Formation of Very Large Conductance Channels by Bacillus cereus Nhe in Vero and GH4 Cells Identifies NheA + B as the Inherent Pore-Forming Structure

Abstract

The nonhemolytic enterotoxin (Nhe) produced by Bacillus cereus is a pore-forming toxin consisting of three components, NheA, -B and -C. We have studied effects of Nhe on primate epithelial cells (Vero) and rodent pituitary cells (GH_4) by measuring release of lactate dehydrogenase (LDH), K^+ efflux and the cytosolic Ca^2+ concentration ([Ca^2+]_i). Plasma membrane channel events were monitored by patch-clamp recordings. Using strains of B. cereus lacking either NheA or -C, we examined the functional role of the various components. In both cell types, NheA + B + C induced release of LDH and K^+ as well as Ca^2+ influx. A specific monoclonal antibody against NheB abolished LDH release and elevation of [Ca^2+]_i. Exposure to NheA + B caused a similar K^+ efflux and elevation of [Ca^2+]_i as NheA + B + C in GH_4 cells, whereas in Vero cells the rate of K^+ efflux was reduced by 50% and [Ca^2+]_i was unaffected. NheB + C had no effect on either cell type. Exposure to NheA + B + C induced large-conductance steps in both cell types, and similar channel insertions were observed in GH_4 cells exposed to NheA + B. In Vero cells, NheA + B induced channels of much smaller conductance. NheB + C failed to insert membrane channels. The conductance of the large channels in GH_4 cells was about 10 nS. This is the largest channel conductance reported in cell membranes under quasi-physiological conditions. In conclusion, NheA and NheB are necessary and sufficient for formation of large-conductance channels in GH_4 cells, whereas in Vero cells such large-conductance channels are in addition dependent on NheC.