Sampathkumar B, Xavier I J, Yu L S, Khachatourians G G
Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Canada.
Int J Food Microbiol. 1999 May 1;48(2):131-7. doi: 10.1016/s0168-1605(99)00037-9.
Early stationary phase cells of Listeria monocytogenes (Scott A) were examined to determine the effect of heat-shock on the production of listeriolysin O (LLO) during and after resuscitation at 37 degrees C. Cells were subjected to a heat-shock at 48 degrees C for 1 h. Intracellular and extracellular proteins of the heat-shocked cells were assayed for LLO using a microtiter plate hemolysis assay and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Our results showed that significant amounts of LLO are synthesized under heat-shock conditions that are not detected in the extracellular medium by a functional assay. This situation is evident by the absence of hemolytic activity immediately after heat-shock, and may be due to either a lack of excretion or inactivation of the LLO at 48 degrees C once outside the cell. By studying the intracellular and extracellular proteins using SDS-PAGE and immunoblots of the heat-shocked cells, we substantiated an absence of excretion as an operating mechanism. Heat-shocked cells resumed LLO production within 2-4 h of resuscitation at 37 degrees C, achieving an activity level 2-fold higher compared to the controls and 4-fold higher compared to cells immediately after heat-shock. Most likely, the LLO excreted must have been from LLO accumulated in the cells during heat-shock.
对单核细胞增生李斯特菌（斯科特A）的早期稳定期细胞进行检测，以确定热休克对其在37℃复苏期间及复苏后产溶血素O（LLO）的影响。将细胞在48℃进行1小时的热休克处理。使用微量滴定板溶血试验对热休克细胞的细胞内和细胞外蛋白质进行LLO检测，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹分析。我们的结果表明，在热休克条件下会合成大量的LLO，但通过功能检测在细胞外培养基中未检测到。热休克后立即没有溶血活性，这种情况很明显，这可能是由于缺乏排泄或LLO一旦在细胞外于48℃失活所致。通过对热休克细胞进行SDS-PAGE和免疫印迹研究细胞内和细胞外蛋白质，我们证实缺乏排泄是一种作用机制。热休克细胞在37℃复苏后2至4小时内恢复LLO产生，其活性水平比对照高2倍，比热休克后立即检测的细胞高4倍。很可能，排出的LLO一定来自热休克期间细胞内积累的LLO。
