【作者】 谢苗苗；

【导师】 吴补领； 闫文娟；

【作者基本信息】 南方医科大学 ， 外科学， 2013， 博士

【摘要】 龋病是人类最常见的细菌感染性疾病之一,发病率高,涉及范围广。变形链球菌(Streptococcus mutans,S. mutans)作为龋病主要的致病菌,拥有复杂的毒力因子,参与了其在牙面的黏附、聚集、菌斑生物膜的形成、产酸耐酸等作用。比较基因组学研究发现一类含有GGDEF/EAL结构域的蛋白在各类细菌中普遍存在,进一步研究发现其功能主要是参与3’-5’环二鸟苷酸(bis-(3’-5’)-cyclic dimeric guanosine monophosphate, c-di-GMP)的合成与分解,该结构域能够催化两个GTPs合成c-di-GMP。大部分的GGDEF结构域在临近N端的活性位点处含有一个Ⅰ位点,该位点可以与非竞争性抑制产物c-di-GMP结合,这种别构抑制作用能够限制c-di-GMP的浓度,阻止GTP的过度消耗。磷酸二酯酶(phosphodiesterases, PDEs)上的EAL结构域具有水解c-di-GMP的作用,其水解产物是线性的5’-磷酸鸟苷酸-(3’-5’)-鸟苷(5’-phosphoguany1y1-(3’-5’)-guanosine,5’-pGpG)分子,这些小分子物质不具备生物活性,在细胞内很快被其它的磷酸二酯酶降解。通常情况下,GGDEF和EAL结构域存在于同一个蛋白的C端,而负责信号感知及传导的结构域则位于蛋白的N端,可以感知一系列信号,例如PAS和GAF结构域,涉及到细胞内小分子的结合或者蛋白与蛋白之间的相互作用。除此之外,还有一小部分GGDEF和EAL结构域蛋白的N端整合在细胞膜上,负责感知信号,但是目前还不清楚这些结构域能够感知什么样的信号。近年来的研究已经证实,c-di-GMP是原核细胞信号转导中一类新的第二信使,是一种小分子复合物,具有热稳定性,能够激活纤维素合成酶,参与多种复杂的生理活动的调控。它的作用涉及细菌细胞的分化、生物膜的形成、细胞间的通讯、毒力因子的表达以及与宿主细胞间相互作用等多个方面,是细菌生存和代谢过程中的关键性调节因子之一。研究发现变形链球菌中存在c-di-GMP信号通路,外源性c-di-GMP的加入和内源性c-di-GMP的丧失均会降低变形链球菌的致龋特性(表现为生物膜形成量减少),但其机制尚不清楚。对这些问题进一步的研究将有助于加深对变形链球菌中c-di-GMP信号途径的作用方式的认识,也为探索新的防龋方法提供了依据。本课题组在前期研究的基础上,构建了变形链球菌内gcp基因敲除菌株,以便更全面、深入的探讨变形链球菌内c-di-GMP的作用机制和信号通路,同时与S. mutans UA159野生菌株进行基因芯片比较,筛选和生物膜有关的基因进行信号通路分析。本论文包括以下三部分内容：第一章：变形链球菌gcp基因突变株的构建本实验根据NCBI提供的S. mutans UA159全基因组序列,采用Primer Premier5.0软件(Premier公司)设计gcp基因的上游引物和下游引物,扩增gcp基因的上游片段和下游片段。采用双酶切及同源重组的方式构建了含有壮观霉素抗性(spe)的载体pFW5-gcp-UP-DOWN。在下一步实验中,采用同源重组的方式构建变形链球菌gcp基因突变菌株。用限制性内切酶Nhe Ⅰ酶(NEB公司)对载体pFW5-gcp-UP-DOWN进行酶切,将环状的质粒变为线性化的片段,胶回收酶切后的线性化片段10ug备用。取1ml变形链球菌感受态细胞菌液,加入30μl线性化的重组载体pFW5-gcp-UP-DOWN,采用同源重组的方式构建变形链球菌gcp基因突变菌株,命名为gcp-KO。通过对转化菌的形态学观察发现,转化菌形态与S. mutans UA159菌基本一致,但是细菌排列更为稀疏,这可能是由于基因缺陷后引起的生长差异所致。对重组载体进行双酶切鉴定结果显示：重组载体pFW5-gcp-UP经过Sal Ⅰ和Bgl Ⅱ双酶切后出现2条带,与质粒pFW5-gcp-UP的大小基本相同。重组载体pFW5-gcp-UP-DOWN经过Nco Ⅰ和Nde Ⅰ双酶切后出现2条带,与质粒pFW5-gcp-UP及gcp-DOWN的大小基本相同。对野生菌与转化菌的gcp基因上下游片段、spe基因进行PCR扩增及电泳鉴定结果显示：野生菌株不能扩增spe基因,而转化菌株能够扩增上、下游基因及spe基因,表明spe基因成功转入菌株基因组,测序结果也显示gcp基因被spe基因所同源替代。第二章：变形链球菌gcp基因突变菌株表达谱基因芯片研究及验证提取变形链球菌gcp-KO及野生菌株的总RNA,参照大连宝生物工程公司1st Strand cDNA Synthesis Kit试剂盒说明书分别合成cDNA。对照组(野生菌株)加入Cy5-CTP*(10mmol/L),实验组(突变菌株)加入Cy3-CTP*(10mmol/L)进行标记,并采用3S柱PCR产物纯化试剂盒纯化,得到杂交混合液。吸取适量杂交混合液,使样品液均匀的覆盖在玻片的表面,检查气泡后将基因芯片字符面朝上放入芯片夹中,将芯片夹置入基因芯片扫描仪NimbleGen MS200中,采用MS200Data Collection软件,由仪器自动平衡红绿荧光的激发光能量及增益值进行扫描,获得双色叠加的杂交结果图,保存该结果。本实验为了确定差异基因表达谱,采用国内外基因芯片实验常用标准Ratio’=2做为差异基因的初步筛选标准。上调基因筛选的转录差异要在2倍以上,即FC≥2。同样下调基因筛选的转录差异也是2倍以上,即FC≤0.5。转录差异在0.5≤FC≤2之间的基因,被认为没有差别,不筛选进入下一步研究,最终有411个基因符合条件。分别比较两组数据中Cy5荧光信号强度和Cy3荧光信号强度,对于同一个探针,如果Cy5信号强度是Cy3的2倍或2倍以上,认为该探针所代表的基因表达降低；如果Cy3的信号强度是Cy5的2倍或2倍以上,说明该探针代表的基因表达升高。初步筛选411个差异基因,表达上调的基因198个,表达下调的基因213个。再结合GO分析、COG分析及KEGG分析的结果,最终确定8个基因进入本课题的Real-time PCR验证研究,其中上调基因和下调基因各4个。表达差异的基因按照功能进行归类,查阅GenBank数据库及相关文献对其结果进行深入的分析及讨论。结合前期实验的结果,选择RgpAc基因作为后续研究的目的基因。第三章：RgpAc基因突变株的构建及功能研究本章主要包括两方面的内容：1.RgpAc基因突变株的构建本实验根据NCBI提供的S. mutans UA159全基因组序列,采用Primer Premier5.0软件设计RgpAc基因的上游引物和下游引物,扩增RgpAc基因的上游片段和下游片段。采用双酶切及同源重组的方式构建了含有壮观霉素抗性(spe)的载体pFW5-RgpAc-UP-DOWN。将载体加入变形链球菌感受态细胞中,采用同源重组的方式构建变形链球菌RgpAc基因突变菌株,命名为RgpAc-KO。通过对转化菌的形态学观察发现,转化菌形态与S. mutans UA159菌基本一致,但是细菌排列更为稀疏,这可能是由于基因缺陷后引起的生长差异所致。实验还对野生菌与转化菌的RgpAc基因及其上下游、spe基因进行了PCR扩增与电泳鉴定。结果显示,野生菌株含有RgpAc基因,不含有spe基因。而转化菌株能够扩增载体的上、下游基因及spe基因,不能扩增RgpAc基因,表明转化菌株有RgpAc基因缺陷。测序结果也显示RgpAc基因被spe基因所同源替代。2.变形链球菌RgpAc基因功能的研究采用SPSS13.0统计学软件进行统计学处理,对比了野生菌株和突变菌株在24h、48h形成的水溶性葡聚糖、水不溶性葡聚糖和胞外多糖的含量,以及野生菌株和突变菌株加入不同浓度的c-di-GMP之后,形成的生物膜含量。采用析因设计的方差分析的方法,各组计量资料数据采用均数±标准差的方式列出,检验水准a=0.05。通过改良蒽酮比色法比较突变菌株和野生菌株胞外多糖的合成能力。实验分为两组,分别为野生菌株和突变菌株,24h的样本量为8例,48h的样本量为5例。野生菌株水溶性葡聚糖和胞外多糖生成量高于突变菌株,差异有统计学意义(F=328.868, P<0.001; F=80.897, P<0.001),突变菌株水不溶性葡聚糖生成量高于野生菌株,差异有统计学意义(F=15.718,P<0.001)。同一菌株不同的时间段相比,野生菌株24h和48h产生的水溶性葡聚糖、水不溶性葡聚糖和胞外多糖差异均有统计学意义(F=36.022,P<0.001；F=15.274, P<0.001; F=-18.241, P<0.001),24h处产生的水溶性葡聚糖高于48h处,水不溶性葡聚糖低于48h处,胞外多糖高于48h处。突变菌株24h和48h产生的水溶性葡聚糖、水不溶性葡聚糖和胞外多糖差异均有统计学意义(F=-7.537, P<0.001; F=18.762, P<0.001; F=-8.587, P<0.001),24h产生的水溶性葡聚糖高于48h处,水不溶性葡聚糖低于48h处,胞外多糖低于48h处。在比较生物膜形成量的实验中,S.mutans UA159与RgpAc-KO加入200μM和400μM的c-di-GMP后,设计了6组,每组有5例,采用析因分析结果发现,两组菌株之间的差异有统计学意义(F=29.411,P<0.001),突变菌株高于野生菌株；三个c-di-GMP浓度间的差异有统计学意义(F=3.945, P=0.033),400μM组最高,空白对照组最低；在不加c-di-GMP干扰的时候,野生菌株和突变菌株之间的差异有统计学意义(t=8.196,P<0.001),野生菌株更高；在加入200μM、400μM的c-di-GMP干扰后,野生菌株和突变菌株之间的差异有统计学意义(t=-4.467, P=0.002;t=-8.038, P<0.001),突变菌株均高于野生菌株；野生菌株加入不同浓度的c-di-GMP干扰后,产生的差异有统计学意义(F=24.766,P<0.001),不加c-di-GMP组最高,加入400μM的c-di-GMP组最低；突变菌株加入不同浓度c-di-GMP干扰后,产生的差异有统计学意义(F=48.322,P<0.001),不加c-di-GMP组最低,加400μM的c-di-GMP组最高;交互效应有统计学意义(F=47.529,P<0.001)。实验结果表明变形链球菌RgpAc基因敲除后,细菌形成生物膜的能力被抑制。在变形链球菌中,各种水溶性和水不溶性的多糖是生物膜形成的重要基质之一。当变形链球菌的RgpAc基因敲除后,合成水溶性葡聚糖和胞外多糖的能力均下降,导致生物膜形成的基质缺乏,最终抑制了生物膜的形成。突变菌株加入不同浓度的c-di-GMP之后,生物膜形成能力增加,推测原因是外源性的c-di-GMP增加后促进了其与下游信号通路受体的结合,最终使生物膜的形成量增加。为了比较两种细菌在牙齿表面形成的生物膜结构的差异,实验模拟体内情况在釉质表面形成人工生物膜,并进行了扫描电镜观察。结果显示,RgpAc-KO与野生菌株培养48h后,产生的生物膜形态不相同。低倍镜下可见S. mutansUA159组产生的生物膜较厚,排列较致密,RgpAc-KO组的生物膜结构较松散,突变菌株分别加入200μM和400μM的c-di-GMP之后,细胞外基质增加。高倍视野下可见S. mutans UA159组细菌之间有较多的细胞外基质,生物膜上的细菌排列规律,形成漩涡状结构；RgpAc-KO组细菌之间的细胞外基质较少,视野内可见少量的细菌。突变菌株分别加入200μM和400μM的c-di-GMP之后,细菌生物膜的结构变致密,细胞数目增多。提示RgpAc基因突变后降低了水溶性葡聚糖的合成,而水溶性葡聚糖既可作为细菌胞外能源物质及代谢底物,又可与细菌表面的Gbp结合,能够促进黏附和聚集。因此在电镜下观察突变菌株的生物膜结构较松散,细胞外基质较少,视野内细菌数量少。加入外源性c-di-GMP后增加了下游信号通路的活性,最终使生物膜的形成增加。综上,本实验通过同源重组的方法构建了变形链球菌gcp基因突变菌株,与野生菌株进行了基因芯片比较,表达差异在2倍以上的基因有411个,包括198个表达上调的基因和213个表达下调的基因。通过查阅资料,最终挑选8个最有可能与变形链球菌生物膜形成及信号通路有关的基因进行验证,发现RgpAc基因表达抑制的程度最大,因此课题组选择该基因作为下一步研究的目的基因,构建了变形链球菌RgpAc基因突变菌株。利用比色、电镜扫描等实验方法初步研究了该基因缺失对变形链球菌生物学特性的影响,为龋病防治提供了新的思路和新的方法。更多还原

【Abstract】 Dental caries is one of the most common chronic diseases afflicting humans and has a polymicrobial etiology. Streptococcus mutans (S. mutans) is one of the human oral caries bacteria, as well as the prime pathogen agents in the development of dental caries. An important virulence property of S. mutans is to form dental plaque biofilm on the tooth surface. Bacteria in the biofilm grow, reproduce, metabolize and constitute a special micro ecological environment.Comparative genomic study found a class of GGDEF/EAL domain protein widespread in various types of bacteria. Further studies showed that its main function is about synthesis and degradation of cyclic di(3’→5’)-guanylic acid (c-di-GMP). The GGDEF/EAL domain could catalyze GTPs to c-di-GMP. The GGDEF and EAL domain families have1,601and1,016members, respectively, in the Pfam protein family database in July2005. Proteins containing those domains are involved in motility and exopolysaccharide-biofilm production in a variety of bacteria. Some GGDEF-domain proteins control biofilm formation and/or cell aggregation by enhancing the levels of the novel second messenger c-di-GMP. Intracellular concentrations of c-di-GMP are regulated by multiple domain proteins. C-di-GMP is synthesized from GTP by diguanylate cyclases (DGCs) proteins containing the amino acid motif GGDEF domain. C-di-GMP is degraded by phosphodiesterases (PDEs) that contain one of two conserved domain families:one defined by the EAL motif and the other by an HD-GYP motif. C-di-GMP plays an important role in the physiology of many bacteria.Recent studies show that c-di-GMP is one of the second messager signaling molecules. It could regulate many processes to adapt to environment in bacteria. It involves in the biofilm formation, virulence, communication and the interaction between the host cells and bacteria. It is one of the key regulatory factors in bacterial survival and metabolism.There is c-di-GMP signaling pathway in Streptococcus mutans. Adding exogenous c-di-GMP or reducing endogenous c-di-GMP will inhibit the biofilm formation of Streptococcus mutans. But the mechanisms are unknown. Further research on these issues will help to deepen the role of c-di-GMP signaling pathways in Streptococcus mutans and explore a new method of anticaries. Construct gcp gene mutation strains of Streptococcus mutans. Then extract the RNA of mutation strain and wild strain. Compare the differences by microarray chip and screen the biofilm-related genes to analysis the signaling passways.This paper includes the following three chapters:Chapter I:Constructed the gcp gene mutation strain of S. mutans UA159Designed the upstream primers and downstream primers of gcp gene by Primer Premeier5.0software according to the whole genome sequence of S. mutans UA159in NCBI. The upstream fragment and downstream fragment were amplified. Constructed a vector containing spectinomycin resistance (spe+) pFW5-gcp-UP-DOWN. Constructed S. mutans UA159gcp gene mutation strain which was named gcp-KO by homologous recombination. Under an optical microscope, S. mutans UA159was G+, arranged in chains with different lengths. The gcp-KO strain was G+too, arranged in short chains and more sparse arrangement compared to the wild strain. This might be due to genetic defects. Gel electrophoresis was used to identify the upstream and downstream of gcp gene and the spectinomycin gene in wild strain and mutation strain. The results showed that the wild strain didn’t contain the spectinomycin gene but contain the gcp gene. The mutation strain could amplify upstream and downstream of gcp gene and amplify the spectinomycin gene, but it couldn’t amplify the gcp gene. Real-time PCR showed gcp gene a down-regulation of the gcp-KO strain. All the results showed the mutation strain was gcp gene defect strain. The sequencing results also showed that the gcp gene was replaced by the spectinomycin gene.Chapter Ⅱ:Microarray chip experiments in gcp-KO strain and wild strain of S. mutans UA159Extracted the RNA of wild strain and gcp-KO strain. Then synthesized "cDNA" by the instructions of "TaKaRa1st Strand cDNA Synthesis Kit" and marked the control group by Cy5-CTP and experimental group by Cy3-CTP. Immediately absorbed the appropriate amount of hybridization mixture on the slide and put the slide into NimbleGen MS200gene chip scanner. Laboratory assistant collected the data with MS200software.Compared the Cy5fluorescence signal strength and Cy3signal strength of the two groups and got the ratio by Cy5than Cy3. For the same probe, if the ratio was2times or more than2times, gene expression of this probe was downregulated. If the ratio was0.5times or less than0.5times, gene expression of this probe was upregulated. Finally got411genes with198gene upregulated and213gene down regulated. After GO analysis, COG analysis and KEGG analysis, choose8genes for Real-time PCR. Think about the previous experiments results, RgpAc gene is selected as the target gene in the following studies.Chapter III Construction and function studies of RgpAc mutation strainThis chapter includes two aspects:"Construction of RgpAc gene mutation strain" and "Function studies of RgpAc gene mutation strain"1. Construction of RgpAc gene mutation strainDesigned the upstream primers and downstream primers of RgpAc gene by Primer Premeier5.0software according to the whole genome sequence of S. mutans UA159in NCBI. The upstream fragment and downstream fragment were amplified. Then constructed a suicide vector containing spectinomycin resistance (spe+) pFW5-RgpAc-UP-DOWN. Finally construct S. mutans UA159RgpAc gene mutation strain which was named RgpAc-KO by homologous recombination. Gel electrophoresis was used to identify the upstream and downstream of RgpAc gene and the spectinomycin gene in wild strain and mutation strain. The experimental results showed that the wild strain contained the RgpAc gene, not the spectinomycin gene. The mutation strain could amplify upstream and downstream of RgpAc gene and the spectinomycin gene, but it couldn’t amplify the RgpAc gene. Real-time PCR showed RgpAc gene downregulation in the RgpAc-KO strain. All the results showed the mutation strain was RgpAc gene defect strain.2. Function studies of RgpAc gene mutation strainThis study compared the extracellular polysaccharide synthesis capacity of RgpAc gene mutation strain and wild strain by modified anthrone colorimetry. SPSS13.0statistical software was used to analysis the results. The formation of water-soluble glucan of RgpAc-KO strain decreased by55%, exopolysaccharide by41%. They were significantly lower than wild strain. But there was no significant difference in the ability to form water-insoluble glucan between RgpAc-KO strain and wild strain. The RgpAc gene was glycosyltransferase in S. mutans UA159. Glycosyltransferases catalyzed the transfer of sugar moieties to specific acceptor molecules and formed glycosidic bonds. This glycosyltransferase mainly relates to water-soluble dextran a-1,6. When RgpAc gene was knocked out, the ability of synthetic dextran a-1,6was inhibited and water-soluble glucan was decreased.Designed the experiment about biofilm formation in different strains. RgpAc-KO strain was experimental group and wild strain was control group. Then added200μM and400μM c-di-GMP into wild strain and RgpAc-KO strain. Both strains grown aerobically at37℃in a5%CO2atmosphere under static conditions for48h. Measured the absorbance value at575nm. The biofilm formation of S. mutans UA159was inhibited after adding c-di-GMP to the culture. However, no major differences were observed between different concentrations of c-di-GMP. The results showed that the RgpAc-KO strain was less efficient55%at forming biofilm than wild strain. The biofilm formation of RgpAc-KO strain was promoted after adding c-di-GMP to the culture. In this study, the biofilm formation of RgpAc-KO was decreased. However, the biofilm formation of RgpAc-KO strain with200μM c-di-GMP and400μM c-di-GMP was increased. Water-soluble and water-insoluble glucan are the biofilm matrix. The ability of the water-soluble glucan decreased after RgpAc gene knocked out. The biofilm formation was inhibited because lack of matrix. When extracellular c-di-GMP was added in the RgpAc-KO strain, it increased the binding of other receptors and promoted the biofilm formation.To observe the biofilm structure of RgpAc-KO strain with the c-di-GMP, prepared the enamel pieces and simulated the vivo situation to form artificial biofilm on the enamel pieces. Then biofilm structure was detected by a combination of scanning electron microscopy. Under low magnification, thick and compact biofilm was observed of sild strain, RgpAc-KO strain with200μM c-di-GMP and RgpAc-KO strain with400μM c-di-GMP. However, thin and loose biofilm was observed of RgpAc-KO strain. Under high magnification, cells with large extracellular matrix arranged regularly of sild strain, RgpAc-KO strain with200μM c-di-GMP and RgpAc-KO strain with400μM c-di-GMP. Cells with little extracellular matrix arranged irregularly of RgpAc-KO strain. RgpAc-KO strain reduced the synthesis of polysaccharide, especially glucan. Glucan and other polysaccharide were energy substances and metabolic substrates, as well as promoted bacteria adhesion and aggregation. When extracellular c-di-GMP was added in the RgpAc-KO strain, it played a role of endogenous second message and finally increased the biofilm formation. But RgpAc-KO strain reduced the addition of extracellular polysaccharide and suppressed the ability of biofilm synthesis. Under scanning electron microscope, loose extracellular matrix and less bacteria were observed.In conclusion, this study constructs gcp gene knockout strain of Streptococcus mutans UA159by homologous recombination. Microarray analysis finds the RgpAc gene is upregulation. Then construct RgpAc gene knockout strain by the same way and design studies about its functions. The results will provide new methods and ideas for caries prevention.更多还原