| 1.C.2 The Channel-forming δ-Endotoxin Insecticidal Crystal Protein (ICP) Family
The ICP family proteins (Cry proteins; δ-Endotoxins) are produced during sporulation by various strains of Bacillus thuringiensis as proteinaceous crystalline inclusions. They have been called ICPs. Each ICP is specific for a different group of related insects or other invertebrates. ICPs are widely used as biopesticides, particularly as insecticides. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with the cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion. Flexiblity is obtained by toxin oligomerization. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab, and this domain is inserted into the membrane (Pardo-Lopez et al., 2006).
Specificity against the following insect orders has been observed: Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Phthiraptera, Mallophaga and Acri. Other invertebrate orders susceptible to their action include Nemathelminthes, Platyhelminthes and Sarcomastigorphora. The ICPs were initially classified according to the host organism in which they act, but close homologues were subsequently found to act on many different hosts. A classification system based exclusively on sequence similarity has recently been proposed. More than 50 sequenced ICPs have been classified into 15 families. However, the phylogenetic tree of the channel-forming domains reveal three primary branches.
Native ICPs (pro-Cry proteins) are 50-140 kDa in size. They are proteolytically activated after ingestion by the host organism to give the toxic protein fragments. Each one probably binds to a glycoprotein receptor in the apical microvillar membranes of epithelial midgut cells. The toxin then undergoes a conformational change, inserting into the membrane to form an oligomeric pore that causes osmotic cell lysis.
The crystal structures of two homologous toxins (Cry3A; Coleopteran-specific) and (Cry1Aa; Lepidopteran-specific) have been reported. They contain three domains. Domain I (N-termini; 220 residues) is the channel-forming domain. It is a seven helix bundle in which a central helix (helix 5) is surrounded by the other six helices. The six helices are amphipathic and long enough to span the 30Å membrane bilayer. Domain II (central 200 residues) is the specificity-determining domain which binds the receptor proteins in the insect midgut membrane. Domain III (C-termini; ~150 residues) is functionally less well defined and may transduce information from domain II (receptor binding domain) to domain I (channel-forming domain). Domain III may also regulate channel activity, stabilize the toxin, and function together with Domain II in receptor binding. Toxicity has been explained by formation of transmembrane oligomeric pores or ion channels and by the ability of the monomeric toxin to subvert cellular signaling pathways. In vitro biophysical studies suggest that helices alpha4 and alpha5 in domain I insert into the lipid bilayer as an alpha-helical hairpin. A trimeric model for the ion conducting channel has been proposed (Torres et al., 2007).
The generalized transport reaction catalyzed by these toxins is:
Small molecules (in)  small molecules (out)
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| References: |
Akiba, T. and S. Okumura. (2016). Parasporins 1 and 2: their structure and activity. J Invertebr Pathol. [Epub: Ahead of Print]
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Andreev, I.M., N.V. Bulushova, I.A. Zalunin, and G.G. Chestukhina. (2009). Effect of entomocidal proteins from Bacillus thuringiensis on ion permeability of apical membranes of Tenebrio molitor larvae gut epithelium. Biochemistry (Mosc) 74: 1096-1103.
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Aronson, A.I. (1993). The two faces of Bacillus thuringiensis: insecticidal proteins and post-exponential survival. Mol. Microbiol. 7:489-496.
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Atsumi, S., K. Miyamoto, K. Yamamoto, J. Narukawa, S. Kawai, H. Sezutsu, I. Kobayashi, K. Uchino, T. Tamura, K. Mita, K. Kadono-Okuda, S. Wada, K. Kanda, M.R. Goldsmith, and H. Noda. (2012). Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori. Proc. Natl. Acad. Sci. USA 109: E1591-1598.
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Bravo, A. (1997). Phylogenetic relationships of Bacillus thuringiensis δ-endotoxin family proteins and their functional domains. J. Bacteriol. 179: 2793-2801.
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Cai, Y., B. Hou, J.A. Fabrick, Y. Yang, and Y. Wu. (2024). The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera. Pestic Biochem Physiol 204: 106068.
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Canton, P.E., A. Cancino-Rodezno, S.S. Gill, M. Soberón, and A. Bravo. (2015). Transcriptional cellular responses in midgut tissue of Aedes aegypti larvae following intoxication with Cry11Aa toxin from Bacillus thuringiensis. BMC Genomics 16: 1042.
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Chen, L., J. Wei, C. Liu, W. Zhang, B. Wang, L. Niu, and G. Liang. (2018). Specific Binding Protein ABCC1 Is Associated With Cry2Ab Toxicity in. Front Physiol 9: 745.
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Crickmore, N., D.R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum and D.H. Dean (1998). Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62: 807-813.
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Dong, X., J. Song, J. Chen, D. Bi, W. Wang, Y. Ren, H. Wang, G. Wang, K.F.J. Tang, X. Wang, and J. Huang. (2019). Conjugative Transfer of the pVA1-Type Plasmid Carrying the Genes Results in the Formation of New AHPND-Causing. Front Cell Infect Microbiol 9: 195.
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Durmaz, E., Y. Hu, R.V. Aroian, and T.R. Klaenhammer. (2015). Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic. Appl. Environ. Microbiol. 82: 1286-1294.
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Fabrick J., Oppert C., Lorenzen MD., Morris K., Oppert B. and Jurat-Fuentes JL. (2009). A novel Tenebrio molitor cadherin is a functional receptor for Bacillus thuringiensis Cry3Aa toxin. J Biol Chem. 284(27):18401-10.
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Groulx, N., M. Juteau, and R. Blunck. (2010). Rapid topology probing using fluorescence spectroscopy in planar lipid bilayer: the pore-forming mechanism of the toxin Cry1Aa of Bacillus thuringiensis. J Gen Physiol 136: 497-513.
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Guo, S., S. Ye, Y. Liu, L. Wei, J. Xue, H. Wu, F. Song, J. Zhang, X. Wu, D. Huang, and Z. Rao. (2009). Crystal structure of Bacillus thuringiensis Cry8Ea1: An insecticidal toxin toxic to underground pests, the larvae of Holotrichia parallela. J Struct Biol 168: 259-266.
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Höfte, H. and H.R. Whiteley (1989). Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53: 242-255.
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Hua, G., R. Zhang, M.A. Abdullah, and M.J. Adang. (2008). Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity. Biochemistry 47: 5101-5110.
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Jia, Y., C. Zhao, Q. Wang, C. Shu, X. Feng, F. Song, and J. Zhang. (2014). A genetically modified broad-spectrum strain of Bacillus thuringiensis toxic against Holotrichia parallela, Anomala corpulenta and Holotrichia oblita. World J Microbiol Biotechnol 30: 595-603.
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Jiménez-Juárez, N., C. Muñoz-Garay, I. Gómez, G. Saab-Rincon, J.Y. Damian-Almazo, S.S. Gill, M. Soberón, and A. Bravo. (2007). Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae. J. Biol. Chem. 282: 21222-21229.
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Knowles, B.H. and J.A.T. Dow (1993). The crystal d-endotoxins of Bacillus thuringiensis: models for their mechanism of action on the insect gut. BioEssays 15: 469-476.
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Lemeshko, V.V., M. Arias, and S. Orduz. (2005). Mitochondria permeabilization by a novel polycation peptide BTM-P1. J. Biol. Chem. 280: 15579-15586.
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Likitvivatanavong, S., G. Katzenmeier, and C. Angsuthanasombat. (2006). Asn183 in alpha5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin. Arch Biochem Biophys 445: 46-55.
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Lopez-Diaz JA., Canton PE., Gill SS., Soberon M. and Bravo A. (2013). Oligomerization is a key step in Cyt1Aa membrane insertion and toxicity but not necessary to synergize Cry11Aa toxicity in Aedes aegypti larvae. Environ Microbiol. 15(11):3030-9.
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Obata, F., S. Tanaka, S. Kashio, H. Tsujimura, R. Sato, and M. Miura. (2015). Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor. BMC Biol 13: 48.
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Pardo-López, L., I. Gómez, C. Muñoz-Garay, N. Jiménez-Juarez, M. Soberón, and A. Bravo. (2006). Structural and functional analysis of the pre-pore and membrane-inserted pore of Cry1Ab toxin. J Invertebr Pathol 92: 172-177.
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Robinson, S.D., A. Mueller, D. Clayton, H. Starobova, B.R. Hamilton, R.J. Payne, I. Vetter, G.F. King, and E.A.B. Undheim. (2018). A comprehensive portrait of the venom of the giant red bull ant, , reveals a hyperdiverse hymenopteran toxin gene family. Sci Adv 4: eaau4640.
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Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler and D.H. Dean (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62: 775-806.
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Thamwiriyasati, N., C. Kanchanawarin, C. Imtong, C.J. Chen, H.C. Li, and C. Angsuthanasombat. (2022). Complete structure elucidation of a functional form of the Bacillus thuringiensis Cry4Ba δ-endotoxin: Insights into toxin-induced transmembrane pore architecture. Biochem. Biophys. Res. Commun. 620: 158-164.
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Torres, J., X. Lin, and P. Boonserm. (2008). A trimeric building block model for Cry toxins in vitro ion channel formation. Biochim. Biophys. Acta. 1778(2): 392-397.
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Xu, L., Z.Z. Pan, J. Zhang, L.Y. Niu, J. Li, Z. Chen, B. Liu, Y.J. Zhu, and Q.X. Chen. (2018). Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure. Cell Microbiol 20: e12827.
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Yang, J., S. Chen, X. Xu, G. Lin, S. Lin, J. Bai, Q. Song, M. You, and M. Xie. (2022). Novel-miR-310 mediated response mechanism to Cry1Ac protoxin in Plutella xylostella (L.). Int J Biol Macromol 219: 587-596.
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.C.2.1.1 | ICP Cry1Aa. Cry1A (Receptors in Lepidoptera are cadherin-like proteins (Fabrick et al., 2009) but can also be ABC-type efflux pumps (Chen et al. 2018). The pore-forming mechanism has been studied by Groulx et al. (2010). This toxin causes necrosis in Drosophila species (Obata et al. 2015). | Bacteria, Firmicute | Cry1Aa of Bacillus thuringiensis (P0A367) |
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| 1.C.2.1.10 | Uncharacterized protein of 617 aas and possibly 3 TMSs, one N-terminal, and two more later on. | | UP of Moniliophthora roreri |
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| 1.C.2.1.11 | RICIN domain-containing protein, putative toxin, of 576 aas and possibly up to 3 central TMSs. | | Putative toxin protein of Pseudolysobacter antarcticus |
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| 1.C.2.1.13 | PirB toxin of 438 aas and possibly two central TMSs. It kills whiteleg shrimp, Penaeus vannamei by causing acute hepatopancreatic necrosis disease (AHPND). It is bourn by a conjugative plasmid (designated as pVA1-type) carrying the pirABvp toxin genes. (Dong et al. 2019). | | PirB of Vibrio parahaemolyticus |
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| 1.C.2.1.14 | Uncharacterized protein of 700 aas and possibly 2 or 3 TMSs near the N-terminus. | | UP of Streptomyces hainanensis |
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| 1.C.2.1.15 | Uncharacterized protein of 639 aas and 2 or 3 possible TMSs. | | UP of Dokdonella sp. (bioanode metagenome) |
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| 1.C.2.1.2 | Pesticide crystal protein Cry4Ba (δ-endotoxin) (1136aas). Cadherin AgCad1 is the receptor for Cry4Ba (Hua et al., 2008). Asn183 in TMS5 is essential for oligomerizatioin of the protein in the midgut membrane of the insect, and therefore for pore formation and toxicity (Likitvivatanavong et al. 2006). The complete structure of a functional form of the Bacillus thuringiensis Cry4Ba delta-endotoxinhas been solved with nsight into the toxin-induced transmembrane pore architecture (Thamwiriyasati et al. 2022). The 2.0 Å crystal structure revealed a wedge-shaped arrangement of three domains: a well-defined N-terminal domain of eight alpha-helices responsible for pore formation, a three-beta-sheet prism displaying two functional motifs and a C-terminal beta-sandwich domain. Two conserved side-chains-Asn(166) and Tyr(170) in the α4-α5 loop were found to interact directly with phospholipid head groups, leading to pore opening and stability (Thamwiriyasati et al. 2022). | Bacteria | Cry4Ba of Bacillus thuringiensis (P05519) |
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| 1.C.2.1.3 |
Pesticidal pre-pore-forming crystal protein, Cry1Ab; insecticidal endotoxin (1155 aas). (90% identical to Cry1Aa; (1.C.2.1.1) Kills Manduca sexta. There are several receptors (Arenas et al., 2010). Also called bt2, Cry1-2, Cry1A(b) and CryIC1. Mutations affecting pre-pore oligomerization and toxin pore formation have been described (Jiménez-Juárez et al. 2007). | Bacteria | Cry1Ab of Bacillus thuringiensis (P0A370) |
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| 1.C.2.1.4 | Cry1Ac (85% identical to Cry1Aa (TC#1.C.2.1.1). May use MRP-4-like ABC transporter as a receptor in Bombyx mori (Atsumi et al., 2012). An miR-310 mediated response to Cry1Ac protoxin in Plutella xylostella (L.) has been documented (Yang et al. 2022). The insecticidal crystalline (Cry) and vegetative insecticidal (Vip)
proteins derived from Bacillus thuringiensis (Bt) are used globally to
manage insect pests, including the cotton bollworm, Helicoverpa
armigera, one of the world's most damaging agricultural pests. Cry
proteins bind to the ATP-binding cassette transporter C2 (ABCC2)
receptor on the membrane surface of larval midgut cells, resulting in
Cry toxin pores, and ultimately leading to cell swelling and/or lysis.
Insect aquaporin (AQP) proteins within the membranes of larval midgut
cells allow the rapid influx of water into enterocytes
following the osmotic imbalance triggered by the formation of Cry toxin
pores (Cai et al. 2024). | Bacteria | Cry1Ac of Bacillus thuringiensis (D3XF72) |
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| 1.C.2.1.5 | Pesticidal crystal protein of 1144 aas, Cry8. | Firmicutes | Cry8 of Bacillus thurengiensis |
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| 1.C.2.1.6 | The Cry8Ea1 toxin of 1164 aas. The 2.2-Å crystal structure has been reported (Guo et al. 2009). Cry8Ea1 is specifically toxic to the underground larvae of Holotrichia parallela (Jia et al. 2014). | Firmicutes | Cry8Ea1 of Bacillus thuringiensis |
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| 1.C.2.1.7 | Pesticidal crystal protein (ICP) Cry3Aa (Andreev et al., 2009). | Bacteria | Cry3Aa of Bacillus thuringiensis (P0A380) |
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| 1.C.2.1.8 | Parasporin 1, PS1 or Cry41Aa of 825 aas (Akiba and Okumura 2016). Also called Cancer cell-killing Cry protein, parasporin-3. | | PS1 of Bacillus thuringiensis |
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| 1.C.2.1.9 | Uncharacterized protein of 502 aas and 3 TMSs, one at the N-terminus, and two further on. | | UP of Fusarium xylarioides |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.C.2.2.1 | Insecticidal crystal protein, Cry11Aa of 643 aas. Cry and Cyt toxins are both oligomeric pore-formers that act synergistically with each other via direct protein-protein interactions (López-Diaz et al. 2013). Target tissue cellular responses to the toxin have been determined (Canton et al. 2015). | Firmicutes | Cry11Aa of Bacillus thuringiensis |
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| 1.C.2.2.2 | Insect pore-forming toxin Cry2Ab (CryB2, CryIIA(b)) of 633 aas. Exposure of helices α4 and α5 is important for the mode of action of Cry2Ab (Xu et al. 2018). It's receptor in the insect membrane is ABCC1 (TC# 3.A.1.208.45) (Chen et al. 2018).
| | CryAb of Bacillus thuringiensis |
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| 1.C.2.2.3 | Bacillus thuringiensis subsp. medellin produces the Cry11Bb protein of 94 kDa (750 aas), which is toxic for mosquito larvae due to permeabilization of the plasma membrane of midgut epithelial cells (Lemeshko et al. 2005). A membrane channel-forming peptide, BTM-P1, has been derived from this protein (Lemeshko et al. 2005). | | Cry11Bb of Bacillus thuringiensis |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.C.2.3.1 | Pesticidal crystal protein (ICP) Cry13Aa | Bacteria | Cry13Aa of Bacillus thuringiensis (Q45755) |
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| 1.C.2.3.2 | Delta-endotoxin, Cry5B, of 1245 aas is lethal to nematodes. Active Cry5B can be expressed intracellularly in and released
extracellularly from Lactococcus lactis via a holin, showing potential for future use as an
anthelminthic that could be delivered orally in a food-grade microbe. (Durmaz et al. 2015). | | Cry5B of Bacillus thuringiensis |
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| 1.C.2.3.3 | Pesticidal crystal protein, Cry5Ac, of 1220 aas. It promotes colloidosmotic lysis by binding to the midgut epithelial cells of hymenopteran species. This and other hymenopteran toxins are the sources of red bull ant venoms that cause pain in mammals (Robinson et al. 2018). | | Cry5Ac of Bacillus thuringiensis |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| Examples: |
| TC# | Name | Organismal Type | Example |