State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
Department of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
Arch Oral Biol. 2019 Mar;99:190-197. doi: 10.1016/j.archoralbio.2019.01.016. Epub 2019 Jan 29.
The goal of this study was to analyze the impact of cas3 gene on the biofilm formation and virulence gene expression in S. mutans, since our previous studies have found a connection between CRISPR/Cas systems and biofilm formation in S. mutans.
The cas3 gene in-frame deletion strains of S. mutans UA159 was constructed by a two-step transformation procedure and the cas3 mutant strain was complemented in trans. The biofilm biomass was measured by crystal violet staining, and the synthesis of exopolysaccharides (EPS) was measured by the anthrone-sulfuric method. Biofilm analysis and structural imaging was using confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) assays. The fluorescence in situ hybridization (FISH) was used to analyze the spatiotemporal interactions between S. mutans and Streptococcus sanguinis. Fluoride sensitivity was determined using fluoride tolerance assays. The expression of biofilm formation related genes was evaluated by qRT-PCR.
Our results showed that S. mutans cas3 deletion strain formed less biofilm and became less competitive when it was co-cultured with S. sanguinis under fluoride treatment. The expression levels of virulence genes including vicR, gtfC, smu0630 and comDE were significantly downregulated.
The cas3 gene in S. mutans could regulate biofilm formation and fluoride resistance, consequently affecting S. mutans competitiveness in a dual-species biofilm model under fluoride treatment. These results also provide a potential strategy for enhancing fluoride specificity, with cas3 gene as a potential genetic target in the modulation of oral microecology and the treatment of dental caries.
本研究旨在分析 cas3 基因对变形链球菌生物膜形成和毒力基因表达的影响，因为我们之前的研究发现 CRISPR/Cas 系统与变形链球菌生物膜形成之间存在关联。
通过两步转化程序构建变形链球菌 UA159 的 cas3 基因缺失株，并通过反式互补 cas3 突变株。通过结晶紫染色测量生物膜生物量，通过蒽酮-硫酸法测量胞外多糖（EPS）的合成。使用共聚焦激光扫描显微镜（CLSM）和扫描电子显微镜（SEM）检测生物膜分析和结构成像。荧光原位杂交（FISH）用于分析变形链球菌和血链球菌之间的时空相互作用。通过氟化物耐量测定来确定氟化物敏感性。通过 qRT-PCR 评估生物膜形成相关基因的表达。
我们的结果表明，变形链球菌 cas3 缺失株在氟化物处理下与血链球菌共培养时形成的生物膜较少，竞争力下降。毒力基因 vicR、gtfC、smu0630 和 comDE 的表达水平显著下调。
变形链球菌中的 cas3 基因可调节生物膜形成和氟化物抗性，从而影响变形链球菌在氟化物处理下双物种生物膜模型中的竞争力。这些结果还为增强氟化物特异性提供了一种潜在策略，将 cas3 基因作为调节口腔微生物群和治疗龋齿的潜在遗传靶标。
