The nematode (Ascaris suum) intestine is a location of synergistic anthelmintic effects of Cry5B and levamisole.

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes.

PRMT-7/PRMT7 activates HLH-30/TFEB to guard plasma membrane integrity compromised by bacterial pore-forming toxins.

Bacterial pore-forming toxins (PFTs) that disrupt host plasma membrane integrity (PMI) significantly contribute to the virulence of various pathogens. However, how host cells protect PMI in response to PFT perforation in vivo remains obscure. Previously, we demonstrated that the HLH-30/TFEB-dependent intrinsic cellular defense (INCED) is elicited by PFT to maintain PMI in Caenorhabditis elegans intestinal epithelium. Yet, the molecular mechanism for the full activation of HLH-30/TFEB by PFT remains elusive. Here, we reveal that PRMT-7 (protein arginine methyltransferase-7) is indispensable to the nuclear transactivation of HLH-30 elicited by PFTs. We demonstrate that PRMT-7 participates in the methylation of HLH-30 on its RAG complex binding domain to facilitate its nuclear localization and activation. Moreover, we showed that PRMT7 is evolutionarily conserved to regulate TFEB cellular localization and repair plasma damage caused by PFTs in human intestinal cells. Together, our observations not only unveil a novel PRMT-7/PRMT7-dependent post-translational regulation of HLH-30/TFEB but also shed insight on the evolutionarily conserved mechanism of the INCED against PFT in metazoans.

An efficient method for viable cryopreservation and recovery of hookworms and other gastrointestinal nematodes in the laboratory.

Hookworms (genera Ancylostoma and Necator) are amongst the most prevalent and important parasites of humans globally. These intestinal parasites ingest blood, resulting in anemia, growth stunting, malnutrition, and adverse pregnancy outcomes. They are also critical parasites of dogs and other animals. In addition, hookworms and hookworm products are being explored for their use in treatment of autoimmune and inflammatory diseases. There is thus a significant and growing interest in these mammalian host-obligate parasites. Laboratory research is hampered by the lack of good means of cryopreservation and recovery of parasites. Here, we describe a robust method for long-term (≥3 year) cryopreservation and recovery of both Ancylostoma and Necator hookworms that is also applicable to two other intestinal parasites that passage through the infective L3 stage, Strongyloides ratti and Heligmosomoides polygyrus bakeri. The key is a revised recovery method, in which cryopreserved L1s are thawed and raised to the infective L3 stage using activated charcoal mixed with uninfected feces from a permissive host. This technique will greatly facilitate research on and availability of gastrointestinal parasitic nematodes with great importance to global health, companion animal health, and autoimmune/inflammatory disease therapies.

Variation in anthelmintic responses are driven by genetic differences among diverse C. elegans wild strains.

Treatment of parasitic nematode infections in humans and livestock relies on a limited arsenal of anthelmintic drugs that have historically reduced parasite burdens. However, anthelmintic resistance (AR) is increasing, and little is known about the molecular and genetic causes of resistance for most drugs. The free-living roundworm Caenorhabditis elegans has proven to be a tractable model to understand AR, where studies have led to the identification of molecular targets of all major anthelmintic drug classes. Here, we used genetically diverse C. elegans strains to perform dose-response analyses across 26 anthelmintic drugs that represent the three major anthelmintic drug classes (benzimidazoles, macrocyclic lactones, and nicotinic acetylcholine receptor agonists) in addition to seven other anthelmintic classes. First, we found that C. elegans strains displayed similar anthelmintic responses within drug classes and significant variation across drug classes. Next, we compared the effective concentration estimates to induce a 10% maximal response (EC10) and slope estimates of each dose-response curve of each strain to the laboratory reference strain, which enabled the identification of anthelmintics with population-wide differences to understand how genetics contribute to AR. Because genetically diverse strains displayed differential susceptibilities within and across anthelmintics, we show that C. elegans is a useful model for screening potential nematicides before applications to helminths. Third, we quantified the levels of anthelmintic response variation caused by genetic differences among individuals (heritability) to each drug and observed a significant correlation between exposure closest to the EC10 and the exposure that exhibited the most heritable responses. These results suggest drugs to prioritize in genome-wide association studies, which will enable the identification of AR genes.

An efficient method for viable cryopreservation of hookworms and other gastrointestinal nematodes in the laboratory.

Hookworms (genera Ancylostoma and Necator ) are amongst of the most prevalent and important parasites of humans globally. These intestinal parasites ingest blood, resulting in anemia, growth stunting, malnutrition, and adverse pregnancy outcomes. They are also critical parasites of dogs and other animals. In addition, hookworms and hookworm products are being explored for their use in treatment of autoimmune and inflammatory diseases. There is thus a significant and growing interest in these mammalian host-obligate parasites. Laboratory research is hampered by the lack of good means of cryopreservation. Here, we describe a robust method for long-term (≥3 year) cryoprotection and recovery of both Ancylostoma and Necator hookworms that is also applicable to two other intestinal parasites that passages through the infective third larval stage, Strongyloides ratti and H eligmosomoides polygyrus bakeri . The key is the use cryo-preserved first-staged larvae raised to the infective third larval stage using activated charcoal mixed with uninfected feces from a permissive host. This technique will greatly facilitate research on and availability of gastrointestinal parasitic nematodes with great importance to global health, companion animal health, and autoimmune/inflammatory disease therapies.

A Caenorhabditis elegans nck-1 and filamentous actin-regulating protein pathway mediates a key cellular defense against bacterial pore-forming proteins.

Pore-forming proteins (PFPs) comprise the largest single class of bacterial protein virulence factors and are expressed by many human and animal bacterial pathogens. Cells that are attacked by these virulence factors activate epithelial intrinsic cellular defenses (or INCEDs) to prevent the attendant cellular damage, cellular dysfunction, osmotic lysis, and organismal death. Several conserved PFP INCEDs have been identified using the nematode Caenorhabditis elegans and the nematicidal PFP Cry5B, including mitogen-activated protein kinase (MAPK) signaling pathways. Here we demonstrate that the gene nck-1, which has homologs from Drosophila to humans and links cell signaling with localized F-actin polymerization, is required for INCED against small-pore PFPs in C. elegans. Reduction/loss of nck-1 function results in C. elegans hypersensitivity to PFP attack, a hallmark of a gene required for INCEDs against PFPs. This requirement for nck-1-mediated INCED functions cell-autonomously in the intestine and is specific to PFPs but not to other tested stresses. Genetic interaction experiments indicate that nck-1-mediated INCED against PFP attack is independent of the major MAPK PFP INCED pathways. Proteomics and cell biological and genetic studies further indicate that nck-1 functions with F-actin cytoskeleton modifying genes like arp2/3, erm-1, and dbn-1 and that nck-1/arp2/3 promote pore repair at the membrane surface and protect against PFP attack independent of p38 MAPK. Consistent with these findings, PFP attack causes significant changes in the amount of actin cytoskeletal proteins and in total amounts of F-actin in the target tissue, the intestine. nck-1 mutant animals appear to have lower F-actin levels than wild-type C. elegans. Studies on nck-1 and other F-actin regulating proteins have uncovered a new and important role of this pathway and the actin cytoskeleton in PFP INCED and protecting an intestinal epithelium in vivo against PFP attack.

Efficient and Scalable Process to Produce Novel and Highly Bioactive Purified Cytosolic Crystals from Bacillus thuringiensis.

Bacillus thuringiensis (Bt) is a Gram-positive soil bacterium that is widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops or are disease vectors. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e.g., Cry5B (formally Cry5Ba1), target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described inactivated bacteria with cytosolic crystal(s) (IBaCC) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here, we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents, yielding purified cytosolic crystals (PCC). Cry5B PCC contains ~95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults in vitro. Cry5B PCC is also highly active in vivo against experimental human hookworm and Ascaris infections in rodents. The process was scaled up to the 100-liter scale to produce PCC for a pilot study to treat two foals infected with the ascarid Parascaris spp. Single-dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new and attractive pharmaceutical ingredient form of Bt Cry proteins. IMPORTANCE Bacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies have shown they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called purified cytosolic crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins is desirable and where these methods will find great utility.

C. elegans monitor energy status via the AMPK pathway to trigger innate immune responses against bacterial pathogens.

Pathogen recognition and the triggering of host innate immune system are critical to understanding pathogen-host interaction. Cellular surveillance systems have been identified as an important strategy for the identification of microbial infection. In the present study, using Bacillus thuringiensis-Caenorhabditis elegans as a model, we found an approach for surveillance systems to sense pathogens. We report that Bacillus thuringiensis Cry5Ba, a typical pore-forming toxin, caused mitochondrial damage and energy imbalance by triggering potassium ion leakage, instead of directly targeting mitochondria. Interestingly, we find C. elegans can monitor intracellular energy status to trigger innate immune responses via AMP-activated protein kinase (AMPK), secreting multiple effectors to defend against pathogenic attacks. Our study indicates that the imbalance of energy status is a prevalent side effect of pathogen infection. Furthermore, the AMPK-dependent surveillance system may serve as a practicable strategy for the host to recognize and defense against pathogens.

Nematode ascarosides attenuate mammalian type 2 inflammatory responses.

Mounting evidence suggests that nematode infection can protect against disorders of immune dysregulation. Administration of live parasites or their excretory/secretory (ES) products has shown therapeutic effects across a wide range of animal models for immune disorders, including asthma. Human clinical trials of live parasite ingestion for the treatment of immune disorders have produced promising results, yet concerns persist regarding the ingestion of pathogenic organisms and the immunogenicity of protein components. Despite extensive efforts to define the active components of ES products, no small molecules with immune regulatory activity have been identified from nematodes. Here we show that an evolutionarily conserved family of nematode pheromones called ascarosides strongly modulates the pulmonary immune response and reduces asthma severity in mice. Screening the inhibitory effects of ascarosides produced by animal-parasitic nematodes on the development of asthma in an ovalbumin (OVA) murine model, we found that administration of nanogram quantities of ascr#7 prevented the development of lung eosinophilia, goblet cell metaplasia, and airway hyperreactivity. Ascr#7 suppressed the production of IL-33 from lung epithelial cells and reduced the number of memory-type pathogenic Th2 cells and ILC2s in the lung, both key drivers of the pathology of asthma. Our findings suggest that the mammalian immune system recognizes ascarosides as an evolutionarily conserved molecular signature of parasitic nematodes. The identification of a nematode-produced small molecule underlying the well-documented immunomodulatory effects of ES products may enable the development of treatment strategies for allergic diseases.

Immune reactivity and host modulatory roles of two novel Haemonchus contortus cathepsin B-like proteases.

BACKGROUND: Haemonchus contortus is a blood-feeding, gastrointestinal nematode (GIN) that causes significant economic losses to the small ruminant industry worldwide. Despite extensive efforts, our understanding of the molecular mechanisms used by GIN to evade host immune responses is limited. Cathepsin B-like proteins (CBPs) are members of the cysteine protease family and are involved in parasite invasion and thus provide viable vaccine candidates. METHODS: In silico comparative analysis was used to identify conserved proteins among a subset of clade V parasitic nematodes with emphasis on blood-feeding worms, among which CBPs appeared prominently. We identified and characterized two novel CBPs designated Hc-CBP-1 and Hc-CBP-2. Rabbit anti-recombinant (r) Hc-CBP-1 and rHc-CBP-2 were used to detect the presence of native proteins in the excretory secretory products (ESP) and in worm tissues of adult H. contortus. Peptide arrays of rHc-CBP-1 and rHc-CBP-2 were screened with the homologous and heterologous anti-sera and with sera from dexamethasone-treated (Dex+) and non-treated (Dex-) H. contortus-infected animals to identify key immunogenic peptides. Gene transcription of Hc-cbp-1 and Hc-cbp-2 was also performed on H. contortus-infected animals treated with Dex+. Finally, the mature recombinant proteins were used to assess their abilities to modulate cell functions. RESULTS: Immunohistochemistry showed that both Hc-CBP-1 and Hc-CBP-2 are present on the brush borders of the intestine; Hc-CBP-2 was also present in the hypodermis of the body wall. Peptide displays screened with rabbit anti-rHc-CBP-1 and anti-rHc-CBP-2 revealed regions within the proteins where dominant and overlapping epitopes prevailed. ELISA results were consistent with only Hc-CBP-1 being present in H. contortus adult ESPs. H. contortus from Dex+ animals exhibited a threefold increase in Hc-cbp-2 transcript while Hc-cbp-1 expression did not change. In contrast, comparisons of immunoreactivities of rHc-CBP-1 and rHc-CBP-2 peptide arrays to sera from Dex+ and Dex- animals primarily showed changes in Hc-CBP-1 binding. Lastly, rHc-CBP-1 suppressed mRNA expression of bovine peripheral blood mononuclear cell cytokines/activation markers, including TNFα, IL-1, IL-6 and CD86. CONCLUSIONS: These results suggest that as secreted and cryptic proteins, respectively, Hc-CBP-1 and Hc-CBP-2 influence cellular and immunological activities that have interesting dynamics during infection and may provide viable immune-related targets for attenuating H. contortus infectivity.

Yeast Particle Encapsulation of Scaffolded Terpene Compounds for Controlled Terpene Release.

Terpenes are naturally occurring compounds produced by plants that are of great commercial interest in the food, agricultural, cosmetic, and pharmaceutical industries due to their broad spectra of antibacterial, antifungal, anthelmintic, membrane permeation enhancement, and antioxidant biological activities. Applications of terpenes are often limited by their volatility and the need for surfactants or alcohols to produce stable, soluble (non-precipitated) products. Yeast particles (YPs) are hollow, porous microspheres that have been used for the encapsulation of terpenes (YP terpenes) by passive diffusion of terpenes through the porous YP cell walls. We here report the development of a second generation YP encapsulated terpene technology that incorporates the stimuli-responsive control of terpene release using biodegradable pro-terpene compounds (YP pro-terpenes). YP terpenes and YP pro-terpenes were both produced, in which high levels of carvacrol, eugenol, thymol and geraniol were encapsulated. The YP pro-terpenes show higher encapsulation stability than YP terpenes due to pro-terpenes being non-volatile solids at room temperature and stable in suspensions at neutral pH. YP pro-terpenes and YP terpenes were evaluated for biological activity in antibacterial, antifungal and anthelmintic assays. The YP pro-terpenes retained the full biological activity of the parent terpene compound.

An inactivated bacterium (paraprobiotic) expressing Bacillus thuringiensis Cry5B as a therapeutic for Ascaris and Parascaris spp. infections in large animals.

Ascaris and Parascaris are important parasites in the family Ascarididae, large, ubiquitous intestinal-dwelling nematodes infecting all classes of vertebrates. Parasitic nematode drug resistance in veterinary medicine and drug recalcitrance in human medicine are increasing worldwide, with few if any new therapeutic classes on the horizon. Some of these parasites are zoonotic, e.g., Ascaris is passed from humans to pigs and vice versa. The development of new therapies against this family of parasites would have major implications for both human and livestock health. Here we tested the therapeutic ability of a paraprobiotic or dead probiotic that expresses the Bacillus thuringiensis Cry5B protein with known anthelmintic properties, against zoonotic Ascaris suum and Parascaris spp. This paraprobiotic, known as IBaCC, intoxicated A. suum larvae in vitro and was highly effective in vivo against intestinal A. suum infections in a new mouse model for this parasite. Fermentation was scaled up to 350 l to treat pigs and horses. Single dose Cry5B IBaCC nearly completely cleared A. suum infections in pigs. Furthermore, single dose Cry5B IBaCC drove fecal egg counts in Parascaris-infected foals to zero, showing at least parity with, and potential superiority to, current efficacy of anthelmintics used against this parasite. Cry5B IBaCC therefore represents a new, paraprobiotic One Health approach towards targeting Ascarididae that is safe, effective, massively scalable, stable, and useful in human and veterinary medicine in both the developed and developing regions of the world.

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

Gastrointestinal nematodes (GINs) of humans, e.g., hookworms, negatively impact childhood growth, cognition, nutrition, educational attainment, income, productivity, and pregnancy. Hundreds of millions of people are targeted with mass drug administration (MDA) of donated benzimidazole anthelmintics. However, benzimidazole efficacy against GINs is suboptimal, and reduced/low efficacy has been seen. Developing an anthelmintic for human MDA is daunting: it must be safe, effective, inexpensive, stable without a cold chain, and massively scalable. Bacillus thuringiensis crystal protein 5B (Cry5B) has anthelmintic properties that could fill this void. Here, we developed an active pharmaceutical ingredient (API) containing B. thuringiensis Cry5B compatible with MDA. We expressed Cry5B in asporogenous B. thuringiensis during vegetative phase, forming cytosolic crystals. These bacteria with cytosolic crystals (BaCC) were rendered inviable (inactivated BaCC [IBaCC]) with food-grade essential oils. IBaCC potency was validated in vitro against nematodes. IBaCC was also potent in vivo against human hookworm infections in hamsters. IBaCC production was successfully scaled to 350 liters at a contract manufacturing facility. A simple fit-for-purpose formulation to protect against stomach digestion and powdered IBaCC were successfully made and used against GINs in hamsters and mice. A pilot histopathology study and blood chemistry workup showed that five daily consecutive doses of 200 mg/kg body weight Cry5B IBaCC (the curative single dose is 40 mg/kg) was nontoxic to hamsters and completely safe. IBaCC is a safe, inexpensive, highly effective, easy-to-manufacture, and scalable anthelmintic that is practical for MDA and represents a new paradigm for treating human GINs.

A new paraprobiotic-based treatment for control of Haemonchus contortus in sheep.

Haemonchus contortus is a critical parasite of goats and sheep. Infection by this blood-feeding gastrointestinal nematode (GIN) parasite has significant health consequences, especially in lambs and kids. The parasite has developed resistance to virtually all known classes of small molecule anthelmintics used to treat it, giving rise in some areas to multidrug resistant parasites that are very difficult to control. Thus, new anthelmintics are urgently needed. Bacillus thuringiensis (Bt) crystal protein 5B (Cry5B), a naturally occurring protein made by a bacterium widely and safely used around the world as a bioinsecticide, represents a new non-small molecule modality for treating GINs. Cry5B has demonstrated anthelmintic activities against parasites of monogastric animals, including some related to those that infect humans, but has not yet been studied in a ruminant. Here we show that H. contortus adults are susceptible to Cry5B protein in vitro. Cry5B produced in its natural form as a spore-crystal lysate against H. contortus infections in goats had no significant efficacy. However, a new Active Pharmaceutical Ingredient (API) paraprobiotic form of Cry5B called IBaCC (Inactivated Bacterium with Cytosolic Crystals), in which Cry5B crystals are encapsulated in dead Bt cell wall ghosts, showed excellent efficacy in vitro against larval stages of H. contortus and relative protein stability in bovine rumen fluid. When given to sheep experimentally infected with H. contortus as three 60 mg/kg doses, Cry5B IBaCC resulted in significant reductions in fecal egg counts (90%) and parasite burdens (72%), with a very high impact on female parasites (96% reduction). These data indicate that Cry5B IBaCC is a potent new treatment tool for small ruminants in the battle against H. contortus.

Anthelmintic Activity of Yeast Particle-Encapsulated Terpenes.

Soil-transmitted nematodes (STN) infect 1-2 billion of the poorest people worldwide. Only benzimidazoles are currently used in mass drug administration, with many instances of reduced activity. Terpenes are a class of compounds with anthelmintic activity. Thymol, a natural monoterpene phenol, was used to help eradicate hookworms in the U.S. South circa 1910. However, the use of terpenes as anthelmintics was discontinued because of adverse side effects associated with high doses and premature stomach absorption. Furthermore, the dose-response activity of specific terpenes against STNs has been understudied. Here we used hollow, porous yeast particles (YPs) to efficiently encapsulate (>95%) high levels of terpenes (52% w/w) and evaluated their anthelmintic activity on hookworms (Ancylostoma ceylanicum), a rodent parasite (Nippostrongylus brasiliensis), and whipworm (Trichuris muris). We identified YP-terpenes that were effective against all three parasites. Further, YP-terpenes overcame albendazole-resistant Caenorhabditis elegans. These results demonstrate that terpenes are broad-acting anthelmintics. Terpenes are predicted to be extremely difficult for parasites to resist, and YP encapsulation provides water-suspendable terpene materials without surfactants and sustained terpene release that could lead to the development of formulations for oral delivery that overcome fast absorption in the stomach, thus reducing dosage and toxic side effects.

Gut microbial signatures associated with moxidectin treatment efficacy of Haemonchus contortus in infected goats.

Parasitic infections are associated with profound changes in the structure and function of the gut microbiome in various host-parasite systems. Here we examined the microbial composition and function in the abomasum, proximal colon and feces of Haemonchus contortus-infected goats after a partial anthelmintic drug clearance. A single-dose treatment of H. contortus-infected goats with Cydectin (moxidectin) resulted in an 83.9 % and 61.8 % reduction in fecal egg counts (EPG) and worm burden, respectively (P < 0.01), and restored abomasal pH to a normal baseline level. The treatment significantly increased the abundance of Proteobacteria, particularly that of Campylobacter, in the proximal colon. It also significantly affected several basic pathways, including bacterial secretion, butyrate metabolism, and LPS biosynthesis, and seemingly reduced the cellulolytic capacity in the colon. Several network modules displayed a strong correlation with EPG and worm burden. The Mantel test indicated a strong correlation between treatment related network topologies of the operational taxonomic units (OTU) belonging to Actinobacteria and Rikenellaceae and EPG and worm burden levels, respectively. Furthermore, microbial signatures that may better predict anthelmintic efficacy were identified. A signature or balance represented by the log ratio of the abundance of Verrucomicrobiaceae and Camplyobacteraceae had a strong correlation with EPG (r = 0.80). These novel insights into the interactions between H. contortus and gut microbiome in the caprine host and the consequence of a partial anthelmintic clearance on animal health and well-being may facilitate the design of more effective next-generation anthelmintics.

Bacillus thuringiensis Cry5B is Active against Strongyloides stercoralis in vitro.

Strongyloidiasis, caused by Strongyloides stercoralis infection, is an important neglected tropical disease that causes significant public health problems in the tropics and subtropics. The disease can persist in hosts for decades and may be life-threatening because of hyperinfection and dissemination. Ivermectin (mostly) and albendazole are the most common anthelmintics used for treatment. Albendazole is suboptimal for this parasite, and although ivermectin is quite effective in immunocompromised patients, a multiple-course regimen is required. Furthermore, reliance on a single drug class for treating intestinal nematodes is a recipe for future failure. Therefore, it is important to discover new anthelmintics to treat or prevent human strongyloidiasis. One promising candidate is the Bacillus thuringiensis crystal protein Cry5B. Cry5B is highly potent against parasitic nematodes, for example, hookworms and Ascaris suum. Here, we investigated the potential of Cry5B against S. stercoralis. Multiple stages of S. stercoralis, including the first larval stage (L1s), infective stage (iL3s), free-living adult stage, and parasitic female stage, were all susceptible to Cry5B as indicated by impairment of motility and decreased viability in vitro. In summary, Cry5B demonstrated strong potential as an effective anthelmintic for treatment and transmission control of human strongyloidiasis, justifying further experiments to investigate in vivo therapeutic efficacy.

Drug Screening for Discovery of Broad-spectrum Agents for Soil-transmitted Nematodes.

Soil-transmitted nematodes (STNs), namely hookworms, whipworms, and ascarids, are extremely common parasites, infecting 1-2 billion of the poorest people worldwide. Two benzimidazoles, albendazole and mebendazole, are currently used in STN mass drug administration, with many instances of low/reduced activity reported. New drugs against STNs are urgently needed. We tested various models for STN drug screening with the aim of identifying the most effective tactics for the discovery of potent, safe and broad-spectrum agents. We screened a 1280-compound library of approved drugs to completion against late larval/adult stages and egg/larval stages of both the human hookworm parasite Ancylostoma ceylanicum and the free-living nematode Caenorhabditis elegans, which is often used as a surrogate for STNs in screens. The quality of positives was further evaluated based on cheminformatics/data mining analyses and activity against evolutionarily distant Trichuris muris whipworm adults. From these data, two pairs of positives, sulconazole/econazole and pararosaniline/cetylpyridinium, predicted to target nematode CYP-450 and HSP-90 respectively, were prioritized for in vivo evaluation against A. ceylanicum infections in hamsters. One of these positives, pararosaniline, showed a significant impact on hookworm fecundity in vivo. Taken together, our results suggest that anthelmintic screening with A. ceylanicum larval stages is superior to C. elegans based on both reduced false negative rate and superior overall quality of actives. Our results also highlight two potentially important targets for the discovery of broad-spectrum human STN drugs.

Identification of small molecule enzyme inhibitors as broad-spectrum anthelmintics.

Targeting chokepoint enzymes in metabolic pathways has led to new drugs for cancers, autoimmune disorders and infectious diseases. This is also a cornerstone approach for discovery and development of anthelmintics against nematode and flatworm parasites. Here, we performed omics-driven knowledge-based identification of chokepoint enzymes as anthelmintic targets. We prioritized 10 of 186 phylogenetically conserved chokepoint enzymes and undertook a target class repurposing approach to test and identify new small molecules with broad spectrum anthelmintic activity. First, we identified and tested 94 commercially available compounds using an in vitro phenotypic assay, and discovered 11 hits that inhibited nematode motility. Based on these findings, we performed chemogenomic screening and tested 32 additional compounds, identifying 6 more active hits. Overall, 6 intestinal (single-species), 5 potential pan-intestinal (whipworm and hookworm) and 6 pan-Phylum Nematoda (intestinal and filarial species) small molecule inhibitors were identified, including multiple azoles, Tadalafil and Torin-1. The active hit compounds targeted three different target classes in humans, which are involved in various pathways, including carbohydrate, amino acid and nucleotide metabolism. Last, using representative inhibitors from each target class, we demonstrated in vivo efficacy characterized by negative effects on parasite fecundity in hamsters infected with hookworms.