超高压对单核细胞增生李斯特氏菌细胞膜损伤及其氧化磷酸化的影响

Effects of High-Hydrostatic Pressure on the Membrane Damage and Oxidative Phosphorylation of Listeria monocytogenes

【目的】研究超高压作用对单核细胞增生李斯特氏菌(LM 54004)细胞膜、氧化磷酸化和F0F1-ATP酶的影响。【方法】用原子吸收法测定LM 54004经超高压作用后细胞内金属离子(K+、Mg2+)的泄漏;以亲脂性阳离子四苯基溴化膦([3H]-TPP+)作为放射性探针标记LM 54004细胞膜,测定经超高压作用后细胞膜电位的变化;用羧基荧光素琥珀酰亚胺酯(cFSE)为荧光探针测定经超高压作用后细胞内pH的变化;用比色法测定超高压对LM 54004 F0F1-ATP酶活性的影响。【结果】150—300 MPa作用10 min,随着压力的增大LM 54004菌落数显著降低,细胞内K+和Mg2+没有泄漏到细胞外,膜电势、跨膜H+浓度梯度和质子动力势大小基本保持不变,F1F0-ATP酶活性降低显著;300 MPa作用10 min,尽管LM 54004菌落数低于检测限,F0F1-ATP酶活性降到0,但细胞膜完整无损,其质子动力势仍然达到最大值。【结论】超高压作用下LM 54004的灭活与F0F1-ATP酶活性的降低之间相关性较好。LM 54004的细胞膜上参与呼吸链有关的酶和电子载体较F0F1-ATP酶耐压,F0F1-ATP酶对超高压敏感,超高压作用下F0F1-ATP酶的失活是超高压灭活的重要原因。

【Objective】 The effects of high-hydrostatic pressure（HHP） on the membrane damage and oxidative phosphorylation were investigated with Listeria monocytogenes（LM 54004） as the test microorganism in this work.【Method】The leakage of intracellular metal ions（K＋,Mg2＋） from LM 54004 subjected to HHP was determined using atomic absorption methods.The cell membrane of LM 54004 was labeled with radioactive probe of lipophilic cation tetraphenylphosphonium chloride（-TPP＋）,and the changes of membrane potential for LM 54004 exposed to HHP were measured.The changes of intracellular pH in LM 54004 treated by HHP were detected using carboxyfluorescein succinimidyl ester（cFSE） as fluorescent probe.The effects of HHP on F0F1-ATPase in LM 54004 were monitored using colorimetric methods.【Result】With increasing pressure at the pressure levels of 150-300 MPa for 10 min,the experimental results showed that survival cells of LM 54004 and the activity of F0F1-ATPase decreased markedly（P0.05）,intracellular metal ions（K＋,Mg2＋） were not leaked from LM 54004 cells,and the membrane potential,transmembrane proton concentration gradient and proton-motive force（PMF） did not change basically（P0.05）.Exposed to 300 MPa for 10 min,although the survival cells on TSAYE medium were below the detection limit and the activity of F0F1-ATPase was significantly decreased to 0%（P0.05）,integral membrane structure was not damaged and proton motive force（PMF） still could reach maximum as compared to these of untreated cells;the protons expelled from LM 54004 by the proton pump could not be returned to the cells of LM 54004.【Conclusion】There was a good relationship between the inactivation of LM 54004 and the decrease of F0F1-ATPase activity by HHP.In summary,it can be concluded that the enzymes and electron carriers involved in respiratory chain in cell membrane were more resistant to high pressure than F0F1-ATPase,and the decrease of F0F1-ATPase activity was highly related to cell reduction during HHP,which provided some theoretical evidences for mechanisms of high-hydrostatic pressure sterilization.The major cause of LM 54004 death by HHP was the inactivation of F0F1-ATPase.HHP can be used as an effective tool to explore the development of microbial energy.