| BackgroundsDental caries is a common oral infectious disease that affect people's health.One of the dominant 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。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. However, its mechanism is still not clearly explained.In 1953 DNA double helix Structure had been diseovered and in 1985 Crick told the genetic information Transmitted to the ceniral dogma, the life seienees have been a great develoPment, genechiP teehnology is the whole—genome analysis of a Power fulteehnology Platform, is a gene sequence and function of large-scale, high-throughput research methods, be able to parallel analysis of a large number of genes, a comprehensive understanding of the various changes in gene expression, thus the rapid access to rich comprehensive biological information, promoting the development of genomics.cDNA microarray is a specific probe thousands of genes or their cDNA fragments fixed to a DNA chip, right from different individuals, organization, cell cycle, developmental stage, differentiation stage,Lesions, stimulation of the cells to detect mRNA expression levels of total and thus the expression of these genes Individual-specific, tissue-specific, developmental stage-specific, differentiation stage-specific, disease-specific, Stimulus-specific conduct comprehensive analysis and judgments could be further studied the interaction between genes and gene Guan Department to provide information on cell expression regulation.Our previous studies have confirmed the existence of S. mutans within the c-di-GMP signaling pathway, the pathway mediated by Streptococcus mutans biofilm formation and in vitro adhesion to enamel surface, exogenous c-di-GMP has reduce Streptococcus mutans biofilm formation, acid, acid, in vitro adhesion and other characteristics. But c-di-GMP regulation of the molecular mechanisms of S. mutans are unknown. S.mutans UA159 strain genome sequencing was completed in 2002. S.mutans genome including the 2030 936 bp, a 1963 open reading frames (open reading frame, ORF), of which 63% of known function,21% of the ORF and the positioning of other bacteria similar but unknown function,16% of ORF is S.mutans unique. The emergence of microarray-based issues and the mechanisms to provide the foundation. In this study, gene chip technology is intended to investigate the c-di-GMP affect the biological properties of Streptococcus mutans molecular ObjectivesUsing gene chips c-di-GMP effects on gene expression profiles of Streptococcus mutans to screen differentially expressed genes. Of c-di-GMP how to participate in a variety of biological characteristics of S. mutans in order to provide research for the efficient and effective clinical method for the theoretical basis for prevention of dental caries.Methods1. S. mutans UA159 was inoculated BHI solid medium,37℃anaerobic culture (80% N2,10% H2, and 10% CO2) 48 hours, pick a single colony was inoculated in freshly prepared BHI broth the monoclonal culture,37℃anaerobic incubation for 24 hours.2。Take the appropriate overnight culture were inoculated to freshly prepared BHI broth, the experimental group by adding appropriate amount c-di-GMP, a final concentration of 200μM; control group, equal volume of normal saline,37℃8 hours of anaerobic incubation. According to the German company Qiagen RNA extraction kit instructions, were extracted from the experimental group and control group of the total bacterial RNA. DU530 biochemical analyzer of total RNA quality and concentration.3. Using reverse transcription kit to test group and control group, the total RNA reverse transcription into cDNA, purified, and the cy3 or cy5 tags. Purification of labeled products, with DU530 biochemical analyzer for quality and concentration, and then with the whole genome microarray. Bioinformatic analysis using c-di-GMP S. mutans UA159 cells before and after changes in gene expression profiles.4. Using real time quantitative PCR method for screening differentially expressed genes microarray validation, select the part of the regulated genes and downregulated genes. RNA extraction and reverse transcription and microarray experiments in the same way, in accordance with the RT-PCR kit (Invitrogen) instructions for reaction system and temperature settings.Results:1. Culture and identification of Streptococcus mutansStreptococcus mutans in BHI agar plate was gray, translucent, smooth, neat edge, 0.5-0.75 mm in diameter, circular protrusions of small colonies. Light microscope for the G+Streptococcus, spherical or oval-shaped, diameter 0.6-1.0μm, were arranged in chains of different lengths, arranged in short chains or dispersions. Rapid trace detection of its biochemical plate can be decomposed mannitol, sorbitol, raffinose, and arginine on Aescin glycosides and no degradation.2. RNA quality controlThe total group of bacteria RNA, in the Beckman DU530 company biochemical analyzer detected in the control group RNA concentration was 223.5μg/ml, OD260/OD280=1.90; treatment RNA concentration was 203.4μg/ml, OD260/OD280=1.96. Gel electrophoresis analysis showed that 23S,16S and 5S in the three bands, in the three-band complex, with or without diffuse tail,23 S RNA 16S band is about twice the brightness, that is more explicitly mentioned in the total RNA integrity, no obvious degradation and no DNA contamination.3. Microarray hybridizationThe gene chip experiment we use a common standard Ratio'=2 and Ratio'=0.5 different genes as the standard,. Chip detected, c-di-gmp gene expression changes before and after treatment are:9 upregulated genes; six genes were downregulated. The data using online tools to GO (http://www.geneontology.org/) and PANTHER (http://www.pantherdb.org/) analysis results show that the main difference between gene and cell tropism and biofilm formation signal transduction, carbohydrate metabolism, nucleic acid metabolism, such as those pathway. 4. Real-time quantitative PCR validation resultsExperiment reflects the upregulation of expression and down, and in the cDNA microarray analysis of differential expression in much the same; can be seen, cDNA microarray analysis of the data is credible.Conclusions:1. Through the bacterial count at different times to determine the c-di-GMP on the rate of growth of Streptococcus mutans, compared with the control group, no significant difference.2. Using cDNA microarray analysis of c-di-GMP effects on gene expression of Streptococcus mutans changes in 9 genes (glgA,glgB,msmF,gftA,lacG,lepB,rgpAc,bacC,apt) expression,6 genes (Hrc,ProC,Hpr,Pdp,Glk,cdsA)was decreased. These differences are mainly Streptococcus mutans genes of energy metabolism, biofilm formation, and adhesion to the tooth surface, the relevant biological characteristics.3 gene glgA, glgB (glycogen synthesis in bacteria in vivo), and its expression in bacteria will increase the excess carbon stored glycogen synthesis, leading to the survival of S. mutans is an important material to reduce the energy needed, which led to Streptococcus mutans decreased pathogenicity. msmf upregulation can reduce the energy consumption of bacteria in other areas, thus indirectly affecting the cariogenic Streptococcus mutans. Gene cdsa (glycerol phospholipid synthesis) reduced expression, the reduced synthesis of glycerol phospholipids, thus affecting the surface of Streptococcus mutans biofilm formation in the amount of organisms, and then to reduce the S. mutans cariogenic properties. Gene pdp (aminmino acid metabolism) down-regulated, the lower the decomposition of cytosine, which affects the conversion of intracellular material in turn affect the vitality of Streptococcus mutans... |
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