Some toxins from several sources for which experimentally determined structures are available in the Protein Data Bank (PDB).
1. Introduction
Insect pests destroy about 18% of crop production each year and transmit disease agents (Oerke & Dehn, 2004). Beetles (order Coleoptera) are the largest and most diverse group of eukaryotes. They contain species of harvest pests that produce major losses around the world (Wang et al., 2007). Some examples of coleopteran pests follow:
The biological control of insect pests is an important alternative to the management of insects (or Integrated Pest Management-IPM). Unfortunately insect pests have been attacked primarily with chemical products, which cause huge environmental losses and adverse effects on human health. However, biological control and IPM-compatible chemicals can be used together [as outlined in a recent review by Gentz et al., (2010). Extensive research has centred on the search for an appropriate insecticidal peptide or polypeptide with toxicity to pest organisms, but not to flora and fauna. Researchers also hope to establish the most appropriate means of delivering the biological molecule to its site of action (De Lima et al., 2007). Recombinant DNA technology allows the exploitation of the insecticidal properties of entomopathogenic organisms. It offers environmentally friendly options for the cost-effective control of insect pests (St Leger & Wang, 2010). Bioinsecticides include microbial agents, natural enemies, plant defences, metabolites, pheromones and genes that transcribe toxic peptides or proteins. The number and variety of toxins is extensive. For example, there are at least 0.5 million insecticidal toxins from arachnids, and evidence suggests that the use of novel toxic factors is likely to be extensive (Whetstone & Hammock 2007).
2. Typical anti-insect toxins
There are two classes of insecticidal toxins: (1) peptide-like toxins (3-10 kDa) from some scorpion and spider venoms and (2) the high molecular mass toxins (
Toxins from arthropod venoms consist of combinations of biologically active compounds (peptides, proteins, nucleotides, lipids and other molecules). They are used for paralysing insects and for defence against natural enemies. They interact with ion channels and/or receptors from neurological systems in the target organism (De Lima et al., 2007).Venom-derived peptide toxins target voltage-gated Na+, K+, Ca2+, or Cl- channels. Proteins, such as neuropeptides and hormones, are analogous. Their effects depend upon their specific activities (Whetstone & Hammock, 2007). Antagonists disrupt and interfere with development and behaviour. Spiders and scorpions maybe the most important arthropods having insecticidal toxins. Many spider venoms contain a complex mixture of both neurotoxic and cytolytic toxins (see: www.arachnoserver.org). Virtually all insecticidal spider toxins contain a cystine-knot motif that provides them with chemical and biological stability (King et al., 2002; Tedford et al., 2004). These types of venoms contain acylpolyamines (from the Araneidae family), cytolytic toxins (from the Zodariidae family) and neurotoxic peptides (J-atratoxins), and neurotoxins (>10 kDa) and enzymes (~35 kDa) in the Sicariidae and Theridiidae families respectively (Vassilevski et al., 2009; Gunning et al., 2008).
Scorpions are a special group of organisms that have interesting toxins. These toxins have 23-78 residues. Generally the conformation has an α-helix packed against a three-stranded β-sheet stabilized by four disulfide bonds. Scorpion toxins recognize the face of voltage-dependent sodium channels and alter their gating. They are defined as α-or β-toxins, based on their mechanism of action (Rodríguez de la Vega et al., 2010; Gurevitz et al., 2007; Karbat et al., 2004). Anti-insect α-toxins bind to voltage-dependent sodium channels with high affinity (Gordon et al., 2007). Scorpion β-toxins change the voltage dependence of channel activation. The first class of entomopathogenic scorpion β-toxins is comprised of excitatory toxins. They are composed of 70-76 amino acids. These toxins may induce spastic paralysis by the activation of sodium flux at negative membrane potential. A second group consists of depress ant toxins, which induce flaccid paralysis by depolarization of the axonal membrane. A third set is composed of active toxins, which act on both insect and mammalian sodium channels, with typical depressant effects on insects (Gurevitz et al., 2007).
Surprisingly, some insects (such as the tobacco hornworm
Microorganisms possess toxins for the biological control of insects. Fungus is an entomopathogenic option.
Plants produce a great variety of toxic compounds that are responsible for insect self-defense mechanisms. Plant cyclotides contain 30 amino acids with acyclic peptide backbone and a knotted alignment of three conserved disulphide bonds connected in a “cystine knot” motif. Members of Lepidoptera and Coleoptera are susceptible to plant cyclotides from the Violaceae, Rubiaceae and Cucurbitaceae families (Gruber et al., 2007). Plant cysteine proteases are accumulated after lepidopteran infestation affecting insect growth (Pechan et al., 2002). Plant defensins are antimicrobial proteins with eight conserved cysteines and four disulfide bridges. Defensins attack lepidopteran α-amylases, causing feeding inhibition (Kanchiswamy et al., 2010; Rayapuram & Baldwin, 2008). Plant glucanases, chitinases, lectins and dehydrins are induced after attack by lepidopteran and coleopteran pests (Ralph et al., 2006).
3. The phylogenetic relationship of insecticidal toxins and their comparison with lepidopteran- and coleopteran-specific molecules
Twenty-seven amino acid sequences from the RCSB Protein Data Bank (PDB) (http://www.pdb.org/pdb/home/home.do) were selected by a bibliographical revision, using the criteria of established insect-specific toxicity. Next a phylogenetic analysis of insect-specific toxins was performed (Figure 1) by means of Phylogeny.fr platform (http://www.phylogeny.fr/) (Dereeeper et al., 2008). The available data from a bibliographical search,show insecticidal protein sequences from a large variety of organisms with toxicity against several orders of targets, including 11 anti-lepidopteran toxins and five coleopteran-specific toxins (Table 1).
1AVB | Arcelin 1 | Coleoptera | Fabre et al., 1998; Mourey et al., 1998 | |
1AXH | ω-ACTX-HV1 | Lepidoptera, Diptera, Ixodida | Chong et al., 2007; Fletcher et al., 1997 | |
1BCG | Bjxtr-IT | Blattaria | Possani et al., 1999; Oren et al., 1998 | |
1BMR | Lqh III | Blattaria | Krimm et al., 1999 | |
1CIY | Cry1Aa | Lepidoptera | Grochulski et al., 1995; López-Pazos & Cerón, 2007 | |
1DLC | Cry3A | Coleoptera | Li et al., 1991; López-Pazos & Cerón, 2007 | |
1EIT | μ-agatoxin | Diptera | Adams, 2004; Omecinsky et al., 1996 | |
1G92 | Poneratoxin | Lepidoptera | Szolajska et al., 2004 | |
1G9P | ω-Atracotoxin-HV2A | Orthoptera | Chong et al., 2007; Wang et al., 2001 | |
1HRL | PP1 | Lepidoptera | Yu et al., 1999; Skinner et al., 1991 | |
1I5P | Cry2Aa | Lepidoptera, Diptera | Morse et al., 2001; López-Pazos & Cerón, 2007 | |
1I6G | CsE-v5 | Blattaria | Jablonsky et al., 2001; Possani et al., 1999; Lee et al., 1994 | |
1JI6 | Cry3Bb1 | Coleoptera | Galitsky et al., 2001; López-Pazos & Cerón, 2007 | |
1LQI | Lqh(α)IT | Diptera | Tugarinov et al., 1997; Zilberberg et al., 1997 | |
1I25 | Huwentoxin-II | Blattaria | Liang., 2004; Shu et al., 2002 | |
1NB1 | Kalata B1 | Lepidoptera | Rosengren et al., 2003; Gruber et al., 2007 | |
1OMY | BmKaIT1 | Diptera, Orthoptera | Ji et al., 1996 | |
1QS1 | VIP2 | Lepidoptera | Han et al., 1999 | |
1TI5 | VrD1 | Coleoptera | Liu et al., 2006 | |
1T0Z | BmK IT-AP | Lepidoptera | Li et al., 2005; Hao et al., 2005 | |
1V90 | δ-palutoxin IT1 | Lepidoptera | De Lima et al., 2007; Ferrat et al., 2005 | |
1WWN | BmK-βIT | It displays toxicity against Diptera and is related with AaIT from against Blattaria, Orthoptera, Diptera and Coleoptera | Pava-Ripoll et al., 2008; Zlotkin et al., 2000 | |
1W99 | Cry4Ba | Diptera | Boonserm et al., 2005; López-Pazos & Cerón, 2007 | |
2C9K | Cry4Aa | Diptera | van Frankenhuyzen, 2009; Boonserm et al., 2006 | |
2E2S | Agelenin | Orthoptera | Yamaji et al., 2007 | |
2I61 | LqhIT2 | Lepidoptera, Diptera | Karbat et al., 2007; De Lima et al., 2007 | |
2JZM | Chymotrypsin inhibitor C1 | Lepidoptera | Schirra et al., 2008; Schirra et al., 2001; Miller et al., 2000 |
The observed toxin phylogenies - specifically active against lepidopteran species - have several relationships among them and are distributed along all of the branches (Figure 1).
4. Insecticidal toxins and site-directed mutagenesis: case reports
Site-directed mutagenesis is a powerful methodology for studying function and protein structure through manipulation at the level of the DNA molecule. Advances in site-directed mutagenesis have allowed the transfer of new or improved gene roles between organisms, such as bacteria, plants and animals (Adair & Wallace, 1998; James & Dickinson, 1998). In this section, we describe several experiences of the application of site-directed mutagenesis on insecticidal toxin sequences.
4.1. Mutagenesis exposes essential residues in the anti-insect toxin Av2 from Anemonia viridis
Sea anemones (Metazoa, Cnidaria, Anthozoa, and Hexacorallia) are sessile predators that are highly dependent on their venom for prospering in a wide range of ecological environments. Venom analysis shows a significant collection of low molecular weight toxins: ~20 kDa pore-forming toxins, 3.5–6.5 kDa voltage-gated potassium channel-active toxins and 3–5 kDa polypeptide toxins active on voltage-gated sodium channels (Navs) (Moran et al., 2009). [A Nav has a central role in the excitability of animals. It functions in the initiation and propagation of action potentials (Goldin, 2002).]
The
4.2. Mutagenesis demonstrates that N183 is a key residue for the mode of action of the Cry4Ba protein
A collection of Cry4Ba mutants (Figure 2), which are modified in polar uncharged residues (Y178, Q180, N183, N185, and N195) within α-helix 5, were developed to observe their effects on biological activity. All mutant toxins were generated using PCR-based site-directed mutagenesis, and each mutant was expressed from the
Other studies indicated that N183 plays a crucial role in both toxic and structural properties. Mutants N183Q and N183K were made so as to be insoluble at alkaline pH. Mutations at N183 using several residues (with different structural characteristics) revealed that substitutions with a polar amino acid still retained lethal activity similar to the Cry4Ba standard. Nevertheless, changes to charged or nonpolar residues suppressed biological activity (Figure 2). In conclusion, N183 polarity and α-helix 5 localization (in the middle of domain I) are very important to the toxicity of the Cry4Ba protein (Likitvivatanavong et al., 2006).
4.3. A Juvenile hormone esterase with a mutated α helix shows improved insecticidal effects
Juvenile hormone (JH) regulates several physiological events in insects (development, metamorphosis, reproduction, diapause, migration, polyphenism and metabolism). JH esterase (JHE) is a hydrolytic enzyme from the α/β-hydrolase fold family, which metabolizes JH (Kamita et al., 2003). When JHE is injected into lepidopteran larval states, it causes a darkening and a decrease in feeding (Hammock et al., 1990; Philpott & Hammock, 1990). JHE is rapidly cleared from the haemolymph following inoculation, suggesting a discriminatory system for its elimination (El-Sayed et al., 2011). In testing, it was revealed that the double histidine mutated JHE [JHE K204H and R208H (in an amphipathic α helix)] is capable of blocking clearance from the haemolymph by reducing its binding to the JHE receptor. These experiments used
Mutant and wild-type JHEs were produced and purified from insect cells, and their activities were found in the culture supernatants of insect cells. The specific activity of mutant JHE was 6.5 nmol of JH III acid (a metabolism product of JH by JHE) formed min-1 mg-1. The specific activity of wild-type JHE was 61.3 nmol of JH III acid formed min-1 mg-1. The K204H and/or R208H alterations, although far-removed from the catalytic site of the protein, induced allosteric properties that led to a decrease in activity. No statistically significant differences were seen in the clearance of JH hydrolysis activity in the fourth instars of
Mutant JHE Wild type JHE | 1.8 (1.0-2.6) (1.8-3.8) | |
Mutant JHE Wild type JHE | 6.3 (3.6-13) 3.3 (2.3-4.6) |
4.4. Predicting important residues responsible for the capacity of scorpion α-toxins to discriminate between insect and mammalian voltage-gated sodium channels
Scorpion toxins are poison molecules (61–67 amino acids). Scorpion α-toxins recognize voltage-gated sodium channels (NaCh). NaChs mediate the temporary increase in sodium ion permeability thereby generating action potentials. The toxin expands the action potential by delaying the inactivation stage (Gordon et al., 2007). LqhαIT, from the scorpion
Mutations in the cDNAs of
LqhαIT | 13 ng |
Aah2 | "/> 10 µg |
Aah2LqhαIT(8–10) | "/> 10 µg |
Aah2LqhαIT(56–64) | "/> 10 µg |
Aah2LqhαIT(8–10, 56–64) | 64 ng |
Aah2LqhαIT(8–10, G17F, 56–64) | 37 ng |
The similar activities of Aah2LqhαIT(8–10, G17F, 56–64) and LqhαIT indicate that their functional NC-domains are equally oriented. This indicates that the increase of insecticidal activity is related to the arrangement of the NC-domain in a structure that projects into the solvent. Remarkably this conformation is universal to all scorpion α-toxins with lethality on insects, in contrast with the flat face in α-toxins that are toxic to mammals (Karbat et al., 2004).
5. Final remarks
5.1. Novel sources?
Whole-genome sequencing projects are a resource of biological functions and their annotation allows for the detection of proteins through orthologous sequences (common ancestry), searches and primary and tertiary structure correlation - a process named “comparative genomics” (Lee et al. 2007; Ellegren, 2008). This theoretical approach makes it possible to find candidate toxins in sequenced genomes. An appropriate criterion for the identification of novel lepidopteran and coleopteran candidate toxins can be understood in terms of the "guilt by association" principle (Gabaldon &Huynen, 2004; Aravind, 2000). For this reason, we applied a very basic protocol (Figure 3). BLAST (tblastn) searches from the National Centre for Biotechnology Information (NCBI) (http://blast.ncbi.nlm.nih.gov /Blast.cgi). Searches were done using each toxin (from Table 1) as a query. The iterative searches were done for proteins larger than 100 aminoacids with an inclusion threshold of 0.01 (the statistical significance limit for inclusion of a sequence in the process) and for proteins smaller than 100 aminoacids with an inclusion threshold of 0.1. The searches used the 881 completely sequenced bacterial and archaeal genomes available on the NCBI Microbial Genomes website at the time of this analysis (January 2011) and the entire NCBI environmental samples database (1.66 million Whole Genome Shotgun reads) (see http://www.ncbi.nlm.nih.gov/). The searches were done until either convergence was achieved or until the last iteration before the first known false positives appeared. Significant hits to proteins encoded in these genomes were further classified as possible insect-specific toxins. The BLAST analysis showed fourteen microbial sequences with a high similarity to insecticidal queries (Table 4). There is a version of Arcelin 1 encoded in the genome of the cyanobacterium
For our trial, the most important organisms harbouring lepidopteran- and coleopteran-active toxins are
We built tertiary (3D) structures of some of the predicted toxins: a lepidopteran-active toxin, a coleopteran-specific toxin and a toxin from a metagenome sequence. Approximately 30% sequence identity in the primary sequence is required for the generation of useful structures (Forster, 2002; Paramasivan et al., 2006). Tertiary models of candidate insecticidal sequences were constructed by homology modelling using the crystal structure of homologous protein from the RCSB PDB database (http://www.pdb.org/pdb/home/home.do). We used SWISS-MODEL (http://swissmodel.expasy.org/) (Arnold et al., 2006) for the identification of templates (Table 4 footnotes). The structural alignments were generated with DeepView Swiss-PdbViewer 4.0 software (http://spdbv.vital-it.ch/) (Guex & Peitsch, 1997).
The final models (Figure 4) have a range of 33% to 37% identity with the templates. The toxins in Figure 4 correspond to the following (A) NCBI ID NC_009925.1 from the
INSECTICIDAL TOXIN (ID PDB) | ||||
1AVBA | NC_009925.1 | 3e-10 | 1669294- 1669911 | |
1CIY, 1DLCB, 1I5P, 1JI6*, 1W99 and 2C9K** | NC_010180 | 8e-97- 2e-10 | 139296-138751 | |
NC_003552.1 | 4e-19- 1e-04 | 3249335-3249832 | ||
NC_013037.1 | 1e-15- 4e-06 | 2869719-2870441 | ||
NC_012491.1 | 5e-16- 0.0261 | 4962833-4963585 | ||
NC_007347.1 | 1e-08- 3.32 | 411729-411409 | ||
1QS1 | ABHF02000033.1 | 2e-41 | 223624-224649 | |
NZ_ABDW01000012.1 | 3e-39 | 66996-65971 | ||
NC_012946.1 | 1e-33 | 103322-104389 | ||
NC_003030.1 | 5e-17 | 398379-398876 | ||
NZ_ACMN01000162.1 | 1e-21 | 17703-17065 | ||
NC_002570.2 | 4e-15 | 3637460-3636978 | ||
NC_003155.4 | 5e-12 | 6590878-6591372 | ||
Listeria monocytogenes FSL R2-561 | AARS01000007.1 | 8e-12 | 72280-71786 | |
NZ_ACGG01000118.1 | 3e-11 | 220449-220006 | ||
NC_008570.1 | 2e-05 | 1214897-1215424 | ||
NC_004668.1 | 2e-05 | 311391-311870 | ||
1BMR | hypothetical protein GOS_4202115 marine metagenome | gb|ECA60195.1 | 0.057 | 88-243 |
1DLC | hypothetical protein GOS_5670768 marine metagenome | gb|ECH33518.1 | 0.014 | 12-142 |
1QS1 | hypothetical protein GOS_355881 marine metagenome | gb|EBA70908.1 | 6e-04 | 102-270 |
hypothetical protein GOS_1734861 marine metagenome | gb|EDJ21677.1 | 8e-04 | 416-584 | |
hypothetical protein GOS_9568803 marine metagenome | gb|EBF61568.1 | 0.003 | 5-173 | |
hypothetical protein GOS_7854205 marine metagenome | gb|EBP79016.1 | 0.004 | 78-232 | |
1W99 | hypothetical protein GOS_6575573 marine metagenome | gb|EBX51304.1 | 0.010 | 29-95 |
the Cry8Ea1 protein, a model of the toxin was obtained; and it corresponds to the general model for a Cry protein (Figure 4). The last structure corresponds to a sequence from the marine metagenome. It was built by homology to a possible transferase of
5.2. B. thuringiensis vs. lepidopteran and coleopteran pests
The entomopathogenic bacterium
5.3. Our experience with lepidopterans
We worked with the tobacco budworm,
We collaborated in the genetic characterization of
Recently we contributed to the determination of Cry1 toxicity against the first instar larvae of
5.4. Our experience with coleopterans
We researched the relationship between ecological niches of the Andean weevil,
Coffee crops are severely affected by the CBB (coffee berry borer,
5.4.1. The modes of action of Cry toxins in coleopterans: the case of CBB
The specific conditions in CBB gut physiology (acidic pH, types of proteases or high proportions of insecticide resistance alleles) are not favourable to the modes of action of the Cry proteins (López-Pazos et al. 2009a). The presence of candidate receptors for Cry proteins in CBB offers evidence for the potential of Cry protein use for the control of this pest. Cadherin-like receptors (CADR) have been studied in lepidopteran and dipteran insects. CADRs were isolated from the coleopterans
Aminopeptidase N (APN) is an N-acetyl-D-galactosamine (GalNAc)-bearing glycoprotein. APN is a receptor for Cry toxins. Different APNs have molecular weights of 90-170-kDa. It was proposed that the Cry-APN interaction has two steps: carbohydrate recognition and irreversible protein-protein interaction (Pigott & Ellar 2007). More than 60 different APNs have been registered in databases. They are from 26% to 65% similar (Herrero et al. 2005, Nakanishi et al. 1999). The 140 kDa protein (from BBMV analysis) is consistent with its being an APN. We do not know if the multiple Cry-binding polypeptides detected in CBB are different proteins or if they are one APN glycosylated differently.
It is also known that CADRs are susceptible to proteolytic digestion and for producing a ~120 kDa fraction. For this reason, CADRs can be confused with APNs in protein-protein interaction blots (Martínez-Rámirez et al. 1994). Cry proteins have multiple binding determinants, possibly specified independently by domains II and III. Moreover, Cry toxins interact with other classes of proteins in the Coleoptera order, such as ALP (molecular weight ~65 kDa), V-ATPase and the Heat-Shock Cognate protein (~ 80 kDa) and the ADAM metalloprotease (~30 kDa) (Hua et al. 2001; Ochoa-Campuzano et al. 2007; Martins et al. 2010; Nakasu et al. 2010). Any signals in the ligand blot for Cry1B and Cry3A would be related with these proteic groups. However, we identified the minor biological activity of Cry1B and Cry3A proteins on CBB larvae (López- Pazos et al. 2009a); and none was seen
with Cry1I, Cry4, Cry9 and SN1917 hybrids. In this sense, there is a correlation between our data and ligand blot observations.
6. Conclusion
Insecticidal toxins are an important option for the biological control of lepidopteran and coleopteran insects. Their use in the genetic engineering of plants could provide a new generation of resistant crops. Such recombinant plants, thanks to their significant environmental and economic benefits, could help agricultural families in poor countries
Acknowledgement
The authors are grateful to the Instituto de Biotecnología de la Universidad Nacional de Colombia. López-Pazos S.A. is grateful to Colciencias for a doctoral fellowship.
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