Mechanistic and Single-Dose In Vivo Therapeutic Studies of Cry5B Anthelmintic Action against Hookworms

Abstract

Background

Hookworm infections are one of the most important parasitic infections of humans worldwide, considered by some second only to malaria in associated disease burden. Single-dose mass drug administration for soil-transmitted helminths, including hookworms, relies primarily on albendazole, which has variable efficacy. New and better hookworm therapies are urgently needed. Bacillus thuringiensis crystal protein Cry5B has potential as a novel anthelmintic and has been extensively studied in the roundworm Caenorhabditis elegans. Here, we ask whether single-dose Cry5B can provide therapy against a hookworm infection and whether C. elegans mechanism-of-action studies are relevant to hookworms.

Methodology/Principal Findings

To test whether the C. elegans invertebrate-specific glycolipid receptor for Cry5B is relevant in hookworms, we fed Ancylostoma ceylanicum hookworm adults Cry5B with and without galactose, an inhibitor of Cry5B- C. elegans glycolipid interactions. As with C. elegans, galactose inhibits Cry5B toxicity in A. ceylanicum. Furthermore, p38 mitogen-activated protein kinase (MAPK), which controls one of the most important Cry5B signal transduction responses in C. elegans, is functionally operational in hookworms. A. ceylanicum hookworms treated with Cry5B up-regulate p38 MAPK and knock down of p38 MAPK activity in hookworms results in hypersensitivity of A. ceylanicum adults to Cry5B attack. Single-dose Cry5B is able to reduce by >90% A. ceylanicum hookworm burdens from infected hamsters, in the process eliminating hookworm egg shedding in feces and protecting infected hamsters from blood loss. Anthelmintic activity is increased about 3-fold, eliminating >97% of the parasites with a single 3 mg dose (∼30 mg/kg), by incorporating a simple formulation to help prevent digestion in the acidic stomach of the host mammal.

Conclusions/Significance

These studies advance the development of Cry5B protein as a potent, safe single-dose anthelmintic for hookworm therapy and make available the information of how Cry5B functions in C. elegans in order to study and improve Cry5B function against hookworms.

Author Summary

Hookworm infections are one of the great parasitic diseases of our time, infecting more than half a billion people worldwide and are a significant source of iron-deficient anemia. Although mass drug administrations to eliminate hookworms from children and pregnant women are being deployed, all the drugs for treatment we have lack full potency against the parasites and are showing signs of reduced efficacy. Crystal proteins, like Cry5B, made by Bacillus thuringiensis are as a class considered safe to vertebrates and have been shown to have efficacy against intestinal roundworms like hookworms. Here we show that the key mechanistic details of how Cry5B functions in hookworms is conserved with that of the model free-living roundworm Caenorhabditis elegans, which has implications for confirming Cry5B safety in vertebrates and for enhancing Cry5B efficacy against roundworms. Furthermore, we show that Cry5B works effectively as a single-dose drug against hookworm infections in hamsters and can be formulated to increase its efficacy, eliminating 97% of the parasites in a single dose. These results advance the development of a novel, safe single-dose therapy for hookworm infections in humans.