Characterize The Function Of TDE2037 In Treponema Denticola

Pulpitis and periodontitis are common clinical infectious oral disease,and oral bacteria are considered as an important causative factor in the development of these diseases[1,2].The detection rate of T.denticola is high in periodontal lesions and infectious root cannel.There is a close relationship between T.denticola and the development of periodontitis and apical period disease.The research on pathogenic mechanisms has been a hot topic for scholars[3].However,due to the diversity of its pathogenic factors,the pathogenic mechanisms and control strategies still require a lot of research and exploration.Proteases are essential for all living organisms to carry out protein quality control and degrade short-lived regulatory proteins[4,5].An analogous system presents in mycobacteria,Bacillus subtilis and closely related species,which is one of the current research hotspots in microbiology filed,is the Clp proteolytic complex in bacteria.Arginine residues are crucial for protein folding and assembly[6].Mcs B,in Bacillus subtilis,is the founding member of a class of protein kinases targeting arginine residues.This kinase catalyzing this reaction,TDE2037 Mcs B,and activate the Clp complexes.However,the function of arginine kinases enzymes and the regulation mechanism of Clp proteases in T.denticola are not yet clear.Our research group found that the structure of arginine kinases4rf6.2.A in sea anemone is similar to the predicted structure of TDE2037 protein in T.denticola,and gene encoding TDE2037 was found in the same operon with gene Clp C[8].To figure out the function of TDE2037 in T.denticola,we construct a prokaryotic expression plasmid for TDE2037.After overexpression and purification of the protein,we made antibody form TDE2037 protein and carried out mass spectrometry(MS).Next,to study the function of TDE2037,our research group made a mutation of TDE2037 gene in T.denticola,and observed the difference of phenotype between wildtype and TDE2037 mutant.Part one: Prokaryotic expression and purification of an Arginine kinase gene TDE2037 of Treponema denticolaObjective1.To construct the a new prokaryotic expression plasmid for TDE2037;2.To express and purify the fusion protein of TDE2037;3.To characterize the fusion protein of TDE2037 by mass spectrometry.Method1.Recover the bacteria of T.denticola ATCC 35405 and extract genomic DNA;2.Use ligase chain reaction connects PCR production of TDE2037 gene and prokaryotic expression plasmid;3.Use IPTG to induce the expression of fusion protein and purify the TDE2037 protein by AKTA prime TM PLUS machine[10];4.Use mass spectrometry to study the fusion protein of TDE2037.Result1.Our research group extracted genomic DNA from T.denticola ATCC 35405,and the size of PCR production of TDE2037 gene is same as expected;2.The new prokaryotic expression plasmid for TDE2037 is correct after sequencing[11];3.The protein of p ET-21a-TDE2037 completely in form of inclusion body and remain insoluble,whereas the expression product of p ET28a-TDE2037-SUMO,partially soluble and partially in form of inclusion body;4.Mass spectrometry experiment results showed that TDE2037 has extremely high similarity with arginine kinase.Conclusion The prokaryotic expression vectors of Treponema denticola gene TDE2037 were successfully constructed and the expressed protein was successfully purified,which may lay a basis for the further study of TDE2037.Mass spectrometry experiment results showed that TDE2037 has extremely high similarity with arginine kinase.Part Two: Making the construct of plasmid for Treponema denticola TDE2037mutantObjective1.Making the construct of plasmid for Treponema denticola TDE2037 mutant and complement;2.To construct Treponema denticola TDE2037 mutant and complement;3.Observing the differences of growth curve and virulaence between Treponema denticola wild type strain and TDE2037 mutant.Method1.The TDE2037 DNA fragment was amplified by primer pairs and cloned into p GEM-T-easy,then a reverse PCR was done by primes and the erythromycin resistant cassette was introduced to replace TDE2037.Use kanamycin resistant cassette for TDE2037 complement;2.The DNA fragment was added in competent cells on ice,followed by electroporation;3.Transformants were picked up in NOS medium with appropriate antibiotics and identified by PCR using bacterial as template to check replace of targeted genes,and observe the differences of growth curve and virulaence between T.denticola wild type strain and TDE2037 mutant[11].Result:1.The new plasmid for TDE2037 mutant and complement are correct;2.Transformants were identified by PCR and Western Blot after the transformation;3.T.denticola wild type,TDE2037 mutant and TDE2037 complement strain were diluted with PBS and added into NOS.Plating and transferring to an anaerobic chamber.We found there is an obvious difference among these three bacteria not only in growth curve but also in virulence.The growth curve of TDE2037 mutant was slower notably.When we fed Drosophila with TDE2037 mutant in starving situation,the survival rate would raise up.Conclusion:According to the results of part 1,TDE2037 has extremely high similarity with arginine kinase.We presume that there is a lack of arginine kinase in the cells of TDE2037 mutant.It will not activate the Clp protease system efficiently.There are lots of “junk” proteins which cannot be degraded.That would lead to a big difference between these three bacteria not only in growth curve but also in virulence.