In vitro anthelmintic effects of cysteine proteinases from plants against intestinal helminths of rodents

Infections with gastrointestinal (GI) nematodes are amongst the most prevalent worldwide, especially in tropical climates. Control of these infections is primarily through treatment with anthelmintic drugs, but the rapid development of resistance to all the currently available classes of anthelmintic means that alternative treatments are urgently required. Cysteine proteinases from plants such as papaya, pineapple and fig are known to be substantially effective against three rodent GI nematodes, Heligmosomoides polygyrus, Trichuris muris and Protospirura muricola, both in vitro and in vivo. Here, based on in vitro motility assays and scanning electron microscopy, we extend these earlier reports, demonstrating the potency of this anthelmintic effect of plant cysteine proteinases against two GI helminths from different taxonomic groups - the canine hookworm, Ancylostoma ceylanicum, and the rodent cestode, Rodentolepis microstoma. In the case of hookworms, a mechanism of action targeting the surface layers of the cuticle indistinguishable from that reported earlier appears to be involved, and in the case of cestodes, the surface of the tegumental layers was also the principal location of damage. Hence, plant cysteine proteinases have a broad spectrum of activity against intestinal helminths (both nematodes and cestodes), a quality that reinforces their suitability for development as a much-needed novel treatment against GI helminths of humans and livestock.     

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

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.     

A Bacillus thuringiensis S-layer protein involved in toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)

The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.     

Anthelmintic profile of methyl 5(6)-(4-methylpiperidin-1-yl) carbonylbenzimidazole-2-carbamate in experimental helminthiases

Biological evaluation of methyl 5(6)-(4-methylpiperidin-1-yl) carbonylbenzimidazole-2-carbamate against Ancylostoma ceylanicum, Nippostrongylus brasiliensis, Syphacia obvelata, Hymenolepis nana, H. diminuta and Cysticercus fasciolaris in experimental animals is reported. The compound (mg/kg) causes 100% elimination of A. ceylanicum (25 x 1), N. brasiliensis (100 x 1), S. obvelata (50 x 1), H. nana (250 x 3) and C. fasciolaris (50 x 10). It was also effective against the developing larvae (L3, L4 and L5) of A. ceylanicum at a single oral dose of 100 mg/kg. Another study indicated that the compound elicits 100% response within 32 hr of drug administration. The drug is well tolerated and LD50 is greater than 4500 mg/kg.     

Bacillus thuringiensis DB27 Produces Two Novel Protoxins,Cry21Fa1 and Cry21Ha1, Which Act Synergistically against Nematodes

Bacillus thuringiensis has been widely used as a biopesticide, primarily for the control of insect pests, but some B. thuringiensis strains specifically target nematodes. However, nematicidal virulence factors of B. thuringiensis are poorly investigated. Here, we describe virulence factors of nematicidal B. thuringiensis DB27 using Caenorhabditis elegans as a model. We show that B. thuringiensis DB27 kills a number of free-living and animal-parasitic nematodes via intestinal damage. Its virulence factors are plasmid-encoded Cry protoxins, since plasmid-cured derivatives do not produce Cry proteins and are not toxic to nematodes. Whole-genome sequencing of B. thuringiensis DB27 revealed multiple potential nematicidal factors, including several Cry-like proteins encoded by different plasmids. Two of these proteins appear to be novel and show high similarity to Cry21Ba1. Named Cry21Fa1 and Cry21Ha1, they were expressed in Escherichia coli and fed to C. elegans, resulting in intoxication, intestinal damage, and death of nematodes. Interestingly, the effects of the two protoxins on C. elegans are synergistic (synergism factor, 1.8 to 2.5). Using purified proteins, we determined the 50% lethal concentrations (LC₅₀s) for Cry21Fa1 and Cry21Ha1 to be 13.6 μg/ml and 23.9 μg/ml, respectively, which are comparable to the LC₅₀ of nematicidal Cry5B. Finally, we found that signaling pathways which protect C. elegans against Cry5B toxin are also required for protection against Cry21Fa1. Thus, B. thuringiensis DB27 produces novel nematicidal protoxins Cry21Fa1 and Cry21Ha1 with synergistic action, which highlights the importance of naturally isolated strains as a source of novel toxins.     

Bacillus thuringiensis-derived Cry5B Has Potent Anthelmintic Activity against Ascaris suum

Ascaris suum and Ascaris lumbricoides are two closely related geo-helminth parasites that ubiquitously infect pigs and humans, respectively. Ascaris suum infection in pigs is considered a good model for A. lumbricoides infection in humans because of a similar biology and tissue migration to the intestines. Ascaris lumbricoides infections in children are associated with malnutrition, growth and cognitive stunting, immune defects, and, in extreme cases, life-threatening blockage of the digestive tract and aberrant migration into the bile duct and peritoneum. Similar effects can be seen with A. suum infections in pigs related to poor feed efficiency and performance. New strategies to control Ascaris infections are needed largely due to reduced treatment efficacies of current anthelmintics in the field, the threat of resistance development, and the general lack of new drug development for intestinal soil-transmitted helminths for humans and animals. Here we demonstrate for the first time that A. suum expresses the receptors for Bacillus thuringiensis crystal protein and novel anthelmintic Cry5B, which has been previously shown to intoxicate hookworms and which belongs to a class of proteins considered non-toxic to vertebrates. Cry5B is able to intoxicate A. suum larvae and adults and triggers the activation of the p38 mitogen-activated protein kinase pathway similar to that observed with other nematodes. Most importantly, two moderate doses of 20 mg/kg body weight (143 nM/kg) of Cry5B resulted in a near complete cure of intestinal A. suum infections in pigs. Taken together, these results demonstrate the excellent potential of Cry5B to treat Ascaris infections in pigs and in humans and for Cry5B to work effectively in the human gastrointestinal tract.     

Efficacy of methyl 5(6)-(alpha-hydroxyphenylmethyl) benzimidazole-2-carbamate, a metabolite of mebendazole, against developing forms of experimental helminth parasites.

Methyl 5(6)-(alpha-hydroxyphenylmethyl) benzimidazole-2- carbamate, a metabolite of mebendazole, was evaluated against metamorphic forms of Ancylostoma ceylanicum in hamsters, Nippostrongylus brasiliensis in rats and cysticercoids of Hymenolepis nana in grain beetles. The test compound offered better action than mebendazole except against H. nana cysticercoids where the activity of the compound and mebendazole was comparable, but was inferior to the standard cestodicidal drug, praziquantel. The results suggest that the action was better by ip route compared to per os route of drug administration.     

The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans

The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.     

Cloning and characterization of a Bacillus thuringiensis serovar higo gene encoding a novel class of the delta-endotoxin protein, Cry27A, specifically active on the Anopheles mosquito

A novel gene encoding a 98-kDa mosquitocidal delta-endotoxin protein, designated Cry27A, was cloned from a Bacillus thuringiensis serovar higo strain. The Cry27A protein contained the five sequence blocks of amino acids commonly conserved in most B. thuringiensis Cry proteins. Relatively high homologies, ranging from 43.0% to 84.4%, existed between the Cry27A protein and several established classes of mosquitocidal Cry proteins (Cry4A, Cry10A, Cry19A, Cry19B, and Cry20A) in the sequence of 51 N-terminal amino acids. The complete sequence of this protein, however, showed low levels (<40%) of amino acid identity to those of the known Cry proteins. Although the expression level of the cry27A gene was low in the transformants under the control of its own promoter, the use of the cyt1A promoter resulted in high-level expression of the gene, leading to the formation of inclusions. The expressed Cry27A protein showed larvicidal activity highly specific for Anopheles stephensi, but lacked the toxicity against Culex pipiens molestus and Aedes aegypti. The results suggest that the Cry27A protein is responsible for the Anopheles-preferential toxicity of the B. thuringiensis serovar higo strain.     

Specific mutations within the alpha4-alpha5 loop of the Bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170

The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry delta-endotoxins suggests that helices alpha4 and alpha5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the alpha4-alpha5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis. As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-beta-Dthiogalactopyranoside induction. When E. coli cells expressing each mutant toxin were assayed against Aedes aegypti mosquito larvae, the activity was almost completely abolished for N166A and Y170A mutations, whereas E171A showed only a small reduction in toxicity. Further analysis of these two critical residues by induction of specific mutations revealed that polarity at position 166 and highly conserved aromaticity at position 170 within the alpha4-alpha5 loop play a crucial role in the larvicidal activity of the Cry4B toxin.     

Mtx toxins synergize Bacillus sphaericus and Cry11Aa against susceptible and insecticide-resistant Culex quinquefasciatus larvae

Two mosquitocidal toxins (Mtx) of Bacillus sphaericus, which are produced during vegetative growth, were investigated for their potential to increase toxicity and reduce the expression of insecticide resistance through their interactions with other mosquitocidal proteins. Mtx-1 and Mtx-2 were fused with glutathione S-transferase and produced in Escherichia coli, after which lyophilized powders of these fusions were assayed against Culex quinquefasciatus larvae. Both Mtx proteins showed a high level of activity against susceptible C. quinquefasciatus mosquitoes, with 50% lethal concentrations (LC(50)) of Mtx-1 and Mtx-2 of 0.246 and 4.13 microg/ml, respectively. The LC(50)s were 0.406 to 0.430 microg/ml when Mtx-1 or Mtx-2 was mixed with B. sphaericus, and synergy improved activity and reduced resistance levels. When the proteins were combined with a recombinant Bacillus thuringiensis strain that produces Cry11Aa, the mixtures were highly active against Cry11A-resistant larvae and resistance was also reduced. The mixture of two Mtx toxins and B. sphaericus was 10 times more active against susceptible mosquitoes than B. sphaericus alone, demonstrating the influence of relatively low concentrations of these toxins. These results show that, similar to Cyt toxins from B. thuringiensis subsp. israelensis, Mtx toxins can increase the toxicity of other mosquitocidal proteins and may be useful for both increasing the activity of commercial bacterial larvicides and managing potential resistance to these substances among mosquito populations.     

A broad spectrum Kunitz type serine protease inhibitor secreted by the hookworm Ancylostoma ceylanicum

Although blood-feeding hookworms infect over a billion people worldwide, little is known about the molecular mechanisms through which these parasitic nematodes cause gastrointestinal hemorrhage and iron deficiency anemia. A cDNA corresponding to a secreted Kunitz type serine protease inhibitor has been cloned from adult Ancylostoma ceylanicum hookworm RNA. The translated sequence of the A. ceylanicum Kunitz type inhibitor 1 (AceKI-1) cDNA predicts a 16-amino acid secretory signal sequence, followed by a 68-amino acid mature protein with a molecular mass of 7889 daltons. Recombinant protein (rAceKI-1) was purified from induced lysates of Escherichia coli transformed with the rAceKI-1/pET 28a plasmid, and in vitro studies demonstrate that rAceKI-1 is a tight binding inhibitor of the serine proteases chymotrypsin, pancreatic elastase, neutrophil elastase, and trypsin. AceKI-1 inhibitory activity is present in soluble protein extracts and excretory/secretory products of adult hookworms but not the infective third stage larvae. The native AceKI-1 inhibitor has been purified to homogeneity from soluble extracts of adult A. ceylanicum using size exclusion and reverse-phase high pressure liquid chromatography. As a potent inhibitor of mammalian intestinal proteases, AceKI-1 may play a role in parasite survival and the pathogenesis of hookworm anemia.     

Metabolism of lipid peroxidation products by the gastro-intestinal nematodes Necator americanus, Ancylostoma ceylanicum and Heligmosomoides polygyrus.

Somatic extracts of the three parasitic nematodes Necator americanus, Ancylostoma ceylanicum and Heligmosomoides polygyrus were able to detoxify a model hydroperoxide and a putative natural peroxide by glutathione-dependent peroxidase activity while cytotoxic carbonyls could be metabolized by NADPH-linked reduction activities. Unlike cestodes and digeneans, the nematodes in this study could not enzymatically conjugate carbonyls with glutathione. The results indicate that the three nematodes can protect themselves against possible host-immune initiated lipid peroxidation of their membranes at the level of the hydroperoxide and at the level of cytotoxic carbonyl, although other protective enzymatic mechanisms are also likely to exist (superoxide dismutase and catalase).     

Antimicrobial activity of different proteins and their fragments from Bacillus thuringiensis parasporal crystals against clostridia and archaea

Proteins of parasporal crystals (Cry proteins) from entomopathogenic bacterium Bacillus thuringiensis (subspecies kurstaki, galleriae, tenebrionis) as well as some fragments of these proteins, obtained by limited proteolysis, are capable of antimicrobial action against anaerobic bacteria and archaea-Clostridium butyricum, Clostridium acetobutylicum and Methanosarcina barkeri. The MICs are 45-150 microg/mL. Electron microscopy showed that lysis of M. barkeri cells in the presence of 49kDa fragment of Cry3Aa toxin is generally similar to the bacterial cell lysis, which has been previously detected in the presence of Cry11A, Cry1Ab and other Cry proteins. The Cry1D-like toxin from crystals of B. thuringiensis subsp. galleriae has been put forward as an example of the supposition that cell wall and some of its components like teichoic acid and N-acetylgalactosamine have possible influence on Cry toxins, enhancing their antimicrobial activity. The possible ecological role of the antimicrobial activity of Cry proteins is also discussed.     

Improvement of Crystal Solubility and Increasing Toxicity against Caenorhabditis elegans by Asparagine Substitution in Block 3 of Bacillus thuringiensis Crystal Protein Cry5Ba

The crystal proteins from Bacillus thuringiensis are widely used for their specific toxicity against insects and nematodes. The highly conserved sequence blocks play an important role in Cry protein stability and flexibility, the basis of toxicity. The block 3 in Cry5Ba subfamily has a shorter sequence (only 12 residues) and more asparagine residues than that of others which harbor about 48 residues but only one asparagine. Based on the theoretical structure model of Cry5Ba, all three asparagines in block 3 are closely located in the interface of putative three domains, implying their probable importance in structure and function. In this study, all three asparagines in Cry5Ba2 block 3 were individually substituted with alanine by site-directed mutagenesis. The wild-type and mutant proteins were overexpressed and crystallized in acrystalliferous B. thuringiensis strain BMB171. However, the crystals formed in one of the mutants, designated N586A, abnormally disappeared and dissolved into the culture supernatant once the sporulation cells lysed, whereas the Cry5Ba crystal and the other mutant crystals were stable. The mutant N586A crystal, isolated from sporulation cells by the ultrasonic process, was found to be easily dissolved at wide range of pH value (5.0 to 10.0). Moreover, the toxicity assays showed that the mutant N586A exhibited nearly 9-fold-higher activity against nematodes and damaged the host''s intestine more efficiently than the native Cry5Ba2. These data support the presumption that the amide residue Asn586 at the interface of domains might adversely affect the protein flexibility, solubility and resultant toxicity of Cry5Ba.     

Interaction of 65- and 62-kD proteins from the apical membranes of the Aedes aegypti larvae midgut epithelium with Cry4B and Cry11A endotoxins of Bacillus thuringiensis

A protein with the molecular weight of 65 kD is the only component of Aedes aegypti larvae BBM capable to specifically bind mosquitocidal toxins Cry4B and Cry11A of Bacillus thuringiensis. This protein lacks the leucine aminopeptidase activity which is characteristic for the toxin-binding proteins from the membranes of caterpillars. Cry-toxins inactive against A. aegypti larvae either fail to bind to the 65-kD protein and to a putative product of its proteolysis with the molecular weight of 62 kD (Cry1Ab), or bind but do not compete for this binding with mosquitocidal proteins (Cry9A). The proteolytic splitting out of the first five -helices in the Cry4B toxin molecule does not affect its binding to the 65- and 62-kD proteins, but an additional removal of 20-30 amino acids from the C-terminal of the molecule sharply spoils this binding. Monosaccharide residues are not involved in the binding of the 65- and 62-kD proteins with Cry4B, Cry11A, and Cry9A.     

The mucosal cellular response to infection with Ancylostoma ceylanicum

Although hookworms are known to stimulate inflammatory responses in the intestinal mucosa of their hosts, there is little quantitative data on this aspect of infection. Here we report the results of experiments conducted in hamsters infected with Ancylostoma ceylanicum. Infection resulted in a marked increase in goblet cells in the intestinal mucosa, which was dependent on the number of adult worms present and was sustained as long as worms persisted (over 63 days) but returned to baseline levels within 7 days of the removal of worms by treatment with ivermectin. Increased mast cell responses were also recorded. Levels were again dependent on the intensity of worm burdens and lasted as long as 63 days after infection. When worms were eliminated, mast cell numbers took over 2 weeks to return to normal. Paneth cell numbers fell soon after infection, the degree of reduction being dependent on the worm burden. After clearance of worms, Paneth cell numbers returned to normal within a week, but then rebounded and numbers rose to higher levels than those in control na?ve animals. The time course of the response was similar whether animals experienced a chronic low-intensity infection without loss of worms or a higher intensity infection during the course of which worm burdens were gradually reduced. Clearly, A. ceylanicum was able to induce a marked inflammatory response in its host's intestine which was sustained for over 9 weeks after infection, and which hamsters appeared able to tolerate well. Our data draw attention to the resilience of hookworms which, unlike many other nematodes, are able to survive for many weeks in a highly inflamed intestinal tract.     

A Bacillus thuringiensis S-Layer Protein Involved in Toxicity against Epilachna varivestis (Coleoptera: Coccinellidae)

The use of Bacillus thuringiensis as a biopesticide is a viable alternative for insect control since the insecticidal Cry proteins produced by these bacteria are highly specific; harmless to humans, vertebrates, and plants; and completely biodegradable. In addition to Cry proteins, B. thuringiensis produces a number of extracellular compounds, including S-layer proteins (SLP), that contribute to virulence. The S layer is an ordered structure representing a proteinaceous paracrystalline array which completely covers the surfaces of many pathogenic bacteria. In this work, we report the identification of an S-layer protein by the screening of B. thuringiensis strains for activity against the coleopteran pest Epilachna varivestis (Mexican bean beetle; Coleoptera: Coccinellidae). We screened two B. thuringiensis strain collections containing unidentified Cry proteins and also strains isolated from dead insects. Some of the B. thuringiensis strains assayed against E. varivestis showed moderate toxicity. However, a B. thuringiensis strain (GP1) that was isolated from a dead insect showed a remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against E. varivestis but not against the lepidopteran Manduca sexta or Spodoptera frugiperda or against the dipteran Aedes aegypti. The gene encoding this protein was cloned and sequenced. It corresponded to an S-layer protein highly similar to previously described SLP in Bacillus anthracis (EA1) and Bacillus licheniformis (OlpA). The phylogenetic relationships among SLP from different bacteria showed that these proteins from Bacillus cereus, Bacillus sphaericus, B. anthracis, B. licheniformis, and B. thuringiensis are arranged in the same main group, suggesting similar origins. This is the first report that demonstrates that an S-layer protein is directly involved in toxicity to a coleopteran pest.