A purified Bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite Ancylostoma ceylanicum

Abstract

Crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis (Bt) are harmless to vertebrates, but they are highly toxic to insects and nematodes. Their value in controlling insects that destroy crops and transmit human diseases is well established. Although it has recently been demonstrated that a few individual Bt Cry proteins, such as Cry5B, are toxic to a wide range of free-living nematodes, the potential activity of purified Cry proteins against parasitic nematodes remains largely unknown. We report here studies aimed at characterizing in vitro and in vivo anthelminthic activities of purified recombinant Cry5B against the hookworm parasite Ancylostoma ceylanicum, a bloodfeeding gastrointestinal nematode for which humans are permissive hosts. By using in vitro larval development assays, Cry5B was found to be highly toxic to early stage hookworm larvae. Exposure of adult A. ceylanicum to Cry5B was also associated with significant toxicity, including a substantial reduction in egg excretion by adult female worms. To demonstrate therapeutic efficacy in vivo, hamsters infected with A. ceylanicum were treated with three daily oral doses of purified Cry5B, the benzimidazole anthelminthic mebendazole, or buffer. Compared with control (buffer-treated) animals, infected hamsters that received Cry5B showed statistically significant improvements in growth and blood hemoglobin levels as well as reduced worm burdens that were comparable to the mebendazole-treated animals. These data demonstrate that Cry5B is highly active in vitro and in vivo against a globally significant nematode parasite and that Cry5B warrants further clinical development for human and veterinary use.

Fig. 1.

A. ceylanicum extracts contain Cry5B-binding glycolipids. Upper-phase glycolipids were extracted from mixed-stage C. elegans (Ce) and adult A. ceylanicum (Ac) and separated by TLC. The separated glycolipids were then subjected to a Cry5B overlay in the presence of 80 mM glucose (Glc) (left lanes) or 80 mM galactose (Gal) (right lanes). Cry5B bound to glycolipids from both nematodes in a galactose-dependent fashion.

Fig. 2.

Effects of Cry5B on A. ceylanicum adults in vitro. (A) Six micrograms of purified Cry5B (lane 2) was subjected to SDS/PAGE and Coomassie blue staining. (B) Exposure to Cry5B impairs motility of adult hookworms. Groups of 10 adult A. ceylanicum worms were cultured in increasing concentrations of purified Cry5B toxin. Motility was monitored at the times indicated, and the data represent the mean values (±SE) of triplicate wells containing each concentration of toxin. Statistically significant (P < 0.001) differences in motility were observed between control worms (buffer only) and worms cultured in 50 or 200 μg/ml Cry5B toxin throughout the observation period (24–120 h). Statistically significant differences (P < 0.001) between control worms (0 μg/ml) and the 5 μg/ml toxin group were detected from 42–120 h in culture. (C) Cry5B toxin reduces A. ceylanicum egg excretion. Adult female hookworms were maintained for 24 h in the presence of increasing concentrations of purified Cry5B toxin. Experimental groups consisted of four replicate wells (three worms per well) at each concentration of toxin. Values represent the mean number of eggs (±SE) counted in each well. P values represent statistical comparisons between each group and the control worms (no toxin).

Fig. 3.

Effects of Cry5B on A. ceylanicum larvae. (A) Cry5B toxin impairs motility of early stage (L1/L2) hookworm larvae. A. ceylanicum eggs were allowed to hatch for 48 h in the presence of increasing concentrations of purified Cry5B toxin. Experimental groups consisted of four replicate wells containing larvae (mean of 20–46 per well) at each concentration of toxin. Values represent the mean percent of motile larvae (±SE) counted in each well. P values represent statistical comparisons between each group and the control worms (buffer only). (B) Cry5B toxicity in early larval (L1/L2) development. Representative photomicrographs show stunted growth and loss of integrity of the tegument and internal structures in A. ceylanicum larvae exposed to purified Cry5B toxin (5 μg/ml) for 48 h. (Magnification: ×200.)

Fig. 4.

Cry5B treatment reduces clinical sequelae of hookworm infection as measured by weight gain (Upper) and blood hemoglobin (Lower). Hamsters were infected with 150 A. ceylanicum L3 on day 0 and treated with Cry5B (Left) or mebendazole (Right) on days 14, 15, and 16 PI as indicated by open arrowheads. All values are the means ± SE. Asterisks indicate statistical significance vs. the infected control group. For numerical values, see Results.

Fig. 5.

Cry5B treatment reduces fecal egg excretion (A) and intestinal hookworm burden (B) in infected hamsters. (A) Fecal samples from infected animals were collected at the times indicated, and hookworm eggs were quantified as described in Materials and Methods. All values are the means ± SE. Asterisks indicate statistical significance (P < 0.05) vs. the infected control group. (B) Individual worm burdens are indicated by closed circles, and the means of each group are shown by horizontal bars. Brackets indicate statistical comparisons between groups, with P values shown.