The Study Of The Function Of PtxA Gene In Phosphotransferase System From Streptococcus Mutans

Background and objectiveStreptococcus mutans (S. mutans) is one of the major causative agent of human dental caries. Its strong cariogenicity is closely related to its ability to acid production, acid resistance and adhesion to the tooth surface. The genome of S.mutans UA159 has been completely sequenced in 2002 and genes associated with carbohydrate metabolism account for almost 15% of the whole genome. Comparison of genomes of several other Gram-positive organisms sequenced to date showed that S. mutans has stronger ability to metabolize carbohydrate.The dominant pathway to transport and concomitantly phosphorylate carbooydrateds in S. mutans is the phosphoenolpyruvate (PEP)-dependent: phosphotransferase systems (PTS). At present, more than 14 unique PTS permeases, including glucose, sucrose, lactose, mannose and so on, have been found in the S. mutans UA159. The PTS is usually composed of three parts:enzyme Ⅰ (EⅠ), HPr and enzyme Ⅱ complex (EⅡ). E Ⅰ and HPr are non-substrate-specific permeases and participate in the transfer of PEP to EⅡ complex. EⅡ complexes which generally consist of three proteins:ⅡA, ⅡB and ⅡC, are substrate-specific and directly responsible for the transportation and concomitant phosphorylation of the substrate. Althouth S. mutans can metabolize carbohydrate by means of ABC transporter or by other means, the leading way is still by PTS. Therefore, the study of relevant proteins of PTS in S. mutans has important implications for the prevention and treatment of dental caries.Previous studies have reported that some enteric bacteria have the ability to obtain life-sustaining carbon source by fermenting L-ascorbate under anaerobic conditions. However, the study of metabolism of L-ascorbate by S. mutans has not been described so far. In 2009, Jian Lei presumed that PtxA protein (encoded by ptxA gene), corresponding to EⅡA of PTS, may involve in the pathway of L-ascorbate metabolism in S. mutans. The analysis of three-dimensional structure and in vitro enzyme activity assay have proved that PtxA protein was specific for the phosphorylation of L-ascorbate.Therefore, PtxA protein which is related to the transport and phosphorylation of L-ascorbate in S. mutans may play a key role in its survival in oral cavity close to anaerobic environment. In this study, I have chosen PtxA as the target protein, constructed ptxA-deficient mutant and studied on its biological characteristics involved in cariogenictity, such as growth, acidogenesis, aciduricity and biofilm formation. This study may help us explore the cariogenictiy mechanism of S. mutans and initiate new ideas for caries prevention.Chapter 1 The construction of ptxA gene mutation strain of S.mutansObjective:To construct ptxA gene mutation strain of S.mutans UA159 and provide the foundation for next study.Methods:The upstream and downstream primers of ptxA gene were designed by Primer Premier 5.0 software according to the whole genome sequence of S.mutans UA159 in NCBI. The upstream fragment and downstream fragment were amplified by PCR. Then the recombinant plasmid pFW5-ptxA-up containing the upstream fragment of ptxA gene was constructed through the process of double restriction enzymes digestion, ligation, transformation and screening. Next, the same process was used to construct the recombinant plasmid pFW5-ptxA-up-down based on the recombinant plasmid pFW5-ptxA-up. Finally, the recombinant plasmid pFW5-ptxA-up-down was transformed into the competent cells from S.mutans and ptxA gene mutation strain which was named SM-ptxA-def was constructed by homologous recombination.Results:1. The gel electrophoresis showed that two band was formed when the recombinant plasmid pFW5-ptxA-up was digested by restriction enzymes BamH Ⅰ and HindⅢ. The size of these two bands was probably the same as the plasmid pFW5 and pFW5-ptxA fragment. The sequencing result showed that the whole recombinant plasmid was 99% consistent with expectation.2. The gel electrophoresis showed that two band was formed when the recombinant plasmid pFW5-ptxA-up-down was digested by restriction enzymes Nco Ⅰ and Nde Ⅰ The size of these two bands was probably the same as the plasmid pFW5-ptxA-up and ptxA-down fragment. The sequencing result showed that the whole recombinant plasmid was 99% consistent with expectation.3. Amplified with the same ptxA-up-F and ptxA-down-R primers, electrophoresis results showed that the size of amplified fragment between SM-ptxA-def strain and recombinant plasmid pFW5-ptxA-up-down was the same, but larger than that of wild-type strain. The sequencing result showed that the fragment amplified from SM-ptxA-def strain was 100% consistent with expectation.Chaper 2 The growth of wild-type S.mutans in L-ascorbate-specific mediumObjective:To explore the best concentration of L-ascorbate in the minimal medium suitable for the growth of wild-type S.mutans UA159. And to further confirm that the ptxA gene was involved in the transport and phosphorylation of L-ascorbate.Methods:1. Explore the best concentration of L-ascorbate. After incubated in BHI liquid medium in an anaerobic atmosphere for 24 hours, S.mutans UA159 was washed by TV medium (containing 3.5% tryptone,0.04μg/mL p-aminobenzoic acid,0.2μg /mL thiamine-HCl,1μg/mL nicotinamide and 0.2μg/mL riboflavin) and the OD600 was adjusted to 1.0. Then diluted 100-fold into TV medium supplemented with 5mM、10mM、5mM、20mM、30 mM L-ascorbate and the cultures were incubated at 37℃ in an anaerobic atmosphere for 48 hours. The value of OD600 of the cultures was monitored every 2 hours using a spectrophotometer. Data were derived from three replicates and the growth curve was drawn.2. The growth of S.mutans UA159 in the medium with two different carbon sources. After incubated in BHI liquid medium in an anaerobic atmosphere for 24 hours, S.mutans UA159 was washed by TV medium and the OD600 was adjusted to 1.0. Then diluted 100-fold into TV medium supplemented with 15mM L-ascorbate or glucose and the cultures were incubated at 37℃ in an anaerobic atmosphere for 48 hours. The value of OD600 of the cultures was monitored every 2 hours using a spectrophotometer. Data were derived from three replicates and the growth curve was drawn.3. The mRNA expression of ptxA gene in the medium with two different carbon sources by qRT-PCR. After incubated in TV medium supplemented with 15mM L-ascorbate or glucose to late-exponential phase, the total RNA from S.mutans UA159 was isolated and cDNA was synthesized by reverse transcription. Using 16S rRNA as a reference gene, qRT-PCR was performed to analyze the mRNA expression of ptxA gene in the medium with two different carbon sources.4. Statistical analysis. T-test was used to analysis the result of qRT-PCR by using SPSS 13.0 software. The statistical significance was defined as P<0.05.Results:1. The value of OD600 increasing over time showed that S.mutans UA159 was growing continuously within 48h. The time of lag phase (about 6h) and the growth rate in the logarithmic phase were similar in the medium with 5mM and lOmM L-ascorbate, but the final growth yield with lOmM is higher than that with 5mM. Although the time of lag phase with 15mM extended (about 12h), the final growth yield is slightly higher than that with 10mM. The growth rate and yield was very low with 20mM and 30mM, especially the apparent growth with 30mM was not observed.2. S.mutans UA159 grew very fast in the medium with 15mM glucose as the sole carbon source. It reached the stationary phase about 14-16h and the final value of OD600 was approximately 1.0. In comparison, S.mutans UA159 grew relatively slowly in the medium with 15mM L-ascorbate as the sole carbon source. It reached the stationary phase about 36h and the final value of OD600 was approximately 0.6.3. The mRNA expression of ptxA gene in the medium with L-ascorbate as the sole carbon source was higher than that with glucose as the sole carbon source. The difference between the two group was statistically significant (P<0.05).Chapter 3 The preliminary study of biological characteristics of SM-ptxA-def strainObjective:To study the effects on biological characteristics of S. mutans UA159, including growth activity, acidogenesis, aciduricity and biofilm formation after mutation in ptxA gene.Methods:1. Comparison of growth activity between wild-type S. mutans UA159 and SM-ptxA-def strain. Two strains were cultured in BHI medium anaerobically for 24h, adjusted to get an equal initial cell density (optical density at 600nm, OD6oo),diluted 20-fold into TV medium supplemented with 15mM L-ascrobate, then incubated at 37℃ in an aerobic environment for 48h. The OD600 of the cultures was monitored every 2 hours. The experiment was performed in triplicate.2. Comparison of the ability of acidogenesis between wild-type S. mutans UA159 and SM-ptxA-def strain. Two strains were cultured in BHI medium anaerobically for 24h, adjusted to get an equal initial cell density, diluted 20-fold into TV medium supplemented with 15mM L-ascrobate, then incubated at 37℃ in an aerobic environment for 48h. The initial and final pH of the mediums were measured. The experiment was performed in triplicate.3. Comparison of the ability of aciduricity between wild-type S. mutans UA159 and SM-ptxA-def strain. Two strains were cultured in BHI medium anaerobically for 24h, adjusted to get an equal initial cell density, diluted 20-fold into TV medium supplemented with 15mM L-ascrobate, then incubated at 37℃ in an aerobic environment. Cultures were harvested (at an OD600 of ≈0.3) by centrifugation, washed twice with 0.1M glycine buffer, pH 7.0, and then incubated in 0.1M glycine buffer, pH 2.8, for 0,15,30 and 45 min. The surviving cells were appropriately diluted, plated on BHI plates, and incubated at 37℃ in an aerobic environment for 48h. The cell viability was expressed as the percentage survival (N/No), with No and N the number of CFU/mL before and after the treatment, respectively. The experiment was performed in triplicate.4. Comparison of the ability of biofilm formation between wild-type S. mutans UA159 and SM-ptxA-def strain. Two strains were cultured in BHI medium anaerobically for 24h, adjusted to get an equal initial cell density, diluted 20-fold into TV medium supplemented with 15mM L-ascrobate, then incubated at 37℃ in an aerobic environment. Cultures were adjusted, diluted 1:40 in fresh medium and 150μL aliquots were dispensed into a 96-well plate. After 24h of incubation (37℃, anaerobically), cell density was measurd (OD595) using a microplate reader, washed with water once, air dried and 30μL of Gram crystal violet was applied for staining for 15min. Plate was washed with water three times and air dried, and crystal violet was solubilized with an ethanol-acetone (4:1) solution. The OD570 was determined from this solution, and the biofilm amount was calculated as the ratio of OD570 to OD595.5. Statistical analysis. The results were analyzed by using SPSS 13.0 software. The statistical significance was defined as P< 0.05. T-test was used to analyze the result of biofilm formation.Results:1. Compared with wild-type S. mutans UA159, SM-ptxA-def strain had longer lag phase, slower growth speed and lower growth yield.2. After incubated in the L-ascorbate-specific medium for 48h, ΔpH from wild-type S. mutans UA159 was significantly higher than that from SM-ptxA-def strain (P< 0.05).3. Wild-type S. mutans UA159 and SM-ptxA-def strain incubated in the L-ascorbate-specific medium had the same killing rate over time (P> 0.05).4. In the L-ascorbate-specific medium, the amount of biofilm for the wild-type 5. mutans UA159 (13.10±0.97) was significantly higher (P<0.05) than that for SM-ptxA-def strain (3.94±0.99).Conclusions:1. The mutation in ptxA gene had affected the growth in the L-ascorbate-specific medium for S. mutans.2. ptxA gene is possibly related to the ability of acidogenesis in L-ascorbate-specific medium for S. mutans.3. No significant relationship between ptxA gene and the ability of aciduricity for S. mutans.4. ptxA gene was possibly related to the biofilm formation in L-ascorbate-specific medium for S. mutans.