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

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. Author summary: The neglected tropical diseases are a diverse set of infectious diseases which are common in low-income populations of Asia, Africa and the Americas. They include soil-transmitted helminth (STH) infections produced by the parasitic nematode Ascaris lumbricoides (= Ascaris suum). There are no effective vaccines, so mass drug administration (MDA) to control and prevent infection is the only practical option. With the limited number of anthelmintic drugs available for treatment, there is an increasing concern about the development of resistance. The use of combinations of anthelmintics, particularly if they are additive, is important for the delay of the onset of resistance. Here we describe synergistic interactions of levamisole and Cry5B mediated by Ca++ in A. suum intestine enterocytes. We have been able to make these new observations because of our access to A. suum parasites and development of our techniques for studying Ca++ signaling in the intestine of this nematode parasite, a site of the interaction of these two anthelmintic drugs. Our study provides new insights for development of effective STH combination therapies of anthelmintic drugs for slowing development of resistance with continued mass drug administration and understanding the nematode parasite intestine as a major target for combinations of anthelmintic drugs. [ABSTRACT FROM AUTHOR]