arends J., Christoffersen J. (1986). " The Nature of Early Caries Lesions in Enamel." Journal of Dental Research 65(1):2-11.
5 Bartlett J. M., Stirling D. (2003). " A short history of the polymerase chain reaction." PCR protocols, Springer:3-6.
6 Bleiweis A.S., Oyston P.C., Brady L.J. (1992). "Molecular, immunological and functional characterization of the major surface adhesin of Streptococcus mutans." Advances in experimental medicine and biology 327:229.
7 Bowen W. (1996). "Vaccine against dental caries-a personal view." Journal of Dental Research 75(8): 1530-1533.
8 Brook I. (1999). "Bacterial interference." Critical reviews in microbiology 25(3):155-172.
9 Burnstock G. (2006). "Purinergic P2 receptors as targets for novel analgesics." Pharmacology & therapeutics 110(3):433-454.
10 Burr B., Burr F. A., Thompson K.H., Albertson MC., Stuber C.W. (1988). "Gene mapping with recombinant inbreds in maize." Genetics 118(3):519-526.
11 Caufield P. W., Cutter G.R., Dasanayake A.P. (1993). "Initial acquisition of mutans streptococci by infants:evidence for a discrete window of infectivity." Journal of Dental Research 72(1):37-45.
12 Chen A., Hillman J.D., Duncan M. (1994). "L-(+)-lactate dehydrogenase deficiency is lethal in Streptococcus mutans." Journal of bacteriology 176(5):1542-1545.
13 Chen Y.Y., Burne R.A. (2003). "Identification and characterization of the nickel uptake system for urease biogenesis in Streptococcus salivarius 57.I." Journal of bacteriology 185(23):6773-6779.
14 Chen Y.Y., Clancy K. A., Burne R.A. (1996). "Streptococcus salivarius urease:genetic and biochemical characterization and expression in a dental plaque streptococcus." Infection and immunity 64(2):585-592.
15 Chikindas M.L., Novak J., Caufield P.W., Schilling K., Tagg J.R. (1997). "Microbially-produced peptides having potential application to the prevention of dental caries." International journal of antimicrobial agents 9(2):95-105.
16 Childers N.K., Zhang S.S., Michalek S.M. (1994). "Oral immunization of humans with dehydrated liposomes containing Streptococcus mutans glucosyltransferase induces salivary immunoglobulin A2 antibody responses." Oral microbiology and immunology 9(3):146-153.
17 Clancy A., Burne R.A. (1997). "Construction and characterization of a recombinant ureolytic Streptococcus mutans and its use to demonstrate the relationship of urease activity to pH modulating capacity." FEMS microbiology letters 151(2):205-211.
18 Clarke J. K. (1924). "On the bacterial factor in the aetiology of dental caries." British journal of experimental pathology 5(3):141.
19 Cvitkovitch D. (2001). "Genetic competence and transformation in oral streptococci." Critical Reviews in Oral Biology & Medicine 12(3):217-243.
20 Dashper S. G., Reynolds E. C. (1996). "Lactic acid excretion by Streptococcus mutans." Microbiology 142(1):33-39.
21 Dunn P. M., Zhong Y., Burnstock G. (2001). "P2X receptors in peripheral neurons." Progress in neurobiology 65(2):107-134.
22 Dunning D. W., McCall L. W., Powell W.F., Arscott W. T., McConocha E.M., McClurg C. J., Goodman S.D., Spatafora G.A. (2008). "SloR modulation of the Streptococcus mutans acid tolerance response involves the GcrR response regulator as an essential intermediary." Microbiology 154(4):1132-1143.
23 Ellwood R.P., Blinkhorn A.S., Davies R.M. (1998). "Fluoride:how to maximize the benefits and minimize the risks." Dental update 25(9):365.
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