Introduction of
            <i>Culex</i>
            Toxicity into
            <i>Bacillus thuringiensis</i>
            Cry4Ba by Protein Engineering

Abdullah, Mohd Amir F.; Álzate, Óscar; Mohammad, Marwan; McNall, Rebecca J.; Adang, Michael J.; Dean, Donald H.

doi:10.1128/aem.69.9.5343-5353.2003

Cited by 76 publications

(84 citation statements)

References 50 publications

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“…Amino acid substitutions in these loops allowed introduction of mosquitocidal activity in Cry4Ba (Abdullah et al, 2003) and Cry19A (Abdullah and Dean, 2004) toxins. Loop 2 seems critical for recognition of Cry receptors (Arenas et al, 2010;Jiménez et al, 2012;Pigott et al, 2008), through interactions proposed to involve hydropathic complementarity (Gómez et al, 2002).…”

Section: Specificity Level Vi: Binding To Receptorsmentioning

confidence: 99%

Specificity determinants for Cry insecticidal proteins: Insights from their mode of action

Jurat-Fuentes

Crickmore

2017

Journal of Invertebrate Pathology

151

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Section: Specificity Level Vi: Binding To Receptorsmentioning

confidence: 99%

Specificity determinants for Cry insecticidal proteins: Insights from their mode of action

Jurat-Fuentes

Crickmore

2017

Journal of Invertebrate Pathology

151

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“…The mutated Cry4Ba has a trypsin cleavage site removed by the replacement of R203 with an A residue (2,29). The R203 to A change is in the loop between α-helices.…”

Section: Preparation Of Cry4ba Toxin and α-Cry4ba Serummentioning

confidence: 99%

“…The Cry4BRA toxin is equal to wild-type Cry4Ba in toxicity to A. quadrimaculatus, Ae. aegypti and Culex pipiens (2). We used Cry4BRA as a surrogate for Cry4Ba due to trypsin stability of the active toxin.…”

Section: Preparation Of Cry4ba Toxin and α-Cry4ba Serummentioning

confidence: 99%

“…We used Cry4BRA as a surrogate for Cry4Ba due to trypsin stability of the active toxin. Production of Cry4BRA (i.e., Cry4Ba) crystals and purification of toxin were as described previously (2). Trypsin digestion of Cry4Ba protoxin according to Abdullah et al (2) produced an ~66 kDa toxin mosquitocidal fragment.…”

Section: Preparation Of Cry4ba Toxin and α-Cry4ba Serummentioning

confidence: 99%

“…Production of Cry4BRA (i.e., Cry4Ba) crystals and purification of toxin were as described previously (2). Trypsin digestion of Cry4Ba protoxin according to Abdullah et al (2) produced an ~66 kDa toxin mosquitocidal fragment. Antiserum against Cry4Ba toxin was prepared in New Zealand White rabbits at the Animal Resources Facility at the University of Georgia.…”

Section: Preparation Of Cry4ba Toxin and α-Cry4ba Serummentioning

confidence: 99%

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Anopheles gambiae Cadherin AgCad1 Binds the Cry4Ba Toxin of Bacillus thuringiensis israelensis and a Fragment of AgCad1 Synergizes Toxicity

et al. 2008

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A midgut cadherin AgCad1 cDNA was cloned from Anopheles gambiae larvae and analyzed for its possible role as a receptor for the Cry4Ba toxin of Bacillus thuringiensis strain israelensis. The AgCad1 cadherin encodes a putative 1735-residue protein organized into an extracellular region of 11 cadherin repeats (CR) and a membrane-proximal extracellular domain (MPED). AgCad1 mRNA was detected in midgut of larvae by polymerase chain reaction (PCR). The AgCad1 protein was localized, by immunochemistry of sectioned larvae, predominately to the microvilli in posterior midgut. The localization of Cry4Ba binding was determined by the same technique, and toxin bound microvilli in posterior midgut. The AgCad1 protein was present in brush border membrane fractions prepared from larvae, and Cry4Ba toxin bound the same-sized protein on blots of those fractions. The AgCad1 protein was expressed transiently in Drosophila melanogaster Schneider 2 (S2) cells. 125 I-Cry4Ba toxin bound AgCad1 from S2 cells in a competitive manner. Cry4Ba bound to beads extracted 200 kDa AgCad1 and a 29 kDa fragment of AgCad1 from S2 cells. A peptide containing the AgCad1 region proximal to the cell (CR11-MPED) was expressed in Escherichia coli. Although Cry4Ba showed limited binding to CR11-MPED, the peptide synergized the toxicity of Cry4Ba to larvae. AgCad1 in the larval brush border is a binding protein for Cry4Ba toxin. On the basis of binding results and CR11-MPED synergism of Cry4Ba toxicity, AgCad1 is probably a Cry4Ba receptor.Mosquitoes in the genus Anopheles vector plasmodia that cause malaria, a major public health problem in the world. In some habitats, Anopheline species are controlled by nonchemical larvicides based on the bacterium Bacillus thuringiensis (Bt) 1 serovariety israelensis de Barjac. The specific toxicity of Bti to Anopheles and Aedes spp. is due to the protein components of the parasporal crystal (reviewed in ref 1).The parasporal crystal of Bti is composed of three major insecticidal Cry proteins (Cry4Aa, Cry4Ba, and Cry11Aa) and cytolytic proteins (Cyt1 and Cyt2). The Cry4Ba insecticidal protein is highly toxic to Anopheles and Aedes larvae but not to Culex larvae (2,3). The Cry4Ba toxin crystal structure shows that it is very similar to other Cry toxins with their three-domain † This research was supported by National Institutes of Health Grant R01 AI 29092 to D. H. Dean (The Ohio State University) and M.J.A. Inhibition of toxicity is accepted evidence for function as a Cry toxin receptor. Typically, a peptide fragment of the receptor (21) or a phage mimic of the receptor (20) attenuates Cry in vivo toxicity to larvae. Recently, we reported an opposite effect in which a fragment of Bt-R 1 cadherin, the Cry1A receptor from Manduca sexta (21,22), not only bound toxin but enhanced Cry1A toxicity against lepidopteran larvae (23). If the binding residues within cadherin repeat 12 (CR) were removed, the resulting peptide lost the ability to bind toxin and lost its function as a toxin synergist.In this work, we descri...

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Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin‐like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins

Endo

Kobayashi²,

Hoshino³

et al. 2014

MicrobiologyOpen

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Directed evolution of a Cry1Aa toxin using phage display and biopanning was performed to generate an increased binding affinity to the Bombyx mori cadherin-like receptor (BtR175). Three mutant toxins (371WGLA374, 371WPHH374, 371WRPQ37425) with 16-, 16-, and 50-fold higher binding affinities, respectively, for BtR175 were selected from a phage library containing toxins with mutations in domain II loop 2. However, the observed toxicities of the three mutants against B. mori larvae and cultured cells expressing the BtR175 toxin-binding region did not increase, suggesting that increased binding affinity to cadherins does not contribute to the insecticidal activity. Affinity maturation of a Cry toxin to a receptor via directed evolution was relatively simple to achieve, and seems to have potential for generating a toxin with increased insecticidal activity.

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Introduction of Culex Toxicity into Bacillus thuringiensis Cry4Ba by Protein Engineering

Cited by 76 publications

References 50 publications

Specificity determinants for Cry insecticidal proteins: Insights from their mode of action

Specificity determinants for Cry insecticidal proteins: Insights from their mode of action

Anopheles gambiae Cadherin AgCad1 Binds the Cry4Ba Toxin of Bacillus thuringiensis israelensis and a Fragment of AgCad1 Synergizes Toxicity

Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin‐like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins

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