Functional characterization of truncated fragments of Bacillus sphaericus binary toxin BinB

Tangsongcharoen, Chontida; Boonserm, Panadda; Promdonkoy, Boonhiang

doi:10.1016/j.jip.2010.10.004

Cited by 9 publications

(7 citation statements)

References 29 publications

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“…The results from the binding data indicate that, excluding the first 32 residues that are removed upon BinB proteolytic cleavage in vivo , the N‐terminal segment encompassing residues from N33 to L158 is required for receptor binding. A recent immunohistochemistry study, which investigated the ability of BinB truncated constructs to bind to midgut sections of C. quinquefasciatus , showed that two N‐terminal N‐25K (N33‐K254), N‐32K (N33‐R318) as well as two C‐terminal proteins, C‐32K (E133‐K408) and C‐18K (M255‐K408), showed specific binding comparable to BinB (Tangsongcharoen et al , 2011). This study indicated that amino acids involved in the receptor‐binding motif are present in both regions between N33‐K254 and M255‐K408; however, it should be noted that these segments represent the entire active core of BinB and do not delimitate specific regions related to this function.…”

Section: Resultsmentioning

confidence: 99%

The N-terminal third of the BinB subunit from the Bacillus sphaericus binary toxin is sufficient for its interaction with midgut receptors in Culex quinquefasciatus

Romão¹,

Neto²,

Silva-Filha³

2011

FEMS Microbiology Letters

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Heterodimeric binary (Bin) toxin, the major insecticidal protein from Bacillus sphaericus, acts on Culex quinquefasciatus larvae through specific binding to the midgut receptor Cqm1, a role mediated by its 448-amino-acid-long BinB subunit. The molecular basis for receptor recognition is not well understood and this study attempted to identify protein segments and amino acid motifs within BinB that are required for this event. First, N- and C-terminally truncated constructs were evaluated for their capacity to bind to native Cqm1 through pull-down assays. These showed that residues N33 to L158 of the subunit are required for Cqm1 binding. Nine different full-length mutants were then generated in which selected blocks of three amino acids were replaced by alanines. In new pull-down assays, two mutants, in which residues (85) IRF(87) and (147) FQF(149) were targeted, failed to bind the receptor. Competition binding assays confirmed the requirements for the N-terminal 158 residues, and the (147) FQF(149) epitope, for the mutant proteins to compete with native Bin toxin when binding to membrane fractions from the insect midgut. The data from this work rule out the involvement of C-terminal segments in receptor binding, highlighting the need for multiple elements within the protein's N-terminal third for it to occur.

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Section: Resultsmentioning

confidence: 99%

The N-terminal third of the BinB subunit from the Bacillus sphaericus binary toxin is sufficient for its interaction with midgut receptors in Culex quinquefasciatus

Romão¹,

Neto²,

Silva-Filha³

2011

FEMS Microbiology Letters

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“…As the strongest interaction with Cry48Aa was observed with the smallest N‐terminal fragment of Cry49Aa, it is most probable that the Cry48Aa ‐binding site is localized between residues N49 and S149. These results are similar to previous work that showed that the receptor binding site of the BinB subunit is located in its N‐terminal region (Romao et al ., ; Tangsongcharoen et al ., ), whereas the C‐terminal region was able to interact with BinA (Limpanawat et al ., ). Thus, we inferred that Cry49Aa plays an important role in the mechanism of action of these two‐component toxins.…”

Section: Discussionmentioning

confidence: 97%

Interaction of Lysinibacillus sphaericus Cry48Aa/Cry49Aa toxin with midgut brush‐border membrane fractions from Culex quinquefasciatus larvae

Guo

Cai

et al. 2016

Insect Molecular Biology

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The Cry48Aa/Cry49Aa mosquitocidal toxin from Lysinibacillus sphaericus was uniquely composed of a three-domain (Cry) toxin and binary (Bin) toxin-like protein, with high toxicity against Culex spp. However, its mode of action against the target mosquitoes is still unknown. In this study, Cry48Aa, Cry49Aa and its N- and C-terminal truncated proteins were expressed and purified, and the binding affinities of the purified proteins with midgut brush-border membrane fractions (BBMFs) from Culex quin-quefasciatus larvae were performed. The results showed that both Cry48Aa and Cry49Aa have specific and high binding affinity to BBMFs, with dissociation constants of 9.5 ± 1.8 and 25.4 ± 3.8 nM, respectively. Competition assays demonstrated that Cry49Aa C-terminal derivatives were able to bind to the BBMFs, whereas Far-Western dot blot analysis revealed that its N-terminal constructs interacted with Cry48Aa. Nevertheless, larvicidal activity was almost lost when Cry49Aa truncated proteins, either individually or in pairs, combined with Cry48Aa. It is concluded that Cry49Aa is responsible for receptor binding and interaction with Cry48Aa and plays an important role in the mechanism of action of these two-component toxins.

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“…The Cry4Aa, Cry4Ba, and Cry11Aa toxins are composed of three domains: domain I is involved in toxin oligomerization and in the pore-formation activity, while domains II and III are involved in receptor binding [ 9 ]. The available crystallographic structures of Cry4Ba and Cry4Aa showed their three-domain structure [ 34 , 37 , 38 , 39 , 40 , 41 , 42 ]. The proposed model of their mode of action is the pore-formation model that was first established for Cry1A toxins in the midgut of the lepidopteran Manduca sexta and involves the sequential binding of the toxin to different receptors.…”

Section: Toxins and Mode Of Actionmentioning

confidence: 99%

Bacterial Toxins Active against Mosquitoes: Mode of Action and Resistance

Silva-Filha

Romão

Rezende

et al. 2021

Toxins

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Larvicides based on the bacteria Bacillus thuringiensis svar. israelensis (Bti) and Lysinibacillus sphaericus are effective and environmentally safe compounds for the control of dipteran insects of medical importance. They produce crystals that display specific and potent insecticidal activity against larvae. Bti crystals are composed of multiple protoxins: three from the three-domain Cry type family, which bind to different cell receptors in the midgut, and one cytolytic (Cyt1Aa) protoxin that can insert itself into the cell membrane and act as surrogate receptor of the Cry toxins. Together, those toxins display a complex mode of action that shows a low risk of resistance selection. L. sphaericus crystals contain one major binary toxin that display an outstanding persistence in field conditions, which is superior to Bti. However, the action of the Bin toxin based on its interaction with a single receptor is vulnerable for resistance selection in insects. In this review we present the most recent data on the mode of action and synergism of these toxins, resistance issues, and examples of their use worldwide. Data reported in recent years improved our understanding of the mechanism of action of these toxins, showed that their combined use can enhance their activity and counteract resistance, and reinforced their relevance for mosquito control programs in the future years.

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Functional characterization of truncated fragments of Bacillus sphaericus binary toxin BinB

Cited by 9 publications

References 29 publications

The N-terminal third of the BinB subunit from the Bacillus sphaericus binary toxin is sufficient for its interaction with midgut receptors in Culex quinquefasciatus

The N-terminal third of the BinB subunit from the Bacillus sphaericus binary toxin is sufficient for its interaction with midgut receptors in Culex quinquefasciatus

Interaction of Lysinibacillus sphaericus Cry48Aa/Cry49Aa toxin with midgut brush‐border membrane fractions from Culex quinquefasciatus larvae

Bacterial Toxins Active against Mosquitoes: Mode of Action and Resistance

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