Bacillus subtilis Strain Engineered for Treatment of Soil-Transmitted Helminth Diseases

Abstract

c Soil-transmitted helminths (hookworms, whipworms, and large roundworms) are agents of intestinal roundworm diseases of poverty that infect upwards of 2 billion people worldwide. A great challenge in treating these diseases is the development of anthelmintic therapeutics that are inexpensive, can be produced in great quantity, and are capable of delivery under varied and adverse environmental conditions. A potential solution to this challenge is the use of live bacteria that are acceptable for human consumption, e.g., Bacillus subtilis, and that can be engineered with therapeutic properties. In this study, we expressed the Bacillus thuringiensis anthelmintic protein Cry5B in a bacterial strain that has been used as a model for live bacterial therapy, Bacillus subtilis PY79. PY79 transformed with a Cry5B expression plasmid (PY79-Cry5B) is able to express Cry5B from the endogenous B. thuringiensis cry5B promoter. During sporulation of PY79-Cry5B, Cry5B is packaged as a crystal. Furthermore, Cry5B produced in PY79 is bioactive, with a 50% lethal concentration (LC 50 ) of 4.3 g/ml against the roundworm Caenorhabditis elegans. PY79-Cry5B was a significantly effective therapeutic in experimental Ancylostoma ceylanicum hookworm infections of hamsters. A single 10-mg/kg (0.071 mol/kg of body weight) dose of Cry5B administered as a Cry5B-PY79 spore crystal lysate achieved a 93% reduction in hookworm burdens, which is superior on a molar level to reductions seen with clinically used anthelmintics. Given that a bacterial strain such as this one can be produced cheaply in massive quantities, our results demonstrate that the engineering and delivery of live bacterial strains have great potential to treat a significant contributor to poverty worldwide, namely, hookworm disease and other soil-transmitted helminthiasis.

Recombinant Paraprobiotics as a New Paradigm for Treating Gastrointestinal Nematode Parasites of Humans

“…We therefore set out to deliver Cry5B, without purification of the protein, as part of a dead bacterial product (or "paraprobiotic" (50)). To make a Cry5B paraprobiotic, we initially turned to and produced Cry5B spore-crystal lysates, which have been used for most published in vivo studies of Cry5B anthelmintic efficacy as well as all Bt insecticidal production and use (31,33,34,43,45). We attempted to inactivate (kill) the spores without losing activity of the crystal protein using gamma irradiation (51).…”

“…Hamsters were infected with A. ceylanicum, and the infestations were allowed to proceed to mature adults with the appearance of parasite eggs (fecundity) excreted into the hamster feces (31,33,44). These hamsters were then treated with IBaCC produced in our laboratory containing 10 mg/kg Cry5B ( Figure 5A) or treated with IBa (vector-only control produced at the same time and processed identically to Cry5B…”

“…More than a dozen different 3D-Cry proteins have been tested and found to be completely safe to vertebrates at doses of >>1000 mg/kg, and are FDA/EPA-approved for ingestion (28,29). The 3D-Cry protein Cry5B is related to the 3D-Cry proteins used as insecticides but targets nematodes instead., When administered orally as spore-crystal lysates or SCLs (a mix of Bt spores and Cry protein crystals as they naturally occur), Cry5B is highly effective against GIN infections in hamsters, pigs, and dogs (30)(31)(32)(33)(34).…”

LyophilizedB. subtilisZB183 Spores: 90-Day Repeat Dose Oral (Gavage) Toxicity Study In Wistar Rats

“…ZB183 is a viable genetically modified B. subtilis strain that constitutively expresses an acetaldehyde dehydrogenase enzyme, AcoD, from C. necator (which has been known by a number of other names, including Alcaligenes eutrophus, Ralstonia eutropha, and Hydrogenomonas eutropha). The parental strain is B. subtilis PY79, a widely used strain in laboratory studies with a published sequence, that is lacking of many mobile genetic elements found in other B. subtilis strains, and has been found to be nontoxic to vertebrates and has been studied in humans [5][6][7].…”

Genetically Engineering Bacillus subtilis with a Heat-Resistant Arsenite Methyltransferase for Bioremediation of Arsenic-Contaminated Organic Waste

“…For example, B. subtilis PY79 was used to express the Bacillus thuringiensis anthelmintic protein Cry5B to control soil-transmitted helminth diseases (19). B. subtilis strain 168 has been well characterized.…”