ComD基因缺陷对变异链球菌生物膜形成的影响

目的观察变异链球菌密度感应信号系统相关基因ComD基因缺失前后变形链球菌生物膜成熟初期的变化情况,探讨ComD基因对变异链球菌形成菌斑生物膜的影响,从而为防治龋病提供实验依据和新方法。方法分别构建标准菌和缺陷菌在离体模型上的培养模型,通过扫描电镜观察两个实验组在6 h、12 h、24 h三个不同时间点的生物膜形成情况。结果在三个不同时间点观察生物膜形成,扫描电镜结果显示培养6 h和12 h两种菌株形成生物膜无明显差异,培养24 h后发现ComD缺陷菌形成的生物膜中细菌定植较少且生物膜孔隙大而疏松。结论与标准菌株相比,ComD基因缺陷的变异链球菌在成熟初期生物膜表型有明显缺陷,因此ComD基因可以作为防治龋病的靶点之一。     

替代失活法构建变形链球菌LuxS基因缺陷株

目的 LuxS基因是变形链球菌生物膜早期形成过程中的关键基因,构建该基因的缺陷菌。方法采用长臂同源多聚酶链反应（LFH-PCR）方法构建含红霉素耐药基因片段的LuxS基因上、下游同源序列的连接片段,转化到变形链球菌中,在红霉素的平板上筛选缺陷菌株,并采用PCR鉴定。结果对变形链球菌LuxS基因缺陷菌株进行PCR和DNA序列测定分析证实构建成功。结论成功构建出变形链球菌LuxS基因的缺陷菌株,为后期针对变形链球菌LuxS基因的相关研究奠定基础。     

变形链球菌UA159及其基因缺陷菌生长特性变化的初步研究

目的探讨变形链球菌密度信号系统相关基因缺陷后其生长特性的变化情况。方法分别配制BHI液体培养基,含2%葡萄糖的BHI液体培养基,含2%蔗糖的BHI液体培养基,然后将细菌接种于上述3个营养环境中生长,采用722S型可见光分光光度计,进行细菌生长曲线的测定。结果在3种不同的营养环境中,变形链球菌UA159△ComD基因缺陷菌株的生长速度最快,变形链球菌UA159菌株变形链球菌UA159△LuxS基因变异株次之,变形链球菌UA159野生菌株最慢。结论在本实验中,无论那种密度感应信号系统被阻断后,均会影响变形链球菌的生长。     

生物膜定量分析仪对不同细菌早期生物膜形成能力的比较研究

目的通过生物膜定量分析仪来观察铜绿假单胞菌(Pseudomonas aeruginosa PAO1),变形链球菌(Streptococcus mutans UA159)以及大肠埃希菌(Escherichia coli MG1655)生物膜形成能力的不同,并以各菌株的吸光度值A600为参考,对3种菌株早期生物膜形成能力进行比较。方法通过向生物膜培养悬液中加入与细菌直径相近的磁性小珠,利用这些小珠在磁场中受到生物膜的位移约束力的原理,采用生物膜定量分析仪,定量比较3种菌株在生物膜形成上的差别。结果实验发现铜绿假单胞菌PAO1和大肠埃希菌MG1655的细菌增长速度基本相同,但铜绿假单胞菌PAO1的生物膜形成明显快于大肠埃希菌MG1655。大肠埃希菌MG1655和变形链球菌UA159的生物膜形成速度基本相同,但大肠埃希菌MG1655的细菌增长速度明显高于变形链球菌UA159。结论不同细菌有各自的生物膜形成模式。生物膜定量分析仪作为一种高效简便的检测手段,可用于生物膜早期形成的动态分析。     

厚朴酚对变形链球菌生物膜致龋毒力因子作用的研究

目的 通过激光共聚焦显微镜观察厚朴酚对变形链球菌生物膜的抑菌效果,并初步了解厚朴酚对变形链球菌生物膜的产酸、耐酸、胞外多糖形成及生物膜形成能力等相关致龋毒力因子的转录表达的影响,为进一步研究厚朴酚防龋的药理作用机制奠定基础.方法 建立变形链球菌生物膜体外模型,激光共聚焦显微镜观察不同药物浓度作用后效果,并进行红绿荧光定量分析;根据GenBank基因库查询ffh、gtfD、pdp等基因序列并设计引物,进行RT-PCR.结果 CLSM观察厚朴酚作用变形链球菌生物膜后可使膜内活菌比例明显下降;RT-PCR结果表明毒力因子ffh、gtfD、pdp的表达水平受到抑制.结论 厚朴酚对变形链球菌生物膜的致龋毒力因子ffh、gtfD、pdp的转录表达有明显的抑制作用.     

唾液乳杆菌ZM06对变形链球菌生物膜形成的抑制作用

变形链球菌是导致龋齿发生最主要的细菌。本实验采用带transwell小室的细胞培养板,从九株不同种属的益生菌中,筛选能有效抑制变形链球菌生物膜形成的菌株。通过筛选获得一株唾液乳杆菌ZM06,并探究其可能的作用机制。实验表明,菌株ZM06并不能直接杀死变形链球菌,但却能抑制变形链球菌生物膜形成。通过生物膜形成实验,扫描电镜观察以及相对定量与生物膜形成直接相关的基因(包括gtf B,gtf C,ftf,gbp B,gbp C)表达证实了这一点。更重要的是,菌株ZM06降低了变形链球菌调控生物膜形成的5条信号通路中关键基因的表达。荧光定量PCR表明,菌株ZM06不仅下调了之前报道的种间群体感应系统中调控生物膜形成的lux S基因的表达,而且还对其他4条调控这些基因的信号通路中的关键基因TCS-1(vic K,vic R),TCS-2(cia H,cia R),TCS-11(hk11,rr11),TCS-13(com D,com E)表达下调。之前还没有报道称益生菌可通过这4条信号通路影响变形链球菌生物膜的形成。本研究为益生菌预防龋齿的潜能提供了理论依据。     

迟缓爱德华菌luxS基因在不同生长时期表达水平的差异

摘要:【目的】探索迟缓爱德华菌LuxS/AI-2型密度感应系统关键基因luxS的分布及在不同生长时期的表达特征和生物功能。【方法】克隆迟缓爱德华菌luxS基因,利用生物信息学工具和网络数据库分析该基因序列的特征及编码蛋白的基本特征和保守结构;原核表达LuxS蛋白,纯化后制备抗LuxS的抗体,运用Western-blot技术分析LuxS在不同毒力、不同来源迟缓爱德华菌中的分布情况及在不同生长时期的表达水平;利用抗体中和方法,分析抗LuxS抗体对迟缓爱德华菌生长的影响,探索LuxS是否为信号分子AI-2的特 异依赖模式。【结果】克隆到迟缓爱德华菌luxS基因,长度为516 bp,序列分析结果表明,该基因在迟缓爱德华菌属中高度保守;对多株迟缓爱德华菌LuxS蛋白的检测结果表明,该基因在该菌属中普遍存在;对不同生长时期LuxS蛋白的检测结果表明,LuxS蛋白的表达量在迟缓期较低,进入对数生长期逐渐增加,在对数生长后期最大,稳定后期逐渐减少;抗体中和生长试验结果表明,1%抗血清(效价1:40000)能延长迟缓爱德华菌生长的平台期,但对细菌生长无显著影响。【结论】LuxS/AI-2介导的密度感应系统在迟缓爱德华菌中普遍存在;关键基因luxS的序列高度保守,在不同生长时期表达量不一致,在对数生长后期达到峰值。     

椰子油对变形链球菌抑菌作用的体外研究

目的研究椰子油对变形链球菌的生长抑制作用,通过观察其对生物膜活性、产酸及粘附的影响,探讨其在口腔中防龋的作用。方法采用96孔微量板液体稀释法进行抑菌试验,并测得最低抑菌浓度(MIC)。体外建立变形链球菌生物膜模型,通过激光共聚焦显微镜(CLSM)扫描生物膜,观察不同浓度药物作用24 h后对生物膜活性的影响。其次测定处理后各组培养基上清液的终末pH值。最后通过玻璃棒粘附试验计算出不同浓度药物作用48 h后对生物膜粘附的影响。结果椰子油对变形链球菌的生长有抑制作用,其对变形链球菌的MIC为3.13%。CLSM观察24 h后生物膜内活菌比例逐渐下降,死菌逐渐增多。培养基上清液的终末pH值随椰子油浓度的增大而升高,且均高于阴性对照组,差异具有统计学意义(P0.05)。实验组变形链球菌的粘附率随椰子油浓度增高而降低,与阴性对照组相比差异有统计学意义(P0.05)。结论椰子油对变形链球菌有抑制作用,并能抑制其生物膜的活性、产酸及粘附等作用。     

原花青素对变形链球菌生物膜的体外抑制作用

目的分析原花青素对变形链球菌生物膜的体外抑制作用。方法采用96孔微量板液体微量稀释法进行抑菌试验,观察对变形链球菌生长的抑制情况。盖玻片上形成变形链球菌生物膜模型,用共聚焦显微镜观察原花青素和对照试剂N-乙酰半胱氨酸对变形链球菌生物膜的影响。结果原花青素对变形链球菌的抑制作用比较强。原花青素在终浓度为2~5mg/mL,N-乙酰半胱氨酸在终浓度为7.5~75mg/mL对变形链球菌生物膜均有明显的影响。N-乙酰半胱氨酸明显减少了生物膜的多糖,而原花青素明显减少了生物膜的蛋白。结论原花青素和对照药物N-乙酰半胱氨酸具有不同的抗生物膜机制,原花青素对生物膜蛋白影响更多一些,而N-乙酰半胱氨酸对生物膜多糖的影响更多一些。     

信号分子AI-2合成关键基因luxS对副溶血性弧菌生物学特性的影响

[背景]副溶血性弧菌是全球范围重要的食源性病原菌,能引起急性肠胃炎。群体感应系统LuxS/AI-2影响细菌的生物学特性,为研究副溶血性弧菌的传播机制和控制技术提供了新的途径。[目的]探讨群体感应信号分子AI-2合成关键基因luxS对海产品中分离的副溶血性弧菌Vp2009027生物学特性的影响。[方法]利用自杀质粒同源重组技术敲除信号分子AI-2合成关键基因luxS,构建副溶血性弧菌Vp2009027的luxS基因缺失株,通过比较野生株与luxS基因缺失株的生长曲线、AI-2活性、运动能力、生物膜形成能力和耐药性,分析LuxS/AI-2系统对副溶血性弧菌生物学特性的影响。[结果]构建了副溶血性弧菌Vp2009027的luxS基因缺失株,野生株和luxS基因缺失株的生长无明显差异,luxS基因的缺失导致AI-2合成受阻、运动能力和生物膜形成能力增强、四环素耐药性降低。[结论]luxS基因对副溶血性弧菌的生物学特性具有重要的调控作用,为进一步研究副溶血性弧菌的传播机制和研发控制技术提供基础。     

LuxS-based signaling affects Streptococcus mutans biofilm formation

Streptococcus mutans is implicated as a major etiological agent in human dental caries, and one of the important virulence properties of this organism is its ability to form biofilms (dental plaque) on tooth surfaces. We examined the role of autoinducer-2 (AI-2) on S. mutans biofilm formation by constructing a GS-5 luxS-null mutant. Biofilm formation by the luxS mutant in 0.5% sucrose defined medium was found to be markedly attenuated compared to the wild type. Scanning electron microscopy also revealed that biofilms of the luxS mutant formed larger clumps in sucrose medium compared to the parental strain. Therefore, the expression of glucosyltransferase genes was examined and the gtfB and gtfC genes, but not the gtfD gene, in the luxS mutant were upregulated in the mid-log growth phase. Furthermore, we developed a novel two-compartment system to monitor AI-2 production by oral streptococci and periodontopathic bacteria. The biofilm defect of the luxS mutant was complemented by strains of S. gordonii, S. sobrinus, and S. anginosus; however, it was not complemented by S. oralis, S. salivarius, or S. sanguinis. Biofilm formation by the luxS mutant was also complemented by Porphyromonas gingivalis 381 and Actinobacillus actinomycetemcomitans Y4 but not by a P. gingivalis luxS mutant. These results suggest that the regulation of the glucosyltransferase genes required for sucrose-dependent biofilm formation is regulated by AI-2. Furthermore, these results provide further confirmation of previous proposals that quorum sensing via AI-2 may play a significant role in oral biofilm formation.     

A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation

In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of S. mutans to initiate biofilm formation. To test this hypothesis, S. mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms. The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in S. mutans is important for the formation of biofilms by this gram-positive organism.     

Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS(Sm)) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.     

Fe(III) Oxide Reduction by Anaerobic Biofilm Formation-DeficientS-Ribosylhomocysteine Lyase (LuxS) Mutant of Shewanella oneidensis

Shewanella oneidensis respires a variety of terminal electron acceptors, including solid phase Fe(III) oxides. S. oneidensis transfers electrons to Fe(III) oxides via direct (outer membrane- or nanowire-localized c-type cytochromes) and indirect (electron shuttling and Fe(III) solubilization) pathways. In the present study, the influence of anaerobic biofilm formation on Fe(III) oxide reduction by S. oneidensis was determined. The gene encoding the activated methyl cycle (AMC) enzyme S-ribosylhomocysteine lyase (LuxS) was deleted in-frame to generate the corresponding mutant ΔluxS. Conventional biofilm assays and visual inspection via confocal laser scanning microscopy indicated that the wild-type strain formed anaerobic biofilms on Fe(III) oxide-coated silica surfaces, while the ΔluxS mutant was severely impaired in anaerobic biofilm formation on such surfaces. Cell-hematite attachment isotherms demonstrated that the ΔluxS mutant was also severely impaired in attachment to hematite surfaces under anaerobic conditions. The S. oneidensis ΔluxS mutant, however, reduced Fe(III) at wild-type rates during anaerobic incubation with Fe(III) oxide-coated silica surfaces or in batch cultures with Fe(III) oxide or hematite as a terminal electron acceptor. Anaerobic biofilm formation by the ΔluxS mutant was restored to wild-type rates by providing a wild-type copy of luxS in trans or by the addition of AMC or transsulfurylation pathway metabolites involved in organic sulfur metabolism. LuxS is thus required for wild-type anaerobic biofilm formation on Fe(III) oxide surfaces, yet the inability to form wild-type anaerobic biofilms on Fe(III) oxide surfaces does not alter Fe(III) oxide reduction activity.     

Novel two-component regulatory system involved in biofilm formation and acid resistance in Streptococcus mutans

The abilities of Streptococcus mutans to form biofilms and to survive acidic pH are regarded as two important virulence determinants in the pathogenesis of dental caries. Environmental stimuli are thought to regulate the expression of several genes associated with virulence factors through the activity of two-component signal transduction systems. Yet, little is known of the involvement of these systems in the physiology and pathogenicity of S. mutans. In this study, we describe a two-component regulatory system and its involvement in biofilm formation and acid resistance in S. mutans. By searching the S. mutans genome database with tblastn with the HK03 and RR03 protein sequences from S. pneumoniae as queries, we identified two genes, designated hk11 and rr11, that encode a putative histidine kinase and its cognate response regulator. To gain insight into their function, a PCR-mediated allelic-exchange mutagenesis strategy was used to create the hk11 (Em(r)) and rr11 (Em(r)) deletion mutants from S. mutans wild-type NG8 named SMHK11 and SMRR11, respectively. The mutants were examined for their growth rates, genetic competence, ability to form biofilms, and resistance to low-pH challenge. The results showed that deletion of hk11 or rr11 resulted in defects in biofilm formation and resistance to acidic pH. Both mutants formed biofilms with reduced biomass (50 to 70% of the density of the parent strain). Scanning electron microscopy revealed that the biofilms formed by the mutants had sponge-like architecture with what appeared to be large gaps that resembled water channel-like structures. The mutant biofilms were composed of longer chains of cells than those of the parent biofilm. Deletion of hk11 also resulted in greatly diminished resistance to low pH, although we did not observe the same effect when rr11 was deleted. Genetic competence was not affected in either mutant. The results suggested that the gene product of hk11 in S. mutans might act as a pH sensor that could cross talk with one or more response regulators. We conclude that the two-component signal transduction system encoded by hk11 and rr11 represents a new regulatory system involved in biofilm formation and acid resistance in S. mutans.     

Effects of oxygen on biofilm formation and the AtlA autolysin of Streptococcus mutans

The Streptococcus mutans atlA gene encodes an autolysin required for biofilm maturation and biogenesis of a normal cell surface. We found that the capacity to form biofilms by S. mutans, one of the principal causative agents of dental caries, was dramatically impaired by growth of the organism in an aerated environment and that cells exposed to oxygen displayed marked changes in surface protein profiles. Inactivation of the atlA gene alleviated repression of biofilm formation in the presence of oxygen. Also, the formation of long chains, a characteristic of AtlA-deficient strains, was less evident in cells grown with aeration. The SMu0629 gene is immediately upstream of atlA and encodes a product that contains a C-X-X-C motif, a characteristic of thiol-disulfide oxidoreductases. Inactivation of SMu0629 significantly reduced the levels of AtlA protein and led to resistance to autolysis. The SMu0629 mutant also displayed an enhanced capacity to form biofilms in the presence of oxygen compared to that of the parental strain. The expression of SMu0629 was shown to be under the control of the VicRK two-component system, which influences oxidative stress tolerance in S. mutans. Disruption of vicK also led to inhibition of processing of AtlA, and the mutant was hyperresistant to autolysis. When grown under aerobic conditions, the vicK mutant also showed significantly increased biofilm formation compared to strain UA159. This study illustrates the central role of AtlA and VicK in orchestrating growth on surfaces and envelope biogenesis in response to redox conditions.     

Effect of Homocysteine on Biofilm Formation by Mycobacteria

Mycobacteria show peculiar aggregated outgrowth like biofilm on the surface of solid or liquid media. Biofilms harbor antibiotic resistant bacteria in a self-produced extracellular matrix that signifies the bacterial fate to sedentary existence. Despite years of research, very little is known about the mechanisms that contribute to biofilm formation. LuxS has been previously known to play a role in biofilm formation in Autoinducer-2 dependent manner. We here show the effect of LuxS product-homocysteine, on the biofilm forming ability of non-tuberculous mycobacteria, Mycobacterium smegmatis and Mycobacterium bovis BCG showing AI-2 independent phenotypic effect of LuxS. Exogenous supplementation of homocysteine in the culture media leads to aberrant cording, pellicle outgrowth, and biofilm formation. Thus, our study contributes to the better understanding of the mechanism of mycobacterial biofilm formation and sheds light on the role of LuxS product homocysteine. In addition, we highlight the contribution of activated methyl cycle in bacterial quorum sensing.