Binding Properties of Bacillus thuringiensis Cry4A Toxin to the Apical Microvilli of Larval Midgut of Culex pipiens

Yamagiwa, Masashi; Kamauchi, Shinya; Okegawa, Takayuki; Esaki, Motoyuki; Otake, Kanao; Amachi, Teruo; Komano, Tohru; Sakai, Hiroshi

doi:10.1271/bbb.65.2419

Cited by 17 publications

(14 citation statements)

References 21 publications

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“…However, no relationship between binding and toxicity was observed, suggesting that the binding assay data may have been obscured by nonspecific binding of GSTCry4Aa. Nonspecific binding of Cry4Aa to BBMV of C. pipiens has been reported previously (40). If such binding occurs, we should rely on bioassay data rather than binding data to search the Cry4Aa molecule for the functional receptor binding site leading to mosquitocidal activity.…”

Section: Discussionmentioning

confidence: 97%

Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa

Howlader

Kagawa

Miyakawa

et al. 2010

Appl Environ Microbiol

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Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.

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Section: Discussionmentioning

confidence: 97%

Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa

Howlader

Kagawa

Miyakawa

et al. 2010

Appl Environ Microbiol

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“…The Cry and Cyt toxins are membrane-perforating proteins although unrelated structurally and differ in their requirement of essential membrane components; the Cry’s bind to membrane receptors [70,71,72,73,74,75] whereas Cyt1Aa binds with high affinities to unsaturated phospholipids [76,77]. …”

Section: δ-Endotoxins Of Btimentioning

confidence: 99%

“…Subsequent steps involve toxin binding to receptors [72,73,74,75,104,105,114,115,116], oligomerization and membrane insertion leading to formation of gated, cation selective channels [117,118]. Lethality is due to collapse of the trans-membrane potential, with subsequent osmotic lysis of cells lining the midgut [119].…”

Section: δ-Endotoxins Of Btimentioning

confidence: 99%

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Bacillus thuringiensis subsp. israelensis and Its Dipteran-Specific Toxins

Ben‐Dov

2014

Toxins

188

149

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Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of 134, 128, 72 and 27 kDa) and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come.

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“…The 60 kDa Cry39A toxin speciWcally bound to the A. stephensi BBMV and did not compete with the Cry4A toxin. Yamagiwa et al (2001) suggested that Cry4A toxin has two binding pathways that lead to irreversible binding to a cell surface. The Wrst pathway depends on a speciWc binding protein and follows the two-step mode of interaction, which results in the correct orientation of the insertion of the toxin molecule into the membrane, leading to the formation of functional channels.…”

Section: Discussionmentioning

confidence: 99%

Activation process of the mosquitocidal δ-endotoxin Cry39A produced by Bacillus thuringiensis subsp. aizawai BUN1-14 and binding property to Anopheles stephensi BBMV

Ito¹,

Bando²,

Asano³

2006

Journal of Invertebrate Pathology

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Most delta-endotoxins produced by Bacillus thuringiensis require proteolytic processing in order to become active. The in vitro and in vivo activation processes of Cry39A, a delta-endotoxin that is highly toxic to Anopheles stephensi, were investigated. Cry39A with a molecular mass of 72 kDa was processed in vitro into a 60 kDa fragment by trypsin and gut extract from A. stephensi larvae. N-terminal amino acid sequencing of the 60 kDa fragment revealed that trypsin and the protease(s) in the gut extract cleaved Cry39A between Arg(61) and Gly(62). In contrast, 40 and 25 kDa polypeptides were generated in vivo by intramolecular cleavage of the 60 kDa fragment in A. stephensi larvae. Further, a co-precipitation assay was used to investigate the binding property of the activated Cry39A to A. stephensi BBMV. Cry39A bound to A. stephensi BBMV specifically and did not compete with the Cry4Aa toxin. This indicated that the binding molecule(s) for Cry39A might differ from those for Cry4A. In addition, Cry39A preferentially bound to the Triton X-100-insoluble membrane fraction.

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Binding Properties of Bacillus thuringiensis Cry4A Toxin to the Apical Microvilli of Larval Midgut of Culex pipiens

Cited by 17 publications

References 21 publications

Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa

Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa

Bacillus thuringiensis subsp. israelensis and Its Dipteran-Specific Toxins

Activation process of the mosquitocidal δ-endotoxin Cry39A produced by Bacillus thuringiensis subsp. aizawai BUN1-14 and binding property to Anopheles stephensi BBMV

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