变形链球菌luxS基因突变对口腔混合细菌生物膜的影响

doi:10.19439/j.sjos.2019.02.001

上海口腔医学 ›› 2019, Vol. 28 ›› Issue (2): 113-117.doi: 10.19439/j.sjos.2019.02.001

变形链球菌luxS基因突变对口腔混合细菌生物膜的影响

何智妍¹, 周薇¹, 黄正蔚², 梁景平^2,*, 姜葳^2,*

1.上海交通大学医学院附属第九人民医院·口腔医学院口腔微生态与系统性疾病实验室,
2.牙体牙髓病科,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室, 上海市口腔医学研究所,上海 200011

收稿日期:2018-05-21 修回日期:2018-08-16 出版日期:2019-04-25 发布日期:2019-06-20
通讯作者: 梁景平,E-mail:liangjpdentist@126.com;姜葳,E-mail:jiangweishanghai@126.com。^*共同通信作者
作者简介:何智妍（1984-）,女,主管技师,E-mail:zyhe23@163.com
基金资助:
国家自然科学基金(81300866); 上海市自然科学基金(18ZR1422300); 上海市口腔医学研究所院级基金(院2017-09)

The effect of Streptococcus mutans luxS gene on mixed-species biofilm communities

HE Zhi-yan¹, ZHOU Wei¹, HUANG Zheng-wei², LIANG Jing-ping², JIANG Wei²

1. Laboratory of Oral Microbiota and Systemic Diseases,
2. Department of Operative Dentistry and Endodontics, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology. Shanghai 200011, China;

Received:2018-05-21 Revised:2018-08-16 Online:2019-04-25 Published:2019-06-20

摘要/Abstract

摘要： 目的研究致龋菌变形链球菌luxS基因在口腔细菌混合培养形成牙菌斑生物膜中的作用。方法将变形链球菌野生株(UA159)及其2种luxS基因突变株（luxS基因高表达株和luxS基因缺陷株）分别与口腔细菌嗜酸乳杆菌（ATCC4356）按照1∶1比例接种于牛心脑浸液培养基,体外混合培养不同时间,包括生物膜形成过程中的初期（4 h）、中期（14 h）、晚期（24 h）,通过MTT法检测混合菌在生物膜形成的量。通过激光共聚焦显微镜观察混合细菌24 h形成的生物膜结构,实时定量PCR检测变形链球菌相关基因（ftf, smu630, brpA, gbpB, gtfB, vicR, comDE和relA）的表达。采用SPSS17.0软件包对数据进行统计学分析。结果变形链球菌野生株及其2种luxS基因突变株与嗜酸乳杆菌混合培养14 h后,生物膜的量分别为0.481±0.024、0.591±0.023和0.279±0.019;24 h后,混合细菌形成生物膜的量趋势与该时间点一致,变形链球菌高表达株高于野生株,而缺陷株明显降低;但4 h后形成的生物膜组间无显著差异。激光共聚焦显微镜结果表明,高表达株和野生株的集聚程度更高,形成生物膜的结构更加紧密;而缺陷株生物膜菌间结构比较稀疏。以变形链球菌野生株和嗜酸乳杆菌混合形成的生物膜中相关基因的表达为标准,高表达株相关基因的表达均增加,缺陷株表达均降低,且各组间存在显著差异（P<0.05）。结论变形链球菌luxS基因影响与具核梭杆菌混合培养形成的牙菌斑生物膜,为进一步研究该基因在生物膜中的作用及其调控机制提供了依据。

关键词: 变形链球菌, 生物膜, luxS基因, 嗜酸乳杆菌

Abstract: PURPOSE: To evaluate the effect of S.mutans luxS gene on mixed-species biofilms communities. METHODS: Biofilms were formed by S. mutans (wild type strain, its luxS overexpression strain and luxS knockout strain) and Lactobacillus acidophilus (ATCC4356) with a ratio of 1:1 at 37℃ for 4 h, 14 h and 24 h. MTT assay was used to detect the quantification of the biofilms formed. The structures of biofilms were observed under confocal laser scanning microscopy after 24 h, and expression of biofilm-related genes (ftf, smu630, brpA, gbpB, gtfB, vicR, comDE and relA) was investigated by real-time PCR. Statistical analysis was performed with SPSS17.0 software package. RESULTS: The results showed that biofilm formed by S. mutans(wild type strain, its luxS overexpression strain and luxS knockout strain) and L.acidophilus after 14 h were 0.481±0.024, 0.591±0.023 and 0.279±0.019, respectively. The same findings were present after 24 h, the biofilm formed by S.mutans overexpression strain with L.acidophilus was higher than wild type strain, and the biofilm formed by knockout strain significantly decreased; but there was no significant difference at 4 h time points. CLSM images revealed that both S.mutans overexpression strain and its wild type strain tended to aggregate into distinct clusters and dense structures, whereas the luxS knockout strain appeared relatively sparse. Compared with wild type strain, all of the genes examined were upregulated in the biofilms formed by the overexpression strain, and were downregulated in the biofilms formed by the luxS mutant strain in mixed-species biofilm. CONCLUSIONS: S.mutans luxS gene can affect mixed-species biofilm formation with L.acidophilus, which provides evidences for further study.

Key words: Streptococcus mutans, Biofilm, luxS gene, Lactobacillus acidophilus

中图分类号:

R780.2

何智妍, 周薇, 黄正蔚, 梁景平, 姜葳. 变形链球菌luxS基因突变对口腔混合细菌生物膜的影响[J]. 上海口腔医学, 2019, 28(2): 113-117.

HE Zhi-yan, ZHOU Wei, HUANG Zheng-wei, LIANG Jing-ping, JIANG Wei. The effect of Streptococcus mutans luxS gene on mixed-species biofilm communities[J]. Shanghai Journal of Stomatology, 2019, 28(2): 113-117.

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变形链球菌luxS基因突变对口腔混合细菌生物膜的影响

The effect of Streptococcus mutans luxS gene on mixed-species biofilm communities

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