Production of listeriolysin O by Listeria monocytogenes (Scott A) under heat-shock conditions

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°C. Cells were subjected to a heat-shock at 48°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°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°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.