2. Clostridium perfringens epsilon toxin is cytotoxic for human renal tubular epithelial cells

FERNANDEZ MIYAKAWA M.E.; IBARRA C.A.; SILBERSTEIN C.

Roma, Italia

Congreso; Clostridia:The Impact of Genomics on Dsease Control 6th ClostPath International Conference.; 2009

Natural disease due to epsilon toxin (ETX) is most frequently seen in ruminant. However, different animal species as mice, rats, sheep, goats and cattle are, at some extent, equally susceptible to the lethal effects of ETX. In humans, there is not information about the susceptibility to ETX although the extreme potency of this toxin also makes it a bioterrorism concern. Since the few cell lines shown to be susceptible to ETX were originally obtained from kidneys, primary cultures of epithelial cells obtained from human kidney were examined for ETX cytotoxicity. Human renal tubular epithelial cells (HRTEC) were isolated from kidneys removed from different adult patients undergoing nephrectomies, characterized and used between three and five passages. The incubation of HRTEC with activated ETX produced a reduction of cell viability in a dose- and time-dependent manner. The binding of ETX to a specific antibody completely inhibited the cytotoxic effect on HRTEC viability. Cells exposed to ETX showed a marked swelling with subsequent large blebs surrounding most cells. The blebs appeared translucent, containing no cell organelle. The pulse with ETX for only 1 min did not change the cell viability compared with control cells, after 1 h of incubation. When cells were pulsed for 3 and 5 min with ETX the cell viability decreased to 63 ± 2% and 55 ± 1% respectively. Samples with ETX-treated and control non-treated HRTEC were analyzed by SDS-PAGE.  A ladder of bands was observed with a major band at about ~160 kDa and a smaller at ~30 kDa. Human umbilical vein endothelial cells (HUVEC) were not sensitive to ETX. Levels of ETX necessary to induce changes in human cells were in the range of concentrations necessary to induce morphological alteration in MDCK cells. These results show that human kidney derived epithelial cells are sensitive to ETX and that cytotoxicity is probably produced by the mulitmeric ETX forminga membrane pore as it was previously described in other cell types.