The Mechanism Of ClpCP Protease Play Roles In Streptococcus Mutans

ObjectiveClp protease is generally present in various organisms, especially in low-GC Gram-positive bacteria. Clp protease is involved in the stress response and the degradation of misfolded proteins in most bacteria. Clp protease complexes are composed of the regulatory subunit Clp ATPases and the catalytic subunit Clp P. Clp P is the central proteolytic core which consists of 14 Clp P serine peptidase subunits stacked in two hetameric rings to from an internal chamer. Narrow axial pores of Clp P monomer that reject the large peoteins but the peptides smaller than six amino acids control access to this proteolytic chamber.To obtain proteolytic activity, the Clp P associates with the Clp ATPases to change the space conformation of Clp P and expose the residues of His-123, Asp-172, Ser-98 to accelerate the process of hydrolysis.Clp C belongs to the conserved Hsp 100/Clp family of AAA+, which can specially associate with Clp P to form the Clp CP complexes. In the majority of gram-positive bacteria, Clp CP proteases are involved in the degradation of heat shock protein Cts R by mediating with the Mcs AB. In addition, Clp CP associates with adaptor protein Mec A to control the competence development by regulating proteolsis and transcription of com K/com S in B. Subtilis. The genome of S. mutans UA159 has been found by Dragana Ajdi? et al in 2002. The Clp ATPases encoded by S. mutans UA159 which have five orthologs, including Clp B, Clp C, Clp E, Clp L, and Clp X. In this paper, we try to construct the markerless clp P/clp C-deletion mutant to detected their roles in morphology, growth kinetics, biofilms, drug-resistance and the expression of competence genes to explore the mechanism of Clp proteases and the roles of them in stress tolerance of S. mutans.Methods(1) The clp P/clp C fragments were amplified from the genome of S. mutans UA159 by PCR, and inserted into the p MD-19 T simple vector, then introduced the kanamycin cassette which was flanked with two lox P sites to obtain the homologous recombination vectors p CKX3 and p CKX4.(2) The linearized p CKX3/p CKX4 plasmids were transformed into S. mutans UA159 induced by the competence-stimulating peptide(CSP), then transformed with the thermosensitive plasmid p Cre PA, then the p Cre PA was removed after over night incubating at 37℃ to obtain the markerless SM-Dclp P and SM-Dclp C.(3) The open reading frame of clp P gene and 5’UTR was amplified by PCR from genomic DNA of S. mutans UA159, then introduced into p Ori23 plasmid and transformed into SM-Dclp P to obtain the complementary strains Sclp P.(4) The growth tendency of wild-type, clp P/clp C and complementary strains were calculated by time as X-axis and changes of OD600 during the observation as Y-axis.(5) The survival ability of wild-type, SM-Dclp P and complementary strains were detected by spot assays at different concentration of sodium chloride and different p H.(6) Each strain was incubated in 0.5% Glucose THY and 0.5% Sucrose THY for 48 hours, then washed 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 elution of dye at 575 nm.(7) Antimicrobial susceptibility test was determined by KB method.(8) The shuttle vector p DL276 were transformed into the S. mutans UA159,and SM-Dclp C strains to determine the effect of clp C gene on competence development induced by the competence-stimulating peptide(CSP).(9) Total RNA of S. mutans UA159 and derivative were extracted with Trizol reagent. The expression of genes(com X, com YA and cin A) analyzed by real-time PCR.Results(1) The results of PCR and sequencing indicated that the homologous recombination vectors p CKX3, p CKX4 and the markerless deletion of SM-Dclp P, SM-Dclp C strains were constructed successfully.(2) The expression vector(p CKX5) and Sclp P strains were constructed successfully, verified by PCR and sequencing.(3) The Δclp P mutant had a slower growth rate than wild strain at 37℃, the growth rate of Sclp P were between wild-type and SM-Dclp P, but it did not restore the growth level to the wild-type. While the Δclp C mutant had a faster growth rate than wild strain at 42℃, the Δclp P mutant had a significant decrease in growth rate.(4) The survival ability of Δclp P mutant was significantly affected by the environment stress to compared with the wild type.(5) In Glucose, the Δclp P mutant showed significantly less biofilm formation than the parent strains which was decreased at 42%. In Sucrose, biofilm formation of the Δclp P mutant was significantly enhanced which was increased 1.74-folds compared to the parent strains. Sclp P could restore the biofilms to the wild-type level, however no significant differences were detected in the Δclp C strains.(6) Antimicrobial susceptibility test showed that the Δclp P mutant was more sensitive to the antibiotics compared with the wild-type, however it has no significant difference between Δclp C mutant and parent strains.(7) In the Δclp P strains, the expression of com YA were increased more than 2-folds, while cin A and com X were decreased at 73% and 58.2% compared to the parent strain. In the Δclp C strains, the expression of com YA, cin A and com X were increased 8.34-folds, 2.99-folds and 1.608-folds compared to the parent strains.(8) The transformation frequency of the parent strains peaking between 50 min and 90 min, then rapidly declined to the basal line, while the clp C-deletion mutant displayed a slow increase in transformation frequency pecking at 120 min to 150 min and maintained a prolonged competence stateConclusionsClp protease complexes play significant roles in maintaining homeostasis and physiological of S. mutans UA159, such as variation in morphology, biofilm formation, fluctuation in pressure stress, alteration in drug-resistance and competence formation.