| Background and objectiveDental caries is one of the most common diseases that affects human oral health and Streptococcus mutans (S. mutans) is one of the primary aetiological agent of dental caries. The pathogenicity of S. mutans relies on the bacterium’s ability to produce organic acids, survive a strongly acidic environment, colonize tooth surfaces and form large amounts of biofilms and extracellular polysaccharides (EPS) by the metabolism of a wide variety of carbohydrates.According to the analysis of whole-genome sequencing of 5. mutans UA159, about 15% of the whole genome is related to carbohydrate metabolism and S. mutans presents a powerful ability to metabolize carbohydrate. Although S. mutans could metabolize carbohydrate in a variety of ways, the dominant, high-affinity, high-capacity mechanism to transport carbohydrates in S. mutans is the phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS).The PTS is usually composed of the non-substrate-specific permeases-enzyme I (EⅠ) and histidine-containing phosphocarrier protein (HPr), and a series of substrate-specific permeases, known as enzyme Ⅱ (EⅡ) complexes, which are directly responsible for the transportation and phosphorylation of the substrate. In most cases, the EⅡ complexes are comprised of three functional domains, A, B, and C. Since PTS plays an important part in carbohydrate transport and metabolism, it attracts lots of domestic and international attention. To date, a number of sugar-specific PTS of S. mutans have been further studied, such as glucose, sucrose, fructose, lactose, maltose, sorbitol, mannitol, etc. Howerer, the study of L-ascorbate-specific PTS of S. mutans is little.The ptxA gene(NCBI GeneID:1027857) and ptxB gene(NCBI GeneID:1027854) encode putative enzyme IIA and enzyme IIB of the L-ascorbate-specific PTS of S. mutans UA159. Some scholars had analyzed the three-dimensional crystal structure of PtxA and PtxB proteins, and performed enzyme activity assay in vitro. Their research showed that PtxA and PtxB proteins were specific for the phosphorylation of L-ascorbate and related to the transport of L-ascorbate in S. mutans.L-ascorbate is abundant in the nature, especially in fruits and vegetables. Some previous studies revealed that some enteric bacteria can ferment and oxidize L-ascorbate under anaerobic conditions, such as Escherichia coli, Lactobacillus and Pneumobacillus. Our previous studies found that S. mutans can obtain energy by fermenting L-ascorbate in an anaerobic atmosphere as well. And deletion of the ptxA gene impaired the L-ascorbate metabolism in S. mutans and influenced its physiology and virulence, including the growth rate, the capacity of acidogenesis and formation of biofilm when using L-ascorbate as the sole carbon source.Since ptxA gene plays an important role in L-ascorbate metabolism, in order to analyze the function of ptxB gene and understand the relation between ptxA gene and ptxB gene, in the present study,ptxB-deletion mutant, ptxA-, ptxB-double deletion mutant and their complemented strains were constructed. The biological characteristics of these strains involved in cariogenicity, such as growth, acidogenesis, aciduricity, biofilm and EPS formation were studied. In addition, PCR and quantitative real-time PCR were performed to characterize the mechanism regulating expression of the ptxA,ptxB and their adjacent genes.Chapter 1 Construction of ptxA-, ptxB-deletion mutants and their complemented strainsObjective:This chapter aims to construct ptxB-deletion mutant, ptxA-, ptxB-double deletion mutant,ptxA-complemented strain,ptxB-complemented strain and ptxAB-complemented strain, laying the foundation for further study.Methods:The upstream and downstream primers were designed with Primer Premier 5.0 software according to the whole genome sequence of S. mutans UA159. The 5’and 3’ regions flanking the target gene were amplified by polymerase chain reaction (PCR). Following proper restriction enzyme digestions, the flanking regions were cloned into two multiple cloning sites of plasmid pFW5 to generate recombinant plasmid. Subsequently, the recombinant plasmid was used to transform the wild-type strain UA159 in the presence of competence-stimulating peptide (CSP) to construct mutant strain. A similar technique was used to construct complemented strain. Plasmid pDL278 was used as the vector to construct complemented plasmid and the complemented plasmid was used for transformation of the mutant strain, generating complemented strain.Results:After PCR identification and sequence confirmation, ptxB-deletion mutant (ptxB-), ptxA-, ptxB-double deletion mutant (ptxAB-), ptxA-complemented strain (CptxA-),ptxB-complemented strain (CptxB-) and ptxB-complemented strain (CptxAB-) were constructed successfully.Chaper 2 Effect of ptxA gene and ptxB gene on biological characteristics of S. mutansObjective:In order to analyze the function of ptxA gene and ptxB gene, the biological characteristics of S. mutans UA159, strain ptxA-, strain ptxB-, strain ptxAB-, strain CptxA-, strain CptxB-and strain CptxAB-involved in cariogenicity, such as growth, acidogenesis, aciduricity, biofilm and EPS formation were studied.Methods:1. Tryptone-vitamin (TV) base medium supplemented with 15mmol/L L-ascorbate was used as the L-ascorbate-specific medium to provide carbon source for S. mutans.2. To measure the growth rates of S. mutans when using L-ascorbate as the sole carbon source, wild-type strain UA159, strain ptxA-, strain ptxB-, strain ptxAB-, strain CptxA-, strain CptxB- and strain CptxAB- were inoculated into fresh L-ascorbate-specific medium and grown at 37℃ for 72 h under anaerobic conditions. Data for plotting growth curves were collected by measuring changes in OD600 at 2 h intervals using a spectrophotometer over a total period of 72 h.3. Acid production assay. Wild-type S. mutans UA159 and its derivatives were inoculated into fresh L-ascorbate-specific medium or fresh BHI medium and then incubated at 37℃ in an anaerobic atmosphere for 48 h. The pH of the supernatant in the media was measured at the beginning, and after 24 h or 48 h of incubation. The acidogenesis ability was calculated as the difference in pH values measured at specific incubation times (ΔpH).4. Acid killing assay. Wild-type S. mutans UA159 and its derivatives were grown in L-ascorbate-specific medium until OD600≈0.3, harvested by centrifugation, and then the cell pellets were resuspended in fresh L-ascorbate-specific medium that was adjusted to pH 5.0 with HC1 to undergo an adaptive acid tolerance response. Following an additional hour of incubation, cells were harvested, washed with 0.1 mol/L glycine, pH 7.0, and subjected to acid killing by incubating the strains in 0.1 mol/L glycine, pH 2.8, for 0,15,30, and 45 min. The surviving cells were appropriately diluted, plated on BHI agar, and incubated in an anaerobic atmosphere at 37℃ for 48 h.5. Biofilm formation analysis. S. mutans UA159 and its derivatives were inoculated into fresh L-ascorbate-specific medium and dispense into 6-well plates with coverslips in each well. After 120 h of 37℃ anaerobic incubation, the formed biofilms were washed, dried and stained with SYTO9. Confocal laser scanning fluorescence microscope was used to examine. At least five independent fields were collected. Image J was used to calculate the area that the biofilms covered.6. EPS formation analysis. S. mutans UA159 and its derivatives were inoculated into fresh L-ascorbate-specific medium and dispense into 6-well plates with coverslips in each well. After 120 h of 37℃ anaerobic incubation, the formed EPS were washed, dried and stained with Calcofluor White. Confocal laser scanning fluorescence microscope was used to examine.7. Statistical Analysis. One-way ANOVA test was used to analysis the result of acid production assay by SPSS 20.0 software and a P value< 0.05 indicated statistically significant differences.Results:1. The growth capacity of the deficient mutants decreased compared with that of the wild-type S. mutans UA159. Strain ptxA-had an extended lag phase and decreased growth yield. Strain ptxB-and strain ptxAB-could hardly grow in the 72 h of incubation. In addition, strain CptxA-and strain CptxAB-restored the wild-type phenotype to some extent, but still could not reach the wild-type level. However, strain CptxB-could not restore the growth.2. The wild-type UA159 and its derivatives grew well and acidified the medium to about the same terminal pH in BHI medium. However, in the case of the L-ascorbate-specific medium, the situation is different. After 24 h of incubation, the terminal pH slightly decreased for wild-type UA159. The three mutants lowered the pH to a level significantly lower than that observed in wild-type UA159 (P< 0.05). Furthermore, complemented strains recovered their acid production capacity, with the exception of strain CptxB-. After 48 h of incubation, all strains had produced more acid, the ApH of the medium was greater than it was at 24 h and the differences among wild-type strain and mutant strains were more significant (P< 0.01).3. After an incubation in L-ascorbate-specific medium with a pH of 5.0 for 1 h to induce an adaptive acid tolerance response and a subjection to acid killing with a low-pH buffer (pH 2.8), none of the mutant strains formed colonies on the assay plates.4. From the results of confocal laser scanning fluorescence microscopy analysis, biofilms stained with the fluorescent dye SYTO9 appeared green. Wild-type UA159 formed large microcolonies and covered 65.93% of the surface. However, the biofilms that strain ptxA-formed were sparser and much thinner than UA159. It covered only 39.61% of the surface, but it still could form network structure. Strain ptxB-and strain ptxAB-formed much less prolific biofilms in which cells were scattered on the surface as chains and the biofilms were too thin to form three-dimensional structure. They covered only 24.24% and 18.57% of the surface respectively. Complementation in strain CptxA-and strain CptxAB-restored biofilms formation to a level similar to that of wild-type UA159. However, strain CptxB-could not restore the wild-type phenotype.5. EPS stained with Calcofluor White appeared blue under confocal laser scanning fluorescence microscope. The situation of EPS formation was similar with that of biofilm formation. The distribution of biofilms was consistent with the distribution of EPS. Compared with wild-type UA159, the mutant strains formed much less EPS.Conclusions:1. ptxA and ptxB genes are associated with the transport and metabolism of L-ascorbate in S. mutans.2. Deletions of the ptxA and ptxB genes impaire the ability of S. mutans in growth, acidogenesis, aciduricity, biofilm and EPS formation.Chapter 3 Transcriptional analysis of ptxA, ptxB and their adjacent genes in S. mutansObjective:In order to explore whether there is an operon associated with L-ascorbate transport and metabolism exist in S. mutans, transcriptional analysis of ptxA,ptxB and their adjacent genes were performed.Methods:1. PCR analysis of ptxA, ptxB and their adjacent genes. Genome DNA and total RNA of S. mutans were extracted and purified, total RNA was reversed into cDNA. PCR was performed on cDNA templates with specific primers that span the sequences SMU.268 to sgaT (a), sgaT to ptxB (b), ptxB to ptxA (c),ptxA to SMU.273 (d), SMU.273 to SMU.274 (e), SMU.274 to SMU.275 (f) and SMU.275 to SMU.277 (g), using DNA of S. mutans UA159 as a positive control. Agarose gel electrophoresis was used to observe the bands.2. To evaluate the expression of ptxA, ptxB and their adjacent genes under the influence of L-ascorbate, S. mutans UA159 was incubated in L-ascorbate-specific medium or glucose -specific medium until late-exponential phase, total RNA were extracted and cDNA were synthesized by reverse transcription. qRT-PCR was performed to analyze the mRNA expression of ptxA, ptxB and their adjacent genes using 16S rRNA as a reference.3. Expression of ptxA gene in wild-type UA159 and strain ptxB-were evaluated by qRT-PCR.4. Statistical Analysis. Independent-samples t-test was used to analysis the results of qRT-PCR by SPSS 20.0 software and a P value< 0.05 indicated statistically significant differences.Results:1. Transcriptional analysis using cDNA templates and primers that spanned the adjacent genes showed amplified bands in b, c, d, e and f regions indicating that ptxA,ptxB, sgaT, SMU.273, SMU.274 and SMU.275 are parts of the same operon. However, SMU.268 and SMU.277 are not parts of it.2. Quantitative real-time PCR demonstrated that, compared with the gene expression in cells grown in medium containing glucose, the transcription level of these genes in cells grown in the presence of L-ascorbate were all elevated significantly (P< 0.01).3. Once ptxB gene was knockout, the expression of ptxA gene decreased significantly (P< 0.01) compared with wild-type UA159.Conclusions:1. ptxA, ptxB, and the upstream gene sgaT, the downstream genes SMU.273, SMU.274 and SMU.275 are parts of the same operon, and L-ascorbate is a potential inducer of the operon.2. The expression of ptxA gene is regulated by upstream gene ptxB. |
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