Bacillus thuringiensis Cry5B protein is highly efficacious as a single-dose therapy against an intestinal roundworm infection in mice

Abstract

Background: Intestinal parasitic nematode diseases are one of the great diseases of our time. Intestinal roundworm parasites, including hookworms, whipworms, and Ascaris, infect well over 1 billion people and cause significant morbidity, especially in children and pregnant women. To date, there is only one drug, albendazole, with adequate efficacy against these parasites to be used in mass drug administration, although tribendimidine may emerge as a second. Given the hundreds of millions of people to be treated, the threat of parasite resistance, and the inadequacy of current treatments, new anthelmintics are urgently needed. Bacillus thuringiensis (Bt) crystal (Cry) proteins are the most common used biologically produced insecticides in the world and are considered non-toxic to vertebrates.

Methods/principal findings: Here we study the ability of a nematicidal Cry protein, Cry5B, to effect a cure in mice of a chronic roundworm infection caused by the natural intestinal parasite, Heligmosomoides bakeri (formerly polygyrus). We show that Cry5B produced from either of two Bt strains can act as an anthelmintic in vivo when administered as a single dose, achieving a approximately 98% reduction in parasite egg production and approximately 70% reduction in worm burdens when delivered per os at approximately 700 nmoles/kg (90-100 mg/kg). Furthermore, our data, combined with the findings of others, suggest that the relative efficacy of Cry5B is either comparable or superior to current anthelmintics. We also demonstrate that Cry5B is likely to be degraded quite rapidly in the stomach, suggesting that the actual dose reaching the parasites is very small.

Conclusions/significance: This study indicates that Bt Cry proteins such as Cry5B have excellent anthelmintic properties in vivo and that proper formulation of the protein is likely to reveal a superior anthelmintic.

Figure 1. Effects of Cry5B on egg production in Heligmosomoides bakeri infected mice.

Shown are the average eggs/gram of feces/mouse for both placebo (n = 10) and Cry5B HD1-(n = 10) treated groups the day before treatment (−1) and then every other day thereafter until the animals were euthanized on day 5 post-treatment.

Figure 2. Effects of Cry5B on intestinal worm burdens in Heligmosomoides bakeri infected in mice.

A. Shown are the intestinal worm burdens in placebo (HD1 SL) and Cry5B (HD1 SCL) treated mice (n = 10 each group). Infected mice were given a single dose of 715 nmoles/kg (100 mg/kg) Cry5B on day 15 P.I. and intestinal worm burdens assessed on day 20 P.I. The worm burdens in each mouse are indicated with a separate symbol. Long horizontal bars represent mean worm burdens; smaller bars indicate sem (standard error of the mean). B. Shown are the intestinal worm burdens in placebo (4Q7 SL) and Cry5B (4Q7 SCL) treated mice (n = 7 per group). Mice were given a single dose of 644 nmoles/kg (90 mg/kg) Cry5B on day 15 P.I. and intestinal worm burdens assessed on day 20 P.I. ** P<0.01, *** P<0.001.

Figure 3. Effects of tribendimidine on Heligmosomoides bakeri infections in mice.

Shown are the intestinal worm burdens in placebo (20 mM citrate buffer pH 7.3) and for various doses of tribendimidine. Data are plotted as in Figure 1. Infected mice (n = 6/group except n = 5 placebo) were given a single dose of tribendimidine on day 15 P.I. and intestinal worm burdens assessed on day 20 P.I. **P<0.01, ***P<0.001 relative to placebo control. Conversion to molar amounts is as follows: 0.125, 0.5, and 2 mg/kg are equivalent to 0.28, 1.1, and 4.4 µmoles/kg respectively.

Figure 4. Behavior of Cry5B in simulated gastric fluids.

Left lane, markers. Next three lanes: Cry5B loading control 10, 1, and 0.1 µg Cry5B in HD1 SCLs. Next two lanes: Cry5B (10 µg/lane) SCLs in water incubated for 0 or 2 h at 37°. No degradation is seen. Next seven lanes: Cry5B (10 µg/lane) SCLs incubated in simulated gastric fluid (SGF) for the time indicated (in minutes). Cry5B has nearly disappeared after four minutes. Right two lanes: simulated gastric fluids (no Cry5B) incubated for 0 and 2 h at 37° to demonstrate where pepsin runs on the gel. 8% SDS polyacrylamide gel stained with Coomassie blue.