The Study Of Protein Expression Differences Between Two Status Of Streptococcus Mutans Clinical Isolates From Different Caries-sensitive Individuals

Caries are the most common human infectious disease,S.mutans(Streptococcus mutans) are recognized as the main cariogenic bacteria.Most of the early research focused on standard S.mutans strains in planktonic satus because of the limitations of research methods and awareness.As research develop,researchers are now aware that oral plaque biofilm is typical biofilm.Comparision with S.mutans of planktonic,there are great differences between growth pattern and gene expression of S.mutans in biofilm with that in planktonic status.As a microenvironment,biofilm play an important role in the formation and development of caries,The biofilm has significant effect on the survival of S.mutans and its cariogenicity.Furthermore,presently research has confirmed that the pathogenicity of different strains of S.mutans are different.Therefore,the study of the differences of cariogenicity and their underlying causes among clinical S.mutans strain isolates from different caries-sensitive individuals has become the research hot spot.At the same time,with the emerge of new techologies,such as two-dimensional eletrophoresis and mass spectrometry techologies,analysis of proteomics bacterica has become possible.The proteomics of S.mutans especially functional proteomics is important for further understanding of caries pathogenesis.Our issue team had been successfully separed the S.mutans clinical strains from different caries-sensitive individuals,593 strains(serum C-type strains,separed from the caries-active individuals) and 18 strains(serum C-type strains,separed from caries-free individuals).Two strains not only show the differences of cariogenicity in vivo,and their genotype which confirmed through genotype analysis and the cariogenic ability which confirmed through a series of in-vitro experiments are different.In this study,based on the results of preliminary studies,by techologies such as one-dimensional electrophoresis,two-dimensional electrophoresis and mass spectrometry techologies,the differences between proteome,especially the surface-associated protein of strain 593 and that of strain 18 in planktonic and biofilm status was compared to investigate the cause of different cariogenic potential of streptococcus mutans strains.The reason for selecting the surface-associated protein expression as the focus of this study is the majority of cariogenic virulence factors are located in the surface of S.mutans,which include all aspects of cariogenicity,that is, bacterial adhesion,biofilm formation,carbohydrate metabolism in bacteria cells and the ability to adapt to constant and fluctuant environment.This study has included two parts:PartⅠ:the biofilm formation of clinical strains of Streptococcus mutans in vitro and the initial analysis of protein expressionObjective:To know biofilm formation law of clinical S.mutans in vitro,and the initial biofilm protein expression.Methods:(1)use the microplate cultivate,dyeing, Spectrophotometry(A630)to map biofilm growth curve of clinical strains 593,18 and the standard strain(ATCC 25175)under different pH conditions(pH7.0～5.0) in vitro. (2)use the polyacrylamide gel(SDS-PAGE) of one-dimensional gel electrophoresis to analysis the expression of soluble protein in biofilm of the standard strains at different time points(16h,18h,20h,22h,24h).Results:(1) the clinical strains 593,18 and the standard strain all can not form a biofilm at pH5.0 in vitro,bacterial biofilm formation as the slow growth of the non-linear at pH7.0,and about 12～24h biofilm begin mature,among 24～36h emerge a relative stagnation of the growth;the growth of S. mutans biofilm which has been formed 12h was significantly inhibited by the ph5.0, but the growth of the biofilm of these three strains are able to restore after the 12h acid shock.(2) at different time points during the 16～24h,the SDS-PAGE electrophoregram of the soluble protein of the standard strain are identical,without specific expression and high expression protein bands.Conclusion:(1) S.mutans form a stable biofilm at 12～24h in vitro at pH7.0,and the biofilm can resistant a certain degree of acid(pH5.0) attacks,and planktonic bacteria can not resistant it.(2) there is no difference about S.mutans biofilm soluble protein expression at different time points of 16～24h.PartⅡ:the differences of the surface-associated proteins between the serotype C S.mutans clinical strains isolated from caries-active and caries-free individuals in planktonic and biofilm status.Objective:(1)analyse the differences of surface-associated protein expression between clinical strains of S.mutans at two different status(planktonic and biofilm). (2)analyse the differences of the surface-associated protein expression of different clinical strains(strains 593 and 18) in the biofilm status.Methods:the surface-associated proteins were extracted from 593 strain and 18 strain in planktonic and biofilm status,identify the differences of these proteins expression between two strains and between different status were compared by SDS-PAGE and 2-DE,then get the the peptide mass fingerprint from matrix-assisted laser desorption time-of-flight mass spectrometry,finally,analyse the peptide mass fingerprint by NCBInr database search,analysis,identification of proteins.Results:(1) In comparison to planktonic, 57.5KDa and 67.9KDa specific band,42.5KDa,38.5KDa and 27.2 KDa high expression band were all observed in biofilm status of clinical strain593 and clinical strain 18 electrophoretic patterns of SDS-PAGE,further analysis by two-dimensional electrophoresis and found that the specific protein 57.5KDa actual strip contains two sites of high expression of protein;42.5KDa and the high expression of 38.5Kda separately strip contains a high expression protein sites and a site-specific protein expression;(2) Biofilm status on the 18 and 593 strains of SDS-PAGE electrophoresis patterns are basically the same,but by the two-dimensional electrophoresis analysis they found that the strains have their own specific and protein highly expressed loci.digestion and identification of these differences by site of protein bacteria found mainly related to energy metabolism, RNA synthesis,protein translation and folding,such as biological function.Conclusion:In this study conditions,the comparison of the expression of surface-associated protein in planktonic and biofilm status of two strains,we found in the biofilm state are both some of the specific and high protein expression,which may be common at the time of changes in biofilm;In addition,in biofilm state the surface-associated protein of strains 593 and 18 is obviously different in two-dimensional map,the identification of high and specific express protein in 593strains which involved in all aspects of Cariogenic power,putative in part that this may be 593 strains showed high Cariogenic.