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1Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel 2Department of Food Sciences, ARO, the Volcani Center, POB 6, Bet Dagan, 50250 Israel
Correspondence Shlomo Sela shlomos{at}volcani.agri.gov.il
Received March 20, 2003
Accepted September 8, 2003
The fate of GAS and epithelial cells following internalization was determined in this study. HEp-2 cells harbouring intracellular bacteria were treated with antibiotics to kill extracellular adherent bacteria, washed, and the fate of bacteria and epithelial cells was assessed up to 24 h post-infection. In the absence of antibiotics, massive bacterial growth was apparent in the cell medium, accompanied by extensive cell death, suggesting that intracellular bacteria had multiplied and damaged the monolayer. Addition of the internalization inhibitor, cytochalasin D, either pre- or post-internalization prevented bacterial growth and cell injury; post-internalization treatment with chloramphenicol had the same effect. Analysis of three apoptotic markers in HEp-2 cells – chromatin condensation, DNA laddering and translocation of phosphatidylserine onto the cell-surface membrane – indicated that HEp-2 cells underwent apoptosis. Taken together, the data presented here support a model in which intenalized bacteria can induce their own externalization into the medium by a process that requires both an intact host-cell cytoskeleton and de novo synthesis of bacterial proteins. Concomitantly, intracellular and, apparently, extracellular free bacteria induce apoptosis through their cytotoxic activity, and release essential nutrients required for their growth.
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