Whole-Genome Analysis of Bacillus thuringiensis Revealing Partial Genes as a Source of Novel Cry Toxins.
ABSTRACT: Despite the successful application of crystal proteins (Cry) from Bacillus thuringiensis as biological control agents against insects, there is an increasing demand to identify new Cry toxins having higher toxicity and broad-spectrum activity against insects and plant-parasitic nematodes. To find novel Cry toxins, we screened 100 whole-genome sequences of B. thuringiensis Surprisingly, in addition to full Cry toxins, we found partial sequences, such as typical N-terminal or C-terminal regions with conserved domains, widely distributed among 20 strains of B. thuringiensis In order to further elucidate the functions of partial genes, here, we selected a partial sequence from strain C15, having 28% similarity with the N terminus of Cry5Ba and lacking a typical C terminus, and denoted it Cry5B-like N terminus. This fragment when coexpressed as a fusion protein with the C terminus of Cry5Ba (N-C fusion protein) produces pyramidal crystals. A recombinant N-C fusion protein having a 50% lethal concentration (LC50) of 23.7 ?g/ml severely affected the life span, growth, and survival rate of nematodes. Light microscopy showed damage to the intestine of nematodes, confirming the pathogenicity of the N-C fusion protein. Last, the green fluorescent protein (GFP)-labeled mutant Caenorhabditis elegans FT63 showed significant damage to the intestine upon feeding N-C fusion toxin compared to the control. These results imply that partial genes can be a source of new Cry toxins, and further understanding about functions of partial cry genes can help in the study of the evolutionary strategy of B. thuringiensis to produce the multidomain toxins.IMPORTANCE Genomic analysis revealed that coding sequences for N termini and C termini of crystal proteins are widely distributed in B. thuringiensis We found Cry5B-like N terminus, lacking typical C terminus, was unable to be expressed in wild-type strain C15. However, its fusion with the C terminus of Cry5Ba not only was successfully expressed but also exhibited activity against the nematodes. This study provides insight into a potential source for novel Cry toxins.
Project description: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 ?g/ml and 23.9 ?g/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.
Project description:Pathogen avoidance behavior protects animal hosts against microbial pathogens. Pathogens have evolved specific strategies during coevolution in response to such host defenses. However, these strategies for combatting host avoidance behavioral defenses remain poorly understood. Here, we used Caenorhabditis elegans and its bacterial pathogen Bacillus thuringiensis as a model and determined that small RNA (sRNA)-mediated Cry toxin silencing allowed pathogens to evade host avoidance behavioral defenses. The B. thuringiensis strain YBT-1518, which encodes three nematicidal cry genes, is highly toxic to C. elegans. However, the expression of the most potent toxin, Cry5Ba, was silenced in this strain when YBT-1518 was outside the host. Cry5Ba silencing was due to the sRNA BtsR1, which bound to the RBS site of the cry5Ba transcript via direct base pairing and inhibited Cry5Ba expression. Upon ingestion by C. elegans, Cry5Ba was expressed in vivo by strain YBT-1518. Cry5Ba silencing may allow B. thuringiensis to avoid nematode behavioral defenses and then express toxins once ingested to kill the host and gain a survival advantage. Our work describes a novel model of sRNA-mediated regulation to aid pathogens in combating host avoidance behavioral defenses.
Project description:Crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis (Bt) are harmless to vertebrates, but they are highly toxic to insects and nematodes. Their value in controlling insects that destroy crops and transmit human diseases is well established. Although it has recently been demonstrated that a few individual Bt Cry proteins, such as Cry5B, are toxic to a wide range of free-living nematodes, the potential activity of purified Cry proteins against parasitic nematodes remains largely unknown. We report here studies aimed at characterizing in vitro and in vivo anthelminthic activities of purified recombinant Cry5B against the hookworm parasite Ancylostoma ceylanicum, a bloodfeeding gastrointestinal nematode for which humans are permissive hosts. By using in vitro larval development assays, Cry5B was found to be highly toxic to early stage hookworm larvae. Exposure of adult A. ceylanicum to Cry5B was also associated with significant toxicity, including a substantial reduction in egg excretion by adult female worms. To demonstrate therapeutic efficacy in vivo, hamsters infected with A. ceylanicum were treated with three daily oral doses of purified Cry5B, the benzimidazole anthelminthic mebendazole, or buffer. Compared with control (buffer-treated) animals, infected hamsters that received Cry5B showed statistically significant improvements in growth and blood hemoglobin levels as well as reduced worm burdens that were comparable to the mebendazole-treated animals. These data demonstrate that Cry5B is highly active in vitro and in vivo against a globally significant nematode parasite and that Cry5B warrants further clinical development for human and veterinary use.
Project description:Cry proteins of Bacillus thuringiensis (Bt) have been successfully used as biopesticides and in transgenic crops throughout the world. However, resources against the most serious agricultural pathogens, plant root-knot nematodes, are limited. The genomes of several highly nematicidal virulent Bt strains from our laboratory have been sequenced, facilitating the identification of novel Cry proteins and other virulence factors. We identified two novel Cry proteins, Cry5Ca1 and Cry5Da1, that exhibit high toxicity against Meloidogyne incognita Using the Caenorhabditis elegans model, the two Cry5 toxins were shown to negatively affect nematode life span, fertility, and survival. The 50% lethal concentrations (LC50s) of Cry5Ca1 and Cry5Da1 were 57.22 ?g/ml and 36.69 ?g/ml, respectively. Moreover, a synergistic effect (synergism factor, 1.61 to 2.04) was observed for nematicidal toxicity of Cry5Ca1 and Cry5Da1, which is accordant with the phylogenetic results suggesting that domain II of the two novel Cry5 toxins evolved into two independent clades. Through comparison of the depressed degree of toxicity in the ?-methylgalactoside detoxification test, we found that the novel toxin Cry5D possesses a different galactose-binding epitope; meanwhile, the finding that Cry5D does not share a motif (GXXXE) in the corresponding loop of domain II with Cry5B could explain the different galactose binding performance. Additionally, low-level cross-resistance of C. elegans bre mutant strains was evident between Cry5B and Cry5D. These results suggest that Cry5D can be used as an alternative to delay the potential resistance of nematodes to Cry5B.IMPORTANCE Although proper gene resources for Bt crops against the most serious agricultural pathogens, plant root-knot nematodes, are limited, we have identified two novel nematicidal toxins, Cry5Ca1 and Cry5Da1, against M. incognita, which have supplied more gene candidates for Bt crops designed against nematodes. Moreover, the association of the dissimilarity between Cry5Da1 and Cry5Ba1 and their low cross-resistance can be attributed not only to a low sequence similarity of domain II but also to the structural difference of the key motif and receptor-binding epitope in the loops. This association facilitates the selection of a proper candidate for the prospective design of pyramided Bt crops that can delay potential resistance.
Project description:Some Bacillus thuringiensis strains have high toxicity to nematodes. Nematicidal activity has been found in several families of crystal proteins, such as Cry5, Cry6, and Cry55. The B. thuringiensis strain YBT-1518 has three cry genes that have high nematicidal activity. The whole genome sequence of this strain contains multiple potential virulence factors. To evaluate the pathogenic potential of virulence factors, we focused on a metalloproteinase called Bmp1. It encompasses a consecutive N-terminal signal peptide, an FTP superfamily domain, an M4 neutral protease GluZincin superfamily, two Big-3 superfamily motifs, and a Gram-positive anchor superfamily motif as a C-terminal domain. Here, we showed that purified Bmp1 protein showed metalloproteinase activity and toxicity against Caenorhabditis elegans (the 50% lethal concentration is 610 ± 9.37 μg/ml). In addition, mixing Cry5Ba with Bmp1 protein enhanced the toxicity 7.9-fold (the expected toxicity of the two proteins calculated from their separate toxicities) against C. elegans. Confocal microscopic observation revealed that Bmp1 protein was detected from around the mouth and esophagus to the intestine. Striking microscopic images revealed that Bmp1 degrades intestine tissues, and the Cry5Ba causes intestinal shrinkage from the body wall. Thus, the B. thuringiensis Bmp1 metalloproteinase is a nematicidal virulence factor. These findings give a new insight into the relationship between B. thuringiensis and its host nematodes.
Project description: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.
Project description:The ability of nano- and microparticles of partially oxidized mesoporous silicon (pSi) to sequester, protect, and deliver the anthelmintic pore-forming protein Cry5B to nematodes is assessed in vitro and in vivo. Thermally oxidized pSi particles are stable under gastric conditions and show relatively low toxicity to nematodes. Fluorescence images of rhodamine-labeled pSi particles within the nematodes Caenorhabditis elegans and Ancylostoma ceylanicum show that ingestion is dependent on particle size: particles of a 0.4 ± 0.2 ?m size are noticeably ingested by both species within 2 h of introduction in vitro, whereas 5 ± 2 ?m particles are excluded from C. elegans but enter the pharynx region of A. ceylanicum after 24 h. The anthelmintic protein Cry5B, a pore-forming crystal (Cry) protein derived from Bacillus thuringiensis, is incorporated into the pSi particles by aqueous infiltration. Feeding of Cry5B-loaded pSi particles to C. elegans leads to significant intoxication of the nematode. Protein-loaded particles of size 0.4 ?m display the highest level of in vitro toxicity toward C. elegans on a drug-mass basis. The porous nanostructure protects Cry5B from hydrolytic and enzymatic (pepsin) degradation in simulated gastric fluid (pH 1.2) for time periods up to 2 h. In vivo experiments with hookworm-infected hamsters show no significant reduction in worm burden with the Cry5B-loaded particles, which is attributed to slow release of the protein from the particles and/or short residence time of the particles in the duodenum of the animal.
Project description: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.
Project description:Bacillus thuringiensis insecticidal Cry toxins break down larval midgut-cells after forming pores. The 3D-structures of Cry4Ba and Cry5Ba revealed a trimeric-oligomer after cleavage of helices ?-1 and ?-2a, where helix ?-3 is extended and made contacts with adjacent monomers. Molecular dynamic simulations of Cry1Ab-oligomer model based on Cry4Ba-coordinates showed that E101 forms a salt-bridge with R99 from neighbor monomer. An additional salt bridge was identified in the trimeric-Cry5Ba, located at the extended helix ?-3 in the region corresponding to the ?-2b and ?-3 loop. Both salt-bridges were analyzed by site directed mutagenesis. Single-point mutations in the Lepidoptera-specific Cry1Ab and Cry1Fa toxins were affected in toxicity, while reversed double-point mutant partially recovered the phenotype, consistent with a critical role of these salt-bridges. The single-point mutations in the salt-bridge at the extended helix ?-3 of the nematicidal Cry5Ba were also non-toxic. The incorporation of this additional salt bridge into the nontoxic Cry1Ab-R99E mutant partially restored oligomerization and toxicity, supporting that the loop between ?-2b and ?-3 forms part of an extended helix ?-3 upon oligomerization of Cry1 toxins. Overall, these results highlight the role in toxicity of salt-bridge formation between helices ?-3 of adjacent monomers supporting a conformational change in helix ?-3.
Project description:Bacillus thuringiensis (Bt) is the most successful, environmentally-friendly, and intensively studied microbial insecticide. The major characteristic of Bt is the production of proteinaceous crystals containing toxins with specific activity against many pests including dipteran, lepidopteran, and coleopteran insects, as well as nematodes, protozoa, flukes, and mites. These crystals allow large quantities of the protein toxins to remain stable in the environment until ingested by a susceptible host. It has been previously established that 135 kDa Cry proteins have a crystallization domain at their C-terminal end. In the absence of this domain, Cry proteins often need helper proteins or other factors for crystallization. In this review, we classify the Cry proteins based on their requirements for crystallization.