Effects of Shiga toxin 2 and Subtilase on human colonic epithelial cell line (HCT-8, Gb3+). Keystone Symposium Malnutrition, gut-microbial interactions and mucosal immunity to vaccines.

AMARAL MARÍA M; GERHARDT ELIZABETH; IBARRA CRISTINA

New Delhi

Simposio; Malnutrition, Gut-Microbial Interactions and Mucosal Immunity to Vaccines. Keystone Symposia; 2011

Keystone Symposia Global Health Series

Post-diarrhea hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children younger than 5 years of age in Argentina. The HUS is associated to Shiga toxin type 1 and 2 (Stx1, Stx2) produced by enterohemorrhagic E. coli (EHEC). Recently, it has been propose that subtilase (SubAB) may contribute with SUH pathogenesis. The receptor of Stx2 is globotriaosylceramide (Gb3), although its presence on human colonic epithelial cells has been discussed. SubAB links to specific sialated glycans (Neu5Gc), a monosaccharide identified in several food products. Our aim is to study the effect of Stx2 and SubAB on the HCT-8 viability in order to know the rol both toxins to intestinal infections by EHEC. We have evaluated the cell viability of HCT-8 at 50% and 100% of confluence in the presence of Stx2 (Phoenix Lab) or SubAB (Dr. J. Paton, Adelaide University). After 72 h, 0.8 µg/ml Stx2 significantly inhibited the cell viability when they were grown at 100% of confluence (16 ± 4 % n=3, p < 0.05) and even more at 50% of confluence (27 ± 1 % n=3, p < 0.05). On the other hand, 0.3 µg/ml SubAB similarly inhibited the cell viability at 50% and 100% of confluence (15 ± 1.3 % vs 19 ± 1.7 %, n=3 NS). The cytotoxic effects induced by Stx2 and SubAB were dependent on dose concentration and time exposition. In addition, a significant inhibition of water absorption across HCT-8 cells was observed when the apical side was exposed to 10 ng/ml Stx2 for 90 min. In summary, these results demonstrated that HCT-8 cells were affected by both toxins although the susceptibility to SubAB was 3-fold higher than Stx2. Moreover, the cell damages were more important under 100% of confluence, suggesting that the cytotoxic effects of Stx2 are dependent of this cell culture condition.