冷冻致亚致死损伤的金黄色葡萄球菌修复机制

Repair mechanism of frozen sublethally damaged Staphylococcus aureus

摘要:

摘要:【目的】研究冷冻致亚致死损伤的金黄色葡萄球菌修复过程中的细胞修复机制。【方法】本文以冷冻致亚致死损伤的金黄色葡萄球菌(Staphylococcus aureus)为研究对象，探讨了不同修复时间细胞的修复情况;利用透射电子显微镜观察修复启动过程中超微结构的变化;通过实时荧光定量PCR(Real-time PCR)方法测定了修复过程中转录弱化子(msrR)、铁离子ABC转运ATP结合蛋白(fhuC)、细胞色素b(cytB)基因表达量的变化，通过紫外分光光度法测定细胞外泄漏物含量、细胞活性氧(ROS)和超氧化物歧化酶( SOD)活性。【结果】修复3 h后，99%以上冷冻致亚致死损伤的金黄色葡萄球菌完成修复，修复后细胞对高盐胁迫抗性恢复。Real-time PCR分析结果表明，msrR和fhuC基因表达量显著下调，而cytB表达量显著上调。修复过程中冷冻致亚致死损伤的金黄色葡萄球菌细胞表面超微结构变化比较明显，细胞表面从光滑透明变得致密结实，细胞内紫外吸收物质泄漏速度也在逐渐变慢，同时细胞中的ROS含量降低，SOD酶活性减弱。【结论】冷冻致亚致死损伤的金黄色葡萄球菌修复过程中，可能是通过细胞膜完整性的修复，细胞恢复对高盐胁迫的抵抗能力;通过基因调控降低细胞内ROS的含量，降低活性氧(O－2)对细胞的毒害作用。同时通过产能代谢相关基因(cytB)的调控为细胞提供修复所需要的能量，最终冷冻致亚致死损伤的细胞得到修复。

Abstract:

Abstract:［Objective］ To study the repair mechanisms of frozen sublethally damaged Staphylococcus aurous cells.［Methods］We resuscitated frozen sublethally damaged S．aureus at 37 ℃ for different time within 3 h． Meanwhile，we compared the morphological changes of the frozen sublethally damaged cells after 1 h of resuscitation using transmission electron microscopy assay (TEM). The expressions of the transcriptional attenuator MsrR (msrR)，iron (Fe3+) ABC transporter ATP-binding protein (fhuC)，and cytochrome b (cytB) genes were quantitatively analyzed by real-time fluorescence quantitative PCR (Real-time PCR) method． The content of cells outside leakage，active oxygen (ROS)，and superoxide dismutase (SOD) activity were also determined by ultraviolet spectrophotometry.［Results］More than 99% of the frozen sublethally damaged S． aureus repaired after 3 h． The resuscitated cells expressed an equal resistance to high concentration of NaCl. Real-time PCR results showed that the msrR and fhuC genes expressions were down-regulated，whereas the cytB gene expression was up-regulated significantly． The frozen sublethally damaged S． aureus cellar surface ultrastructure significant changed during resuscitation． The cell surface became compact and sturdy from smooth and transparent． The cell leakage rate of ultraviolet absorption material gradually decreased． Meanwhile，the intracellular ROS level declined along with the decrease of SOD activity． ［Conclusion］Frozen sublethally damaged cells may regain the capability of resistance to high salt stress by repairing cell membrane integrity，reducing the content of ROS through gene regulation，inhibiting the toxicity of active oxygen to the cells． Meanwhile，the regulation of metabolism related genes (cytB) provides the energy for the requirement of cells，therefore，the frozen sublethally damaged cells were repaired finally.