Discovery of a highly synergistic anthelmintic combination that shows mutual hypersusceptibility

Abstract

The soil-transmitted helminths or nematodes (hookworms, whipworms, and Ascaris) are roundworms that infect more than 1 billion of the poorest peoples and are leading causes of morbidity worldwide. Few anthelmintics are available for treatment, and only one is commonly used in mass drug administrations. New anthelmintics are urgently needed, and crystal (Cry) proteins made by Bacillus thuringiensis are promising new candidates. Combination drug therapies are considered the ideal treatment for infectious diseases. Surprisingly, little work has been done to define the characteristics of anthelmintic combinations. Here, by means of quantitative assays with wild-type and mutants of the roundworm Caenorhabditis elegans, we establish a paradigm for studying anthelmintic combinations using Cry proteins and nicotinic acetylcholine receptor (nAChR) agonists, e.g., tribendimidine and levamisole. We find that nAChR agonists and Cry proteins, like Cry5B and Cry21A, mutually display what is known in the HIV field as hypersusceptibility--when the nematodes become resistant to either class, they become hypersensitive to the other class. Furthermore, we find that when Cry5B and nAChR agonists are combined, their activities are strongly synergistic, producing combination index values as good or better than seen with antitumor, anti-HIV, and insecticide combinations. Our study provides a powerful means by which anthelmintic combination therapies can be examined and demonstrate that the combination of nAChR agonists and Cry proteins has excellent properties and is predicted to give improved cure rates while being recalcitrant to the development of parasite resistance.

Fig. 1.

nAChR agonist–resistant mutants are hypersusceptible to crystal proteins. (A) Dose-dependent mortality response of wild-type N2 nematodes and ben-1, lev-8, and bre-5 mutant nematodes to purified Cry5B. (B) Dose-dependent mortality response of wild-type N2 and unc-50(ye494) animals to purified Cry5B. (C) Dose-dependent developmental inhibition response of wild-type N2 nematodes and ben-1, lev-8, and bre-5 mutant nematodes to E. coli–expressing Cry5B. (D) Dose-dependent developmental inhibition response of wild-type N2 nematodes and unc-63(ye492) and unc-50(ye494) mutant nematodes to E .coli–expressing Cry5B. The smaller effect seen with unc-63 may be due to the possible nonnull nature of this allele. (E) Effect of 10 μg/mL purified Cry5B on the 64-h brood sizes of N2 wild-type, lev-8, ben-1, and bre-5 mutant nematodes. For A–D and in similar figures below, each data point represents mean ± SEM. LC₅₀ and IC₅₀ values for exposures to Cry5B can be found in Table S1. For E, each bar shows 64-h broods from 15 worms; error bars represent SD. For A–E and in all similar figures below, *P < 0.05 relative to N2; **P < 0.01 relative to N2; and ***P <0.001 relative to N2. The alleles used here and in all other figures are ben-1(e1880), lev-8(ye493), and bre-5(ye17).

Fig. 2.

Effects of Cry21A on H. bakeri and on C. elegans wild-type and mutant strains. (A) In vivo anthelmintic activity of Cry21A against the small intestinal parasite H. bakeri. Shown are intestinal roundworm burdens normalized to average in the placebo control (41.7 worms) following treatment of infected mice with placebo (spore lysates, no Cry21A) or Cry21A spore crystal lysate (SCL). Normalized worm burdens in each mouse are shown (n = 6 for placebo; n = 5 for Cry21A; one animal in the Cry21A group died with a tumor before treatment). Long horizontal bar shows mean worm burdens; smaller bars indicate SEM. (B) Dose-dependent mortality response of wild-type N2 and nAChR agonist-resistant lev-8 mutant nematodes to Cry21A SCLs at 20 ° for 3 days. LC₅₀ values for exposure to Cry21A can be found in Table S1.

Fig. 3.

Cry5B-resistant mutants are hypersusceptible to nAChR agonists. (A) Dose-dependent mortality response of wild-type N2 animals and ben-1, lev-8, and bre-5 mutant animals to tribendimidine. (B) Dose-dependent mortality response of wild-type N2 nematodes and ben-1, lev-8, and bre-5 mutant nematodes to levamisole. (C) Dose-dependent mortality response of wild-type N2 animals and bre-2(ye31) mutant animals to tribendimidine. (D) Dose-dependent mortality response of wild-type N2 animals and bre-2(ye31) mutant animals to levamisole. (E) Dose-dependent developmental inhibition response of wild-type N2 nematodes and ben-1, lev-8, and bre-5 mutant nematodes to tribendimidine. (F) Effect of 25 μg/mL tribendimidine on 64-h brood sizes of N2 wild-type, lev-8, ben-1, and bre-5 nematodes.

Fig. 4.

Cry5B and nAChR agonist combinations are strongly synergistic. (A) Dose-dependent mortality response of wild-type N2 animals to purified Cry5B, tribendimidine, and 1:1 ratio of Cry5B:tribendimidine based on mass. The x axis is plotted in terms of total dose (e.g., 1 μg/mL = 1 μg/mL of Cry5B, 1 μg/mL of tribendimidine, or combination of 0.5 μg/mL of each). (B) Dose-dependent mortality response of wild-type N2 animals to purified Cry5B, levamisole, and 1:1 ratio of Cry5B:levamisole based on mass. (C) Dose-dependent mortality response of wild-type N2 animals to purified Cry5B, tribendimidine, and 1:1 ratio of Cry5B:tribendimidine based on LC₅₀ values. (D) Dose–response curve of wild-type N2 animals to tribendimidine without and with a low dose of Cry5B (2 μg/mL), which normally gives ~5% mortality. LEV, levamisole; Trib, tribendimidine.