Project Summary/Abstract Soil-transmitted helminth or soil-transmitted nematode (STN) infections are intestinal parasitic nematodes, mainly Ascaris, hookworms, and whipworms. They are amongst the most prevalent parasites on earth and cause severe morbidity in children, including growth stunting, intellectual and educational impairment, malnutrition, anemia, and lower future earning;; they also have significant impacts on pregnant women and worker productivity. Single dose mass drug administration (MDA) to treat STNs relies on a single drug class, the benzimidazoles (BZs). BZs have poor efficacy against whipworms and highly variable efficacy against hookworms. BZ resistance alleles have been detected in STNs and there are clear examples of low BZ efficacy against all parasites. New mechanism-of-action and broadly potent therapies for STNs are urgently needed. The soil- bacterium Bacillus thuringiensis (Bt) is the number one biological insecticide agent in the world. The insect- active components are crystal (Cry) proteins that kill insects but that are harmless to vertebrates (no effect >1000 mg/kg). Insecticidal Bt Cry proteins are expressed in transgenic food crops (e.g., >80% of USA corn) and are FDA approved for ingestion. The nematode-active Cry protein, Cry5B (related to insecticidal Cry proteins), cures hookworm and Ascaris infections in large animals and can kill whipworms in vitro. Novel Cry5B microbiologically- derived forms called IBaCC and PCC, which are compatible with MDA, have been developed. The Cry protein, Cry21A, has also emerged as a potentially highly potent anthelmintic against STN parasites and an excellent stacking partner for Cry5B. Cry5B and Cry21A make a combination predicted to be difficult for nematodes to resist. This proposal aims to optimize Cry5B and Cry21A against all STNs by 1. taking advantage of the tremendous resource of natural variants of these proteins found in the environment as part of Bt’s evolution and survival, 2. optimizing acid neutralization and bacterial expression systems, and 3. testing key hypotheses about how best to target whipworms, the most difficult of the parasites to treat. Moderate throughput in vitro assays will be used to efficiently inform in vivo assays for all three parasite classes. Efficacy of Cry proteins against multidrug resistant hookworms will be studied. Potential resistance against Cry proteins will be addressed upfront by studying how Cry5B and Cry21A proteins can be combined to provide resistance-busting efficacy;; stacking of Cry proteins is now the norm for insecticidal application. This proposal is unique in 1. applying a safe and validated natural resource, Bt, to a wholly new application, contr...