Cry6Aa

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Cry6Aa
Crystal structure of Cry6Aa toxin.gif
Protein schematic of Cry6Aa toxin
Identifiers
Organism Bacillus thuringiensis
SymbolCry6Aa
UniProt Q45757
Search for
Structures Swiss-model
Domains InterPro

Cry6Aa (Pesticidal crystal protein Cry6Aa) is a toxic crystal protein generated by the bacterial family Bacillus thuringiensis during sporulation. [1] This protein is a member of the alpha pore forming toxins family, which gives it insecticidal qualities advantageous in agricultural pest control. Each Cry protein has some level of target specificity; Cry6Aa has specific toxic action against coleopteran insects and nematodes. The corresponding B. thuringiensis gene, cry6aa, is located on bacterial plasmids. Along with several other Cry protein genes, cry6aa can be genetically recombined in Bt corn and Bt cotton so the plants produce specific toxins. Insects are developing resistance to the most commonly inserted proteins like Cry1Ac. [2] Since Cry6Aa proteins function differently than other Cry proteins, they are combined with other proteins to decrease the development of pest resistance. Recent studies suggest this protein functions better in combination with other virulence factors such as other Cry proteins and metalloproteinases. [3] > [4]

Contents

Structure

Cry6Aa proteins are unrelated to other insecticidal crystal proteins in primary amino acid structure; it is a member of the Tripartite Haemolysin BL family (TCDB). The protein is rod-shaped, with a diameter of 25 Å and height of 95 Å. It contains 475 residues, not including the N-terminal tail. [5] Most Cry proteins have 3 main domains with functional homology across proteins, domain I contains an alpha helix bundle, domain II is composed of three antiparallel beta sheets in a Greek key motif, and domain III forms a beta sandwich responsible for catalyzing pore formation. [6] However, Cry6Aa, a nine turn protein, consists of bipartite head and tail domains composed mainly of alpha helices. Secondary structure conformation is 71-72% alpha helices and 1-2% beta sheets in most pH conditions. The remaining regions are either bends, turns, or 3/10 helices. [7] The trypsin resistant core is composed of long amphipathic alpha helices and fuels toxic function. The hydrophobic regions of the helices interact with each other, while the hydrophilic portions have increased exposure to the outside environment. Some of the helices are interrupted by loops that have variable positions in the structure. The head domain folds over the helices and contains a beta tongue group, which may trigger pore formation. There is a strong disulfide bond between the C terminal region and a portion of the core that isn't disrupted by trypsin. The protein has structural similarities to other toxins, including haemolysin E and B. cereus toxins HlbB and NheA. [8] No other members of the Cry family utilizing alpha pore toxin structure have been discovered.

Mechanism of action

Coleoptera

Cry6Aa has pore-forming action that destroys insect intestinal epithelial cells. Most Cry proteins have 3 domains, but Cry6Aa is composed largely of alpha helices, which indicates different membrane insertion methods. Cry6Aa has catalytic head domains regulated by hydrophobic residues. When Cry6Aa is first ingested, it remains a pro-toxin until intestinal proteases cleave the protein into active particles. After activation, the beta tongue head domain binds with the target membranes on brush border membrane cells similar to Haemolysin E. [8] [9] Typical Cry proteins are enhanced by interactions with cadherin, but Cry6Aa receptors remain unknown. Experimental data suggests the proteins embed in the membrane and form oligomeric pores, but the complete mechanism has not been deduced in 2016. [10]

Nematodes

Presence of Cry6Aa in nematodes triggers a regulated necrosis pathway via an aspartic protease (ASP-1). In order for the toxin to be activated, it must be partially digested inside the organism’s intestine after ingestion. ASP-1 proteases are highly concentrated in nematode intestinal cells and protect Cry6Aa proteins from over-degradation during activation. They are also members of the cathepsin family and can digest lysosomes. Cry6Aa triggers a magnesium dependent adenylyl cyclase/protein kinase A signaling pathway, which releases calcium ions into the cell from inositol triphosphate ion channels. Ca2+ activates calpain, a cysteine protease, which promotes lysosome rupture. The lysosome is further digested by ASP-1, which leads to cell degradation by cytosolic acidification. Alterations of apoptosis or autophagy proteins do not affect the action of Cry6Aa. Mutations in required proteins for necrosis inhibit Cry6Aa, but not other Cry proteins, revealing a rare mechanism in Cry6Aa. Necrosis isn't promoted in mammalian cells since they express ASP-3 and ASP-4 proteases at higher rates than ASP-1, which is necessary for toxic action by Cry6Aa. The cell receptor for Cry6Aa has not been identified. [11] < [12] Additionally, nematocidal activity is enhanced by Bmp1 metalloproteinase, which degrades the intestinal cell wall of the organism. This either speeds death by loss of intestinal function or by increased cell wall perforation easing protein insertion. [4]

Significance

Agriculture

In order to combat growing pest resistance, Cry6Aa is implemented in transgenic plants because it targets pests differently, increasing susceptibility. DNA shuffling is the process of selecting genes of compatible Cry proteins to transfer into crops. Although the binding site of Cry6Aa is unknown, several sites have been ruled out, allowing successful Cry protein stacking. Because an organism has to be resistant to both expressed Cry proteins to survive, the chances of developing and vertically transferring resistance is lower, granting more time for pesticide research. In 2013, Cry6Aa and Cry3Aa combination transgenic plants were patented to prevent resistance in the western corn rootworm. [13] Additionally, Cry6Aa has been layered with Cry34Ab1/Cry35Ab1, a binary toxin. [9] Pyramiding Cry proteins can enhance the effect of toxins. Cry6Aa and Cry55Aa both can reduce brood size of the root-knot nematode Meloidogyne incognita , but when they are combined, these two proteins are five times more effective. Synergy between Cry proteins comes from either improved toxin docking, membrane insertion, or advanced degradation of the midgut protein matrix which increases action of the slower acting toxin. [3]

Necrosis research

Cry6Aa can induce necrosis in laboratories without risking cell damage through heat or other triggers. Since necrosis results in swelling and damage to surrounding cell areas, it can be more effective in treating cancers than induced apoptosis. [14] Although Cry6Aa has no action against mammals, many essential cell pathways are conserved throughout eukaryotes. C. elegans is a groundbreaking model nematode affected by Cry6Aa, which can be used to understand the activation of the necrosis pathway. Understanding the role of the aspartic protease may allow scientists to engineer other necrosis-inducing proteins which act through ASP-3 and ASP-4 in order to target mammalian cancer cells. [11]

Related Research Articles

<i>Bacillus thuringiensis</i> Species of bacteria used as an insecticide

Bacillus thuringiensis is a gram-positive, soil-dwelling bacterium, the most commonly used biological pesticide worldwide. B. thuringiensis also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well on leaf surfaces, aquatic environments, animal feces, insect-rich environments, flour mills and grain-storage facilities. It has also been observed to parasitize moths such as Cadra calidella—in laboratory experiments working with C. calidella, many of the moths were diseased due to this parasite.

<span class="mw-page-title-main">Transmembrane protein</span> Protein spanning across a biological membrane

A transmembrane protein is a type of integral membrane protein that spans the entirety of the cell membrane. Many transmembrane proteins function as gateways to permit the transport of specific substances across the membrane. They frequently undergo significant conformational changes to move a substance through the membrane. They are usually highly hydrophobic and aggregate and precipitate in water. They require detergents or nonpolar solvents for extraction, although some of them (beta-barrels) can be also extracted using denaturing agents.

<i>Bacillus thuringiensis israelensis</i> Subspecies of bacterium

Bacillus thuringiensis serotype israelensis (Bti) is a group of bacteria used as biological control agents for larvae stages of certain dipterans. Bti produces toxins which are effective in killing various species of mosquitoes, fungus gnats, and blackflies, while having almost no effect on other organisms. The major advantage of B. thuringiensis products is that they are thought to affect few non-target species. However, even though Bti may have minimal direct effects on non-target organisms, it may potentially be associated with knock-on effects on food webs and other ecosystem properties, including biodiversity and ecosystem functioning.

<span class="mw-page-title-main">Enterotoxin</span> Toxin from a microorganism affecting the intestines

An enterotoxin is a protein exotoxin released by a microorganism that targets the intestines. They can be chromosomally or plasmid encoded. They are heat labile (>60⁰), of low molecular weight and water-soluble. Enterotoxins are frequently cytotoxic and kill cells by altering the apical membrane permeability of the mucosal (epithelial) cells of the intestinal wall. They are mostly pore-forming toxins, secreted by bacteria, that assemble to form pores in cell membranes. This causes the cells to die.

A latrotoxin is a high-molecular mass neurotoxin found in the venom of spiders of the genus Latrodectus as well as at least one species of another genus in the same family, Steatoda nobilis. Latrotoxins are the main active components of the venom and are responsible for the symptoms of latrodectism.

<span class="mw-page-title-main">Diphtheria toxin</span> Exotoxin

Diphtheria toxin is an exotoxin secreted mainly by Corynebacterium diphtheriae but also by Corynebacterium ulcerans and Corynebacterium pseudotuberculosis, the pathogenic bacterium that causes diphtheria. The toxin gene is encoded by a prophage called corynephage β. The toxin causes the disease in humans by gaining entry into the cell cytoplasm and inhibiting protein synthesis.

<span class="mw-page-title-main">Anthrax toxin</span> Tripartite protein complex secreted by virulent strains of Bacillus anthracis

Anthrax toxin is a three-protein exotoxin secreted by virulent strains of the bacterium, Bacillus anthracis—the causative agent of anthrax. The toxin was first discovered by Harry Smith in 1954. Anthrax toxin is composed of a cell-binding protein, known as protective antigen (PA), and two enzyme components, called edema factor (EF) and lethal factor (LF). These three protein components act together to impart their physiological effects. Assembled complexes containing the toxin components are endocytosed. In the endosome, the enzymatic components of the toxin translocate into the cytoplasm of a target cell. Once in the cytosol, the enzymatic components of the toxin disrupt various immune cell functions, namely cellular signaling and cell migration. The toxin may even induce cell lysis, as is observed for macrophage cells. Anthrax toxin allows the bacteria to evade the immune system, proliferate, and ultimately kill the host animal. Research on anthrax toxin also provides insight into the generation of macromolecular assemblies, and on protein translocation, pore formation, endocytosis, and other biochemical processes.

<span class="mw-page-title-main">Pore-forming toxin</span> Protein-produced toxins that create pores in cell membrane

Pore-forming proteins are usually produced by bacteria, and include a number of protein exotoxins but may also be produced by other organisms such as apple snails that produce perivitellin-2 or earthworms, who produce lysenin. They are frequently cytotoxic, as they create unregulated pores in the membrane of targeted cells.

<span class="mw-page-title-main">Hemolysin</span> Molecule destroying the membrane of red blood cells

Hemolysins or haemolysins are lipids and proteins that cause lysis of red blood cells by disrupting the cell membrane. Although the lytic activity of some microbe-derived hemolysins on red blood cells may be of great importance for nutrient acquisition, many hemolysins produced by pathogens do not cause significant destruction of red blood cells during infection. However, hemolysins are often capable of lysing red blood cells in vitro.

The Membrane Attack Complex/Perforin (MACPF) superfamily, sometimes referred to as the MACPF/CDC superfamily, is named after a domain that is common to the membrane attack complex (MAC) proteins of the complement system and perforin (PF). Members of this protein family are pore-forming toxins (PFTs). In eukaryotes, MACPF proteins play a role in immunity and development.

<i>Staphylococcus aureus</i> alpha toxin

Alpha-toxin, also known as alpha-hemolysin (Hla), is the major cytotoxic agent released by bacterium Staphylococcus aureus and the first identified member of the pore forming beta-barrel toxin family. This toxin consists mostly of beta-sheets (68%) with only about 10% alpha-helices. The hly gene on the S. aureus chromosome encodes the 293 residue protein monomer, which forms heptameric units on the cellular membrane to form a complete beta-barrel pore. This structure allows the toxin to perform its major function, development of pores in the cellular membrane, eventually causing cell death.

<span class="mw-page-title-main">Delta endotoxins</span> Insecticide

Delta endotoxins (δ-endotoxins) are a family of pore-forming toxins produced by Bacillus thuringiensis species of bacteria. They are useful for their insecticidal action and are the primary toxin produced by the genetically modified (GM) Bt maize/corn and other GM crops. During spore formation the bacteria produce crystals of such proteins that are also known as parasporal bodies, next to the endospores; as a result some members are known as a parasporin. The Cyt (cytolytic) toxin group is another group of delta-endotoxins formed in the cytoplasm. VIP toxins are formed at other stages of the life cycle.

<span class="mw-page-title-main">Phospholipase C</span> Class of enzymes

Phospholipase C (PLC) is a class of membrane-associated enzymes that cleave phospholipids just before the phosphate group (see figure). It is most commonly taken to be synonymous with the human forms of this enzyme, which play an important role in eukaryotic cell physiology, in particular signal transduction pathways. Phospholipase C's role in signal transduction is its cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which serve as second messengers. Activators of each PLC vary, but typically include heterotrimeric G protein subunits, protein tyrosine kinases, small G proteins, Ca2+, and phospholipids.

<span class="mw-page-title-main">Heat-stable enterotoxin</span> Class of bacterial toxins

Heat-stable enterotoxins (STs) are secretory peptides produced by some bacterial strains, such as enterotoxigenic Escherichia coli which are in general toxic to animals.

<i>Clostridium septicum</i> Species of bacterium

Clostridium septicum is a gram positive, spore forming, obligate anaerobic bacterium.

The RTX toxin superfamily is a group of cytolysins and cytotoxins produced by bacteria. There are over 1000 known members with a variety of functions. The RTX family is defined by two common features: characteristic repeats in the toxin protein sequences, and extracellular secretion by the type I secretion systems (T1SS). The name RTX refers to the glycine and aspartate-rich repeats located at the C-terminus of the toxin proteins, which facilitate export by a dedicated T1SS encoded within the rtx operon.

The thiol-activated Cholesterol-dependent Cytolysin(CDC) family is a member of the MACPF superfamily. Cholesterol dependent cytolysins are a family of β-barrel pore-forming exotoxins that are secreted by gram-positive bacteria. CDCs are secreted as water-soluble monomers of 50-70 kDa, that when bound to the target cell, form a circular homo-oligomeric complex containing as many as 40 monomers. Through multiple conformational changes, the β-barrel transmembrane structure is formed and inserted into the target cell membrane. The presence of cholesterol in the target membrane is required for pore formation, though the presence of cholesterol is not required by all CDCs for binding. For example, intermedilysin secreted by Streptococcus intermedius will bind only to target membranes containing a specific protein receptor, independent of the presence of cholesterol, but cholesterol is required by intermedilysin for pore formation. While the lipid environment of cholesterol in the membrane can affect toxin binding, the exact molecular mechanism that cholesterol regulates the cytolytic activity of the CDC is not fully understood.

Host microbe interactions in <i>Caenorhabditis elegans</i>

Caenorhabditis elegans- microbe interactions are defined as any interaction that encompasses the association with microbes that temporarily or permanently live in or on the nematode C. elegans. The microbes can engage in a commensal, mutualistic or pathogenic interaction with the host. These include bacterial, viral, unicellular eukaryotic, and fungal interactions. In nature C. elegans harbours a diverse set of microbes. In contrast, C. elegans strains that are cultivated in laboratories for research purposes have lost the natural associated microbial communities and are commonly maintained on a single bacterial strain, Escherichia coli OP50. However, E. coli OP50 does not allow for reverse genetic screens because RNAi libraries have only been generated in strain HT115. This limits the ability to study bacterial effects on host phenotypes. The host microbe interactions of C. elegans are closely studied because of their orthologs in humans. Therefore, the better we understand the host interactions of C. elegans the better we can understand the host interactions within the human body.

Cry34Ab1 is one member of a binary Bacillus thuringiensis (Bt) crystal protein set isolated from Bt strain PS149B1. The protein exists as a 14 kDa aegerolysin that, in presence of Cry35Ab1, exhibits insecticidal activity towards Western Corn Rootworm. The protein has been transformed into maize plants under the commercialized events 4114 (DP-ØØ4114-3) by Pioneer Hi-Bred and 59122 (DAS-59122-7) by Dow AgroSciences. These events have, in turn, been bred into multiple trait stacks in additional products.

RopB transcriptional regulator, also known as RopB/Rgg transcriptional regulator is a transcriptional regulator protein that regulates expression of the extracellularly secreted cysteine protease streptococcal pyrogenic exotoxin B (speB) [See Also: erythrogenic toxins] which is an important virulence factor of Streptococcus pyogenes and is responsible for the dissemination of a host of infectious diseases including strep throat, impetigo, streptococcal toxic shock syndrome, necrotizing fasciitis, and scarlet fever. Functional studies suggest that the ropB multigene regulon is responsible for not only global regulation of virulence but also a wide range of functions from stress response, metabolic function, and two-component signaling. Structural studies implicate ropB's regulatory action being reliant on a complex interaction involving quorum sensing with the leaderless peptide signal speB-inducing peptide (SIP) acting in conjunction with a pH sensitive histidine switch.

References

  1. ↑ Adang MJ, Crickmore N, Jurat-Fuentes JL (January 2014). "Diversity of Bacillus thuringiensis crystal toxins and mechanism of action.". Advances in insect physiology. Vol. 47. Academic Press. pp. 39–87. doi:10.1016/B978-0-12-800197-4.00002-6. ISBN   9780128001974.
  2. ↑ Tabashnik BE, Brévault T, Carrière Y (June 2013). "Insect resistance to Bt crops: lessons from the first billion acres". Nature Biotechnology. 31 (6): 510–21. doi:10.1038/nbt.2597. PMID   23752438. S2CID   205278530.
  3. 1 2 Peng D, Chai L, Wang F, Zhang F, Ruan L, Sun M (November 2011). "Synergistic activity between Bacillus thuringiensis Cry6Aa and Cry55Aa toxins against Meloidogyne incognita". Microbial Biotechnology. 4 (6): 794–8. doi:10.1111/j.1751-7915.2011.00295.x. PMC   3815414 . PMID   21923640.
  4. 1 2 Luo X, Chen L, Huang Q, Zheng J, Zhou W, Peng D, Ruan L, Sun M (January 2013). "Bacillus thuringiensis metalloproteinase Bmp1 functions as a nematicidal virulence factor". Applied and Environmental Microbiology. 79 (2): 460–8. Bibcode:2013ApEnM..79..460L. doi:10.1128/AEM.02551-12. PMC   3553750 . PMID   23124228.
  5. ↑ "Pesticidal crystal protein Cry6Aa". UniProt. CR6AA_BACTU.
  6. ↑ Pigott CR, Ellar DJ (June 2007). "Role of receptors in Bacillus thuringiensis crystal toxin activity". Microbiology and Molecular Biology Reviews. 71 (2): 255–81. doi:10.1128/MMBR.00034-06. PMC   1899880 . PMID   17554045.
  7. ↑ PDB: 5KUC ​; Dementiev A, Board J, Sitaram A, Hey T, Kelker MS, Xu X, et al. (August 2016). "The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins". BMC Biology. 14 (1): 71. doi: 10.1186/s12915-016-0295-9 . PMC   5004264 . PMID   27576487.
  8. 1 2 Dementiev A, Board J, Sitaram A, Hey T, Kelker MS, Xu X, et al. (August 2016). "The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins". BMC Biology. 14 (1): 71. doi: 10.1186/s12915-016-0295-9 . PMC   5004264 . PMID   27576487.
  9. 1 2 Li H, Olson M, Lin G, Hey T, Tan SY, Narva KE (2013). "Bacillus thuringiensis Cry34Ab1/Cry35Ab1 interactions with western corn rootworm midgut membrane binding sites". PLOS ONE. 8 (1): e53079. Bibcode:2013PLoSO...853079L. doi: 10.1371/journal.pone.0053079 . PMC   3537739 . PMID   23308139.
  10. ↑ Tzokov SB, Wyborn NR, Stillman TJ, Jamieson S, Czudnochowski N, Artymiuk PJ, et al. (August 2006). "Structure of the hemolysin E (HlyE, ClyA, and SheA) channel in its membrane-bound form". The Journal of Biological Chemistry. 281 (32): 23042–23049. doi: 10.1074/jbc.M602421200 . PMID   16754675.
  11. 1 2 Zhang F, Peng D, Cheng C, Zhou W, Ju S, Wan D, et al. (January 2016). "Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)". PLOS Pathogens. 12 (1): e1005389. doi: 10.1371/journal.ppat.1005389 . PMC   4721865 . PMID   26795495.
  12. ↑ Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, et al. (January 2009). "Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009". Cell Death and Differentiation. 16 (1): 3–11. doi:10.1038/cdd.2008.150. PMC   2744427 . PMID   18846107.
  13. ↑ US 2013263331,Narva KE, Meade T, Fencil K, Li H, Hey T, Woosley A, Olsen M,"Combinations including cry3aa and cry6aa proteins to prevent development of resistance in corn rootworms (diabrotica spp.)",issued 4 September 2017, assigned to Corteva Agriscience LLC, Indiana.
  14. ↑ Long JS, Ryan KM (December 2012). "New frontiers in promoting tumour cell death: targeting apoptosis, necroptosis and autophagy". Oncogene. 31 (49): 5045–60. doi: 10.1038/onc.2012.7 . PMID   22310284. S2CID   31689965.
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