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

by Paul D. E. Williams, Matthew T. Brewer, Raffi V. Aroian, Alan P. Robertson, Richard J. Martin

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus 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 cholinergic 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 at the whole-worm level 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 in the intestine of Ascaris suum using 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 the synergism is due to the cytoplasmic Ca²⁺ overload that is induced by the combination of levamisole opening Ca²⁺ permeable L-subtype nAChRs and the Ca²⁺ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca²⁺ overloads that accelerates cell-death of the enterocytes.