[Effect of csn2 gene deficiency on the starvation tolerance and the synthesis of extracellular polysaccharides in oligotrophic environment of Streptococcus mutans].

Objective: To explore the effect of csn2 gene deficiency on starvation tolerance and extracellular polysaccharides (EPS) synthesis in an oligotrophic environment of Streptococcus mutans (Sm). Methods: The csn2 gene deletion strains and complementary strains of Sm were cultivated and then an oligotrophic growth environment for Sm growth by setting different concentration gradient media were created. Cell growth in oligotrophic environment was detected by growth curve. Biofilm volume was measured by crystalline violet staining. Scanning electron microscopy (SEM) and laser confocal microscope were performed to observe the biofilm structure of Sm. The synthesis of EPS was measured by the anthrone-sulfuric acid method. The expression of genes related to EPS synthesis was evaluated by quantitative real-time PCR (qRT-PCR). Results: The growth curve results showed that the deletion of csn2 gene inhibited the growth of Sm under starvation stress. Furthermore, the results of laser confocal microscope showed that the biofilm EPS/bacteria ratios produced by the wild-type strain, csn2 gene-deficient strain and complement strains under nutrient sufficient culture conditions were 0.44±0.07, 1.05±0.13 and 0.57±0.08 respectively, while the ratios of EPS/bacteria in an oligotrophic environment were 0.93±0.24, 3.05±0.21 and 1.32±0.46 respectively, indicating that the deletion of csn2 gene enhanced the ability of extracellular polysaccharide synthesis of Sm in the oligotrophic environment. The expression levels of EPS synthesis-related genes gtfB and gtfC were up-regulated by 2.5 fold and 1.8 fold respectively and the expression level of gtfD was down-regulated by two-thirds. Conclusions: The csn2 gene deficiency showed multiple effects on the physiological functions and virulence characteristics of Sm, including starvation tolerance and EPS synthesis. These changes might be related to the shift of the complex regulative network caused by csn2 gene deletion.目的： 探索csn2基因缺失对变异链球菌（Streptococcus mutans，Sm）饥饿耐受和寡营养环境下胞外多糖合成的影响。 方法： 培养Sm csn2基因的缺失菌株及回补菌株，通过设置不同浓度梯度培养基创造寡营养生长环境供其生长。生长曲线检测寡营养生长环境下Sm的生长，结晶紫染色，扫描电镜、激光共聚焦显微镜检测寡营养生长环境下Sm的生物膜表型，蒽酮硫酸法检测Sm生物膜中胞外多糖的量，实时荧光定量PCR检测胞外多糖合成相关基因的表达。 结果： 生长曲线结果显示csn2基因缺失抑制了饥饿胁迫下Sm的生长，激光扫描共聚焦显微镜检测结果显示野生型菌株、csn2基因缺陷株、回补菌株在营养充足培养条件下所得生物膜胞外多糖/细菌比值为0.44±0.07、1.05±0.13和0.57±0.08，在寡营养条件下所得生物膜胞外多糖/细菌比值为0.93±0.24、3.05±0.21和1.32±0.46，表明csn2基因缺失增强了Sm在寡营养环境下胞外多糖的合成能力；在饥饿胁迫下，胞外多糖合成相关基因gtfB、gtfC的表达水平分别显示出2.5和1.8倍的增加，gtfD的表达水平下调2/3。 结论： csn2基因对Sm的生理功能及毒力特性表现出多种影响，包括饥饿耐受和胞外多糖合成，这些改变可能与csn2基因缺失引发复杂的调控网络相关。.