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.

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.

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.

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.

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.

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.

Recombinant subunit vaccines for soil-transmitted helminths.

Soil-transmitted helminths (STHs) collectively infect one fourth of all human beings, and the majority of livestock in the developing world. These gastrointestinal nematodes are the most important parasites on earth with regard to their prevalence in humans and livestock. Current anthelmintic drugs are losing their efficacies due to increasing drug resistance, particularly in STHs of livestock and drug treatment is often followed by rapid reinfection due to failure of the immune system to develop a protective response. Vaccines against STHs offer what drugs cannot accomplish alone. Because such vaccines would have to be produced on such a large scale, and be cost effective, recombinant subunit vaccines that include a minimum number of proteins produced in relatively simple and inexpensive expression systems are required. Here, we summarize all of the previous studies pertaining to recombinant subunit vaccines for STHs of humans and livestock with the goal of both informing the public of just how critical these parasites are, and to help guide future developments. We also discuss several key areas of vaccine development, which we believe to be critical for developing more potent recombinant subunit vaccines with broad-spectrum protection.

The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins.

BACKGROUND: The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action. RESULTS: The two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action. CONCLUSIONS: Cry6 proteins are members of the alpha helical pore-forming toxins - a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.

Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic.

The Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production, cry5B was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in Lactococcus lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain L. lactis KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from L. lactis cultures expressing both Cry5B and tCry5B, in vivo challenges of Caenorhabditis elegans worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from L. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe.

Novel role for the yceGH tellurite resistance genes in the pathogenesis of Bacillus anthracis.

Bacillus anthracis, the causative agent of anthrax, relies on multiple virulence factors to subvert the host immune defense. Using Caenorhabditis elegans as an infection model, we screened approximately 5,000 transposon mutants of B. anthracis Sterne for decreased virulence. One of the attenuated mutants resulted in loss of expression of yceG and yceH, the last two genes in a six-gene cluster of tellurite resistance genes. We generated an analogous insertional mutant to confirm the phenotype and characterize the role of yceGH in resistance to host defenses. Loss of yceGH rendered the mutants more sensitive to tellurite toxicity as well as to host defenses such as reactive oxygen species and the cathelicidin family of antimicrobial peptides. Additionally, we see decreased survival in mammalian models of infection, including human whole blood and in mice. We identify a novel role for the yceGH genes in B. anthracis Sterne virulence and suggest that C. elegans is a useful infection model to study anthrax pathogenesis.

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.

Global functional analyses of cellular responses to pore-forming toxins.

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Bacillus subtilis strain engineered for treatment of soil-transmitted helminth diseases.

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 (LC50) of 4.3 µg/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 µmol/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.

Discovery of a highly synergistic anthelmintic combination that shows mutual hypersusceptibility.

The soil-transmitted helminths or nematodes (hookworms, whipworms, and Ascaris) are roundworms that infect more than 1 billion of the poorest peoples and are leading causes of morbidity worldwide. Few anthelmintics are available for treatment, and only one is commonly used in mass drug administrations. New anthelmintics are urgently needed, and crystal (Cry) proteins made by Bacillus thuringiensis are promising new candidates. Combination drug therapies are considered the ideal treatment for infectious diseases. Surprisingly, little work has been done to define the characteristics of anthelmintic combinations. Here, by means of quantitative assays with wild-type and mutants of the roundworm Caenorhabditis elegans, we establish a paradigm for studying anthelmintic combinations using Cry proteins and nicotinic acetylcholine receptor (nAChR) agonists, e.g., tribendimidine and levamisole. We find that nAChR agonists and Cry proteins, like Cry5B and Cry21A, mutually display what is known in the HIV field as hypersusceptibility--when the nematodes become resistant to either class, they become hypersensitive to the other class. Furthermore, we find that when Cry5B and nAChR agonists are combined, their activities are strongly synergistic, producing combination index values as good or better than seen with antitumor, anti-HIV, and insecticide combinations. Our study provides a powerful means by which anthelmintic combination therapies can be examined and demonstrate that the combination of nAChR agonists and Cry proteins has excellent properties and is predicted to give improved cure rates while being recalcitrant to the development of parasite resistance.

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.

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.

Structure and glycolipid binding properties of the nematicidal protein Cry5B.

Crystal (Cry) proteins are globally used in agriculture as proteinaceous insecticides. They have also been recently recognized to have great potential as anthelmintic agents in targeting parasitic roundworms (e.g., hookworms). The most extensively characterized of the anthelmintic Cry proteins is Cry5B. We report here the 2.3 Å resolution structure of the proteolytically activated form of Cry5B. This structure, which is the first for a nematicidal Cry protein, shows the familiar three-domain arrangement seen in insecticidal Cry proteins. However, domain II is unusual in that it more closely resembles a banana lectin than it does other Cry proteins. This result is consistent with the fact that the receptor for Cry5B consists of a set of invertebrate-specific glycans (attached to lipids) and also suggests that domain II is important for receptor binding. We found that not only galactose but also N-acetylgalactosamine (GalNAc) is an efficient competitor for binding between Cry5B and glycolipids. GalNAc is one of the core arthroseries tetrasaccharides of the Cry5B receptor and galactose an antennary sugar that emanates from this core. These and prior data suggest that the minimal binding determinant for Cry5B consists of a core GalNAc and two antennary galactoses. Lastly, the protoxin form of Cry5B was found to bind nematode glycolipids with a specificity equal to that of activated Cry5B, but with lower affinity. This suggests that the initial binding of Cry5B protoxin to glycolipids can be stabilized at the nematode cell surface by proteolysis. These results lay the groundwork for the design of effective Cry5B-based anthelmintics.

Eosinophils in the zebrafish: prospective isolation, characterization, and eosinophilia induction by helminth determinants.

Eosinophils are granulocytic leukocytes implicated in numerous aspects of immunity and disease. The precise functions of eosinophils, however, remain enigmatic. Alternative models to study eosinophil biology may thus yield novel insights into their function. Eosinophilic cells have been observed in zebrafish but have not been thoroughly characterized. We used a gata2:eGFP transgenic animal to enable prospective isolation and characterization of zebrafish eosinophils, and demonstrate that all gata2(hi) cells in adult hematopoietic tissues are eosinophils. Although eosinophils are rare in most organs, they are readily isolated from whole kidney marrow and abundant within the peritoneal cavity. Molecular analyses demonstrate that zebrafish eosinophils express genes important for the activities of mammalian eosinophils. In addition, gata2(hi) cells degranulate in response to helminth extract. Chronic exposure to helminth- related allergens resulted in profound eosinophilia, demonstrating that eosinophil responses to allergens have been conserved over evolution. Importantly, infection of adult zebrafish with Pseudocapillaria tomentosa, a natural nematode pathogen of teleosts, caused marked increases in eosinophil number within the intestine. Together, these observations support a conserved role for eosinophils in the response to helminth antigens or infection and provide a new model to better understand how parasitic worms activate, co-opt, or evade the vertebrate immune response.

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.