Molecular Mechanism Of Injury In Seafood-Borne Vibrio Parahaemolyticus Caused By Ultra-High Pressure

In the present work, we investigated the effects of ultra high pressure on the morphology and microstructural damage relevant to the cell wall and membrane of Vibrio Parahaemolyticus, and explored the potential mechanisms of damage by high hydrostatic pressure on the cellular level. Moreover we established a proper system of two-dimensional gel electrophoresis (2-DE) to differentiate the total protein expression profiling of Vibrio parahaemolyticus under high pressure stress, in order to reveal the potential molecular mechanisms of microbial damage caused ultra-high pressure. This work could lay the foundation for the application of ultra-high pressure technique in seafood sterilization.The different pressure and hold time on contribution to the death of Vibrio parahaemolyticus cells were investigated. The effects of ultra high pressure on the microstructural damage of cell wall and membrane, membrane permeability, fluidity and Na+/K+-ATPase activity were examined. Moreover, we explored the characteristic changes of cell wall and cell surface potential of Vibrio parahamolyticus treated by ultra high pressure. The results showed that no viable cell counts were detected by pressure treatment at200MPa and20℃for10min. High pressure treatment caused cell wall and membrane damage in the microstructure of Vibrio par ahamolyticus, which produced an enormous impact on cell membrane permeability and fluidity, enzyme activity, the characteristic of cell wall, cell surface potential as well as the senior structure of the bacterial protein. This study indicated that ultra high pressure can cause the microstructural damage, changes of structure and characteristics of cell wall and membrane of Vibrio par ahamolyticus, leading to the leakage of cell contents and lethal effect.We established and optimized a proper two-dimensional gel electrophoresis (2-DE) system for the study of Vibrio parahaemolyticus total protein. The total proteins extracted from Vibrio parahaemolyticus were separated by carrierampholytes pH gradients based immobilized isoelectric focusing gel electrophoresis for first and the vertical SDS-polyacrylamide gel electrophoresis for second to the line of bidirectional gel electrophoresis2-DE. The gels were dyed and scanned to gain gel image, then PD-Quest software was used for gel image analysis. The2-DE related techniques were constructed and optimized by comparative tests on some important factors including different protein extraction methods, lysis buffer components, length of IPG strips, pH range of IPG strips, isoelectric focusing programs, sample volume, salt bridge, staining methods, gel concentrations, and so on. The results showed that the resolution and reproducibility of2-DE profiles was significantly improved by adding Tris and TBP in lysis buffer, the combination of sonication and trichloroacetic acid/acetone (TCA) precipitation for sample preparation, active rehydration of17cm (pH4-7) IPG gel strips, loading the sample100μg with sample volume of300μl, prolonging the time of desalting (2.5h) and isoelectric focusing time (80000vhr), preparing12.5%SDS-PAGE gel, and dying the gels by the silver stained. This work provided a technical basis for the further study on differential proteomics of V. parahaemolyticus.By combination of2-DE and mass spectrometry techniques, we identified24typical proteins in Vibrio parahaemolyticus under different pressures and holding times. After analysis of peptide fingerprinting obtained by MALDI-TOF-MS, four differential proteins spots related to cell wall and membrane were identified to be alanine racemase, murein hydrolase, phospholipase A2and amino-acid ABC transporter binding protein. Of which, alanine racemase is a main component in the biosynthesis of the peptidoglycan layer in bacterial cell walls. The main function of the murein hydrolase is effectively hydrolysis peptidoglycan of the bacterial cell wall; the bond broken may cause damage to the bacterial cell wall. The substrate of phospholipase A2is phospholipids that are a major component of the cell membranes and organelle membrane. Amino-acid ABC transporter binding protein involves in the transporter of lipid molecules in the cell membrane. Therefore, it was confirmed that after ultra-high pressure treatment, the injury or death of Vibrio parahaemolyticus mostly attributed to the morphology and microstructural damage happened in the cell wall and membrane, especially the expression changes of cell wall and membrane-related proteins.