谷胱甘肽对嗜热链球菌和德氏乳杆菌协同生长作用的机制研究

Streptococcus thermophilus is traditionally used in combination with Lactobacillus delbrueckii subsp.bulgaricus in yoghurt production. The two partners share the same ecological environment and form a complex symbiotic relationship proto-cooperation. Our previous study has shown that S. thermophilus can accumulate glutathione intracellularly and the produced glutathione could protect cells against oxygen and acid challenge. Moreover, the glutathione could be secreted into the medium and improve the survival of L..bulgaricus. We speculate glutathione play an important role in a novel proto-cooperation between S. thermophilus and L. bulgaricus. The role of glutathione in the synergistic growth of two strains has theoretical significance for the comprehensive analysis of the role of glutathione in lactic acid bacteria and further reveal the mechanism of synergistic growth of the two strains.Here we investigated the function and the secretion of glutathione by site-directed gene knock-out and isothermal titration calorimetry. The transfer of glutathione between S. thermophilus and L. bulgaricus was detected by the Transwell technology. We also elucidated the regulation mechanism of glutathione and explored the potential function of glutathione in L. bulgaricus through transcriptome analysis、RT-PCR and cell survival assay. Our results provide new insights in the mechanism of proto-cooperation between S. thermophilus and L. bulgaricus mediated by glutathione.

嗜热链球菌和德氏乳杆菌是酸奶生产的主要菌种，长期共生于牛乳环境形成了多种协同关系。前期研究发现，嗜热链球菌产生的谷胱甘肽除赋予菌株自身抗氧、酸等胁迫能力外，还能将其转运至胞外并促进德氏乳杆菌的生长和存活，由此我们推测谷胱甘肽在两菌种协同生长中发挥了重要作用。研究谷胱甘肽在两菌种协同生长中的作用，对全面分析谷胱甘肽在乳酸菌中的功能及进一步揭示两菌种协同生长的机制具有理论指导意义。基于此，本项目拟采用比较基因组分析、基因敲除、等温滴定量热等方法筛选确定嗜热链球菌SDMCC18内源性谷胱甘肽转运蛋白；通过Transwell技术分析谷胱甘肽在嗜热链球菌SDMCC18和德氏乳杆菌ATCC11842细胞间的传递；进而结合转录组学、荧光定量PCR和关键酶酶活分析探索谷胱甘肽对德氏乳杆菌抵抗氧、酸、低温胁迫相关基因和蛋白的影响，最终为揭示谷胱甘肽促进嗜热链球菌和德氏乳杆菌协同生长的新作用机制提供理依据。

本项目首先通过比较基因组分析、基因插入失活、荧光定量PCR等方法筛选得到了嗜热链球菌 ST-1内源性谷胱甘肽转运蛋白Ptrp-2，这是嗜热链球菌首次报道的GSH转运蛋白；其次结合Transwell技术，构建了嗜热链球菌ST-1和德氏乳杆菌ATCC11842共培小室，证明了嗜热链球菌 ST-1 合成并分泌的 GSH运输到菌株德氏乳杆菌 ATCC11842 胞内，并对德氏乳杆菌 ATCC11842的生长、存活和抵抗氧、酸等环境胁迫起到重要作用；最后，结合嗜热链球菌GSH合成酶基因缺失突变株ST-1△gshF，分析了GSH对酸奶发酵过程中凝乳时间、酸度、黏度、脱水收缩性、发酵乳蛋白、发酵乳脂肪的影响，并最终为GSH促进嗜热链球菌和德氏乳杆菌协同生长的新作用机制提供理依据。