Cytopathological Effects of
            <i>Bacillus sphaericus</i>
            Cry48Aa/Cry49Aa Toxin on Binary Toxin-Susceptible and -Resistant
            <i>Culex quinquefasciatus</i>
            Larvae

Melo, Janaina Viana de; Jones, Gareth Wyn; Berry, Colin; Vasconcelos, Romero Henrique Teixeira; Oliveira, Cláudia Maria Fontes de; Furtado, André Freire; Peixoto, Christina Alves; Silva-Filha, Maria Helena Neves Lobo

doi:10.1128/aem.00811-09

Cited by 22 publications

(17 citation statements)

References 31 publications

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“…quinquefasciatus larvae resistant to the Bin and Cry48Aa1/Cry49Aa1 toxins were also observed using transmission electron microscopy (de Melo et al, 2008; 2009). These columnar epithelial cells were characterized by pronounced production of lipid inclusions throughout the 4 th instar.…”

Section: Resistance and Receptorsmentioning

confidence: 99%

Properties and applied use of the mosquitocidal bacterium, Bacillus sphaericus

Park¹,

Bideshi²,

Federici³

2010

Journal of Asia-Pacific Entomology

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Strains of Bacillus sphaericus exhibit varying levels of virulence against mosquito larvae. The most potent strain, B. sphaericus 2362, which is the active ingredient in the commercial product VectoLex®, together with another well-known larvicide Bacillus thuringiensis subsp. israelensis, are used to control vector and nuisance mosquito larvae in many regions of the world. Although not all strains of B. sphaericus are mosquitocidal, lethal strains produce one or two combinations of three different types of toxins. These are (1) the binary toxin (Bin) composed of two proteins of 42 kDa (BinA) and 51 kDa (BinB), which are synthesized during sporulation and co-crystallize, (2) the soluble mosquitocidal toxins (Mtx1, Mtx2 and Mtx3) produced during vegetative growth, and (3) the two-component crystal toxin (Cry48Aa1/Cry49Aa1). Non-mosquitocidal toxins are also produced by certain strains of B. sphaericus, for examples sphaericolysin, a novel insecticidal protein toxic to cockroaches. Larvicides based on B. sphaericus-based have the advantage of longer persistence in treated habitats compared to B. thuringiensis subsp. israelensis. However, resistance is a much greater threat, and has already emerged at significant levels in field populations in China and Thailand treated with B. sphaericus. This likely occurred because toxicity depends principally on Bin rather than various combinations of crystal (Cry) and cytolytic (Cyt) toxins present in B. thuringiensis subsp. israelensis. Here we review both the general characteristics of B. sphaericus, particularly as they relate to larvicidal isolates, and strategies or considerations for engineering more potent strains of this bacterium that contain built-in mechanisms that delay or overcome resistance to Bin in natural mosquito populations.

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Section: Resistance and Receptorsmentioning

confidence: 99%

Properties and applied use of the mosquitocidal bacterium, Bacillus sphaericus

Park¹,

Bideshi²,

Federici³

2010

Journal of Asia-Pacific Entomology

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“…israelensis cultures were kept under agitation (200 rpm) at 30°C for 72 h until sporulation (Ն80%). Bin spores/crystals were centrifuged, sequentially washed, and stored at 4°C (76). Cyt1Aa, Cry11Aa, and Cry1Ab spore/crystals were washed three times with acidic distilled water adjusted at pH 4 using 5 M HCl, six times with 300 mM NaCl-10 mM EDTA (pH 8), and three times with a 1 mM concentration of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and then stored at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Functional Bacillus thuringiensis Cyt1Aa Is Necessary To Synergize Lysinibacillus sphaericus Binary Toxin (Bin) against Bin-Resistant and -Refractory Mosquito Species

Nascimento

Torres-Quintero

Molina

et al. 2020

Appl Environ Microbiol

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The binary (Bin) toxin from Lysinibacillus sphaericus is effective to mosquito larvae, but its utilization is threatened by the development of insect resistance. Bin toxin is composed of the BinB subunit required for binding to midgut receptors and the BinA subunit that causes toxicity after cell internalization, mediated by BinB. Culex quinquefasciatus resistance to this toxin is caused by mutations that prevent expression of Bin toxin receptors in the midgut. Previously, it was shown that the Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis restores Bin toxicity to Bin-resistant C. quinquefasciatus and to Aedes aegypti larvae, which are naturally devoid of functional Bin receptors. Our goal was to elucidate the mechanism involved in Cyt1Aa synergism with Bin in such larvae. In vivo assays showed that the mixture of Bin toxin, or its BinA subunit, with Cyt1Aa was effective to kill resistant larvae. However, no specific binding interaction between Cyt1Aa and the Bin toxin, or its subunits, was observed. The synergy between Cyt1Aa and Bin toxins is dependent on functional Cyt1Aa, as demonstrated by using the nontoxic Cyt1AaV122E mutant toxin affected in oligomerization and membrane insertion, which was unable to synergize Bin toxicity in resistant larvae. The synergism correlated with the internalization of Bin or BinA into anterior and medium midgut epithelial cells, which occurred only in larvae treated with wild-type Cyt1Aa toxin. This toxin is able to overcome failures in the binding step involving BinB receptor by allowing the internalization of Bin toxin, or its BinA subunit, into the midgut cells. IMPORTANCE One promising management strategy for mosquito control is the utilization of a mixture of L. sphaericus and B. thuringiensis subsp. israelensis insecticidal toxins. From this set, Bin and Cyt1Aa toxins synergize and display toxicity to resistant C. quinquefasciatus and to A. aegypti larvae, whose midgut cells lack Bin toxin receptors. Our data set provides evidence that functional Cyt1Aa is essential for internalization of Bin or its BinA subunit into such cells, but binding interaction between Bin and Cyt1Aa is not observed. Thus, this mechanism contrasts with that for the synergy between Cyt1Aa and the B. thuringiensis subsp. israelensis Cry toxins, where active Cyt1Aa is not necessary but a specific binding between Cry and Cyt1Aa is required. Our study established the initial molecular basis of the synergy between Bin and Cyt1Aa, and these findings enlarge our knowledge of their mode of action, which could help to develop improved strategies to cope with insect resistance.

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“…The theoretical model of the Cry49Aa1 protein missing the 40 amino acids closest to the N‐terminal showed significant similarity to Cry35Aa1, particularly in the C‐terminal β‐sheet region (Kelker et al ., ). Previous reports have demonstrated that the original steps of the action mode of Cry48Aa/Cry49Aa crystals were similar to that of the Bin toxin, both including the ingestion of crystals and their solubilization under alkaline pH, followed by proteolytic activation of the protoxin into toxin in the midgut, destroying the cells and killing the larvae (Baumann et al ., ; Jones et al ., ; de Melo et al ., ). Further research found that Cry49Aa processed by C. quinquefasciatus gut extract produced a band of about 47 kDa, which was identified as the site of a chymotrypsin‐like cleavage between F48 and N49.…”

Section: Introductionmentioning

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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Cytopathological Effects of Bacillus sphaericus Cry48Aa/Cry49Aa Toxin on Binary Toxin-Susceptible and -Resistant Culex quinquefasciatus Larvae

Cited by 22 publications

References 31 publications

Properties and applied use of the mosquitocidal bacterium, Bacillus sphaericus

Properties and applied use of the mosquitocidal bacterium, Bacillus sphaericus

Functional Bacillus thuringiensis Cyt1Aa Is Necessary To Synergize Lysinibacillus sphaericus Binary Toxin (Bin) against Bin-Resistant and -Refractory Mosquito Species

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

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