The Mechanism And Function Of ClpXP Protease In The Stress And Biofilms Of Streptococcus Mutans

ObjectiveStreptococcus mutans mainly causes the human dental decay. In addition to causing dental decay, S. mutans alse can cause dental pulp disease, periodontal disease,infective endocarditis and so on, which seriously affect people?s health and living quality. Clp protease of S. mutans participates in the regulation of the stress endurance and the degradation of abnormal proteins. Clp protease complexe are consist of the catalytic subunit Clp P including serine protease active domain and the highly conserved regulatory subunit Clp ATPases.To enhance proteolytic activity, the Clp P combined with the Clp ATPases to change the spatial conformation of Clp P and expose the residues to speed up the process of hydrolysis. Clp X is one of the Clp ATPases, which can specially associate with Clp P to form the Clp XP complexes. The Clp XP proteases are involved in the degradation of protein by specific recognition of tagged protein through Clp ATPases in B.Subtilis and S. aureus, thus, the biological character, stress endurance and the biofilms of the bacterias were regulated by degrading abnormal proteins. S. mutans UA159 have five Clp ATPases, containing Clp X, Clp B, Clp C, Clp E and Clp L. In this study, we would construct the clp X/clp Pgene mutation to observe their roles in the phenotype, growth curve, biofilms and so on to explore the mechanism of Clp XP proteases complexes and the roles of them in stress tolerance and biofilms formation of S. mutans. At the same time, the induction of Clp XP proteases in vitro can explore the mechanism of the substrate recognized by Clp XP proteases complexes thus the mechanism of virulence adjusted by Clp XP proteases complexes was verifed.Methods(1) The clp X fragment was amplified from the genome of S. mutans UA159 by PCR,and inserted into the p MD-20 T vector, then introduced the kanamycin cassette which was flanked with lox71 and lox66 to obtain the homologous recombination vectors p CKS3.(2) The linearized p CKS3 plasmid was transformed into S. mutans UA159 and S(35)clp P induced by the competence-stimulating peptide(CSP), then transformed with the thermosensitive plasmid p Cre PA, the kan gene was deleted incubating at30? and then the p Cre PA was removed after over night incubating at 37? to obtain the markerless S(35)clp X ? S(35)clp XP.(3) The gfp gene amplification was performed by PCR from p EGFP-N1 plasmid, and inserted into the p DLP containing promoter to obtain the fluorescent expression vector p CKS6 in vivo and it?s strain SCKS6. The fluorescent expression of SCKS6 was observed by fluorescence microscope.(4) The open reading frame of clp X gene was amplified by PCR from genomic DNA of S. mutans UA159, then introduced into p DLP plasmid and transformed into S(35)clp X to obtain the complementary strains Sclp X.(5) The growth curve of WT, S(35)clpP, S(35)clpXP, S(35)clpX and complementary strain Sclp X were drawed by time as X-axis and changes of OD600 during the observation as Y-axis.(6) Comparing the survival ability by spot assays at different concentration of sodium chloride and different p H among WT, S(35) clp X, Sclp X, S(35) clp P and S(35)clp XP with the survival ability on THY(p H 7.4) as reference.(7) Each strain was incubated in 0.5% glucose THY and 0.5% sucrose THY for 48 hours, then washed it with distilled water, dried and stained using crystal violet to observe the biofilm formation using the fluorescence microscopy, and obtain the absorbance values of the dyed elution at OD570.(8) Each strain was incubated in 0.5% glucose THY and 0.5% sucrose THY for 48 hours, The biofilms were handled by fixing, drying dehydrating sealing and metal spraying, and then the biofilm EPS was observed by the scanning electron microscope(SEM).(9) Each strain was incubated in 0.5% sucrose THY for 48 hours, the Dextran, Alexa Fluor? 647 was added in 24 h and the SYTO? 9 Green Fluorescent Nucleic Acid Stain was added in 48 h. and then the biofilm and EPS was observed by the confocal laser scanning microscope(CLSM).(10)The clp P and clp X gene open reading flames were amplified from the genome of S. mutans UA159 by PCR, and inserted into the p ET-21(b) and p GEX-4T-1expression vector respectively to obtain the recombination expression vectors p CKS1 and p CKS5, and then converted into competent E. coli BL21(DE3) to obtain SCKS1 and SCKS5.(11)Clp X and Clp P proteases were inducing by SCKS1 and SCKS5. The strains were crushed by ultrasonic to obtain total protein. The purified protein was purified by Ni-NTA SefinoseTMand Glutathione Sefinose kit.(12) The protein was analyzed by means of SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and then dyed by coomassie brilliant blue. The Clp X and Clp P proteases were observed after decoloration.Results(1) The results of PCR, restrictive endonuclease digest and DNA sequencing indicated that the homologous recombination vectors p CKS3 and mutation of S(35)clp X and S(35)clp XP strains were constructed successfully.(2) The fluorescent expression vector p CKS6 in vivo and it?s strain SCKS6 were constructed successfully, and the fluorescence light was observed by fluorescence microscope.(3) Verified by PCR and sequencing, the complemental vector p CKS4 and complemental strain Sclp X were constructed successfully.(4) Compared with the WT, the mutation strains S(35) clp X and S(35) clp P displayed several phenotypes in common: tendency to aggregate in culture and form long chain and the bacteria is bulky.(5) The mutation strains S(35)clpP, S(35)clpXP and S(35)clpX had a slower growth rate than WT strain at 37?, while the growth rate of the mutation strains and the complemental strain have no significant difference.(6) The tolerance test of WT, S(35)clp X, Sclp X, S(35)clp P and S(35)clp XP was significantly influenced by the stress condition compared with the WT.(7) S. mutans UA159 resulted in enhanced biofilm formation in THY supplemented with sucrose, while reduced in THY supplemented with glucose. In 0.5% glucose,the clp X and clp P mutant strains showed significantly reduce biofilm formation than the WT strain. In 0.5% sucrose, biofilm formation of the clp X and clp P mutant strains were significantly enhanced compared to the parent strains.complemental strain Sclp X could restore the ability to the WT level.(8) S. mutans UA159 resulted in enhanced EPS in THY supplemented with sucrose,while reduced in THY supplemented with glucose. In 0.5% glucose, the clp X and clp P mutant strains showed nearly the same as the WT strain. In 0.5% sucrose,EPS formation of the clp X and clp P mutant strains were significantly enhanced compared to the WT strain, and the Sclp P could restore the ability to the WT level.(9) Recombinat expression vectors pCKS1 and pCKS5 were constructed successfully, as well as the relevant expression strains SCKS1 and SCKS5. The target protein Clp X and Clp P were induced and purified successfully.ConclusionsClp XP protease complexes played a significant role in keeping homeostasis and regulating virulence of S. mutans UA159, such as the transformation of phenotype,biofilm, stress endurance and so on. The induction and purification of Clp X and Clp P proteases offered an experimental basis to the further study of the recognition of the substrate and the verification of mechanism of the virulence regulated by Clp XP protease complexes.