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

et al. (2012) Mechanistic and Single-Dose In Vivo Therapeutic Studies of Cry5B Anthelmintic Action against Hookworms. PLoS Negl Trop Dis 6(11): e1900. doi:10.1371/journal.pntd.0001900 Mechanistic and Single-Dose In Vivo Therapeutic Studies of Cry5B Anthelmintic Action against Hookworms Yan Hu 0 Bin Zhan 0 Brian Keegan 0 Ying Y. Yiu 0 Melanie M. Miller 0 Kathryn Jones 0 Raffi V. Aroian 0 Timothy G. Geary, McGill University, Canada 0 1 Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America, 2 Section of Tropical Medicine, Department of Pediatrics, Baylor College of Medicine , Houston, Texas , United States of America 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. - Funding: This work was funded by National Institutes of Health/National Institute of Allergy and Infectious grant 2R01AI056189 to R.V.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Hookworms (Ancylostoma duodenale, Necator americanus, and, less commonly, Ancylostoma ceylanicum) are major soil-transmitted helminths (nematodes, roundworms) that parasitize humans, infecting 576740 million people globally and are the leading source of iron-deficient anemia in endemic areas [1]. Hookworms are estimated by some to be second only to malaria in terms of disease burden by a parasite ([2,3] where they are associated with ,22 million disability adjusted life years). Hookworm infections lead to blood loss and anemia that most significantly impacts children and pregnant women leading to growth and cognitive stunting in children and increased risk of death or low birthweight babies during pregnancy. For mass drug administration against soil-transmitted helminths like hookworms, the current drug of choice is albendazole [4]. The single-dose cure rate with albendazole for hookworm infection is variable with an average of about 70%, leaving clear room for improvement [5]. More worrisome are increasing numbers of reports with lower efficacies of albendazole and possible resistance [6,7]. Resistance to the class of drugs to which albendazole belongs is already rampant in veterinary medicine [8,9]. Past experiences with infectious diseases (e.g., malaria) has taught us that reliance on one drug for treatment of hundreds of millions of infected peoples inevitably leads to treatment failure. One promising group of alternative anthelmintics is roundworm-active crystal proteins, in particular Cry5B, made by Bacillus thuringiensis [10]. Three 14 mg/kg Cry5B doses administered once/day for three days resulted in an 89% reduction in Ancylostoma ceylanicum hookworm burdens in hamsters [11]. A. ceylanicum is a zoonotic hookworm species, closely related to the major human parasite Ancylostoma duodenale, and is emerging as an important human parasite in Southeast Asia [12]. A. ceylanicum infections in hamsters are also considered a good model for 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 irondeficient 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. hookworm disease in humans [13]. Cry5B is also effective against Heligmosomoides bakeri (polygyrus) roundworm infections in mice as a single 90 mg/kg dose was able to reduce parasite burdens by 70% [14]. It was furthermore shown that Cry5B rapidly degrades in simulated gastric fluids, suggesting that protection of Cry5B from gastric fluids should increase its efficacy [14]. The response of the free-living roundworm Caenorhabditis elegans to Cry5B has been extensively studied and analyzed, including via genetic screens, microarray analyses, whole-genome RNAi analyses, and (...truncated)