Effect of luxS overexpression on biofilm formation by Streptococcus mutans
10.3760/cma.j.issn.1002-0098.2015.09.011
- VernacularTitle:变形链球菌luxS基因高表达对生物膜形成的影响
- Author:
Zhiyan HE
1
;
Yuxia WANG
;
Zhengwei HUANG
Author Information
1. 200011,上海交通大学医学院附属第九人民医院口腔医学院上海市口腔医学研究所上海市口腔医学重点实验室
- Keywords:
Streptococcus mutans;
Biofilm;
Gene expression
- From:
Chinese Journal of Stomatology
2015;50(9):554-560
- CountryChina
- Language:Chinese
-
Abstract:
Objective To evaluate the effect of quorum sensing luxS gene on biofilm formation through construction of a luxS overexpression strain by Streptococcus mutans(Sm).Methods In order to construct pIB-luxS plasmid,the luxS gene fragment amplified by PCR was inserted into the shuttle plasmid pIB169 by corresponding double digests.The pIB-luxS plasmid was linearized electro-transformed into Sm cell and the overexpression strain was selected on chloramphenicol plate and testified by electrophoresis and western blot.The growth rate of both Sm wild type strain and its luxS overexpression strain were observed.Methyl thiazolyl tetrazolium(MTT) assay method was used to compare the biofilm formation quantificationally by both strains at different time points and containing different sucrose.The structures of the biofilms were observed by using confocal laser scanning microscopy,and biofilm-related gene expressions were investigated by real-time PCR.All experiments were performed in triplicate.Results The luxS overexpression strain was successfully constructed and confirmed by electrophoresis and Western blotting.The planktonic growth mode of the wild-type and luxS overexpression strain showed no difference,but biofilm formed by Sm overexpression strain was 0.400 ±0.009 and 0.609±0.041 at 14 and 24 h,higher than the wild type strain biofilm at the same time point(0.352 ±0.028 and 0.533 ±0.014,respectively,P＜ 0.05).After adding 0.125％ sucrose,biofilm formed by Sm overexpression strain raised to 1.041 ±0.038,higher than that by the wild type strain(0.831 ±0.020,P＜0.05).The biofilm formed by both strains were also increased with the sucrose concentration increase,but there was no difference between them.The overexpression strain aggregated into distinct clusters on structure,genes expression including gtfB,ftf,gbpB,relA,brpA,smu630,comDE,vicR were increased(6.10±0.12,3.34±0.07,8.75±0.13,2.96±0.04,5.20± 0.19,2.20±0.06,2.32±0.07 and 10.67±0.57 fold) compared to the wild-type strain(P＜0.05).Conclusions Quorum sensing luxS gene can promote the biofilm formation of Sm.
