Physiological Changes And Proteomic Analysis Of Frozen Sublethally Damaged Staphylococcus Aureus During The Repair Starting Process

In present study, the frozen sublethally damaged Staphylococcus aureus ATCC6538 cells were used as materials, the pattern of resuscitation, the changes of ultrastructure, as well as the changes of physiological indexes at different time points were determined. Meanwhile, high performance liquid chromatography-mass spectrometry(HPLC-MS) technique was employed to analyze the difference between the proteomics of the frozen sublethally damaged cells during resuscitation. The results of the experiment provide an important basis for the further research upon the resuscitation mechanisms of the frozen sublethally damaged S. aureus. The research results are as follows,We resuscitated the frozen sublethally damaged S. aureus at 37 °C for different time within 3 h, more than 99% of the cells is in sublethally damaged state after 90 d at-18℃. The mainly frozen sublethally damaged S. aureus cells could complete the resuscitation after 3 h. We compared the morphological changes during resuscitation using transmission electron microscopy assay(TEM). The frozen sublethally damaged S. aureus cellar surface ultrastructure significant changes during resuscitation. The cell surface becomes compact sturdy from smooth and transparent. The content of cells outside leakage, active oxygen(ROS) and superoxide dismutase(SOD) activity were also determined by ultraviolet spectrophotometry, as well. The intracellular ROS level declined along with the decrease of SOD activity, which were conducive to the repair of sublethally damaged cells. 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 provides the energy for the requirement of cells, therefore, the frozen sublethally damaged cells were repaired finally.We also compared the differential proteomes during resuscitation using High Performance Liquid Chromatography–Mass Spectrometry(HPLC-MS). The differentially expressed proteins were identified using the Uniprot database. Our research has shown that, compared with frozen survived S. aureus cells, those undergoing the 1 h resuscitation have 118 distinctly expressed proteins, including 45 upregulated and 73 downregulated. Gene Ontology(GO) analysis of the differentially expressed proteins indicates that the resuscitation of frozen survived S. aureus is related to plasma membrane transporters(Gnt P, Atp C, Mod A, Mtl F, Fab D), transcriptional factors(Nus B, Fap R, Gre A, Rsb V), Redox activate protein Mtl D, the cell wall synthesis-related protein Drp35, and the ribosome function-related protein Rbf A, etc.In conclusion, S. aureus is under sublethally damaged state during frozen storage. The cell membrane has a certain degree of damage, then the cell’s osmotic regulation ability is impaired and is sensitive to the high concentration of Na Cl. When the frozen sublethally damaged cells were resuscitated at a suitable temperature, under the regulation of related proteins, the cells may regain the capability of resistance to high salt stress by repairing cell membrane integrity, reducing the content of ROS, inhibiting the toxicity of active oxygen to the cells. Meanwhile, the regulation of metabolism related genes provides the energy for the requirement of cells, therefore, the frozen sublethally damaged cells were repaired finally.