The nematode (Ascaris suum) intestine is a location of synergistic anthelmintic effects of Cry5B and levamisole

Abstract

AbstractA novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced byBacillus thuringiensis(Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinomimetic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+levels in enterocyte cells from the intestine ofAscaris suumusing fluorescence imaging. The peak Ca2+responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that there is an irreversible cytoplasmic Ca2+overload that leads to necrotic cell-death in the enterocyte that is induced by levamisole opening Ca2+permeable L-subtype nAChRs and the development of Ca2+permeable Cry5B toxin pores in enterocyte plasma membranes. The effects of levamisole potentiate and speed the actions of Cry5B.