Ingestion, dissolution, and proteolysis of the Bacillus sphaericus toxin by mosquito larvae

Aly, Christoph; Mulla, Mir S.; Federici, Brian A.

doi:10.1016/0022-2011(89)90068-2

Cited by 33 publications

(13 citation statements)

References 14 publications

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“…In addition, derivatives of the 51-kDa protein which have this deletion have unexpected migration properties in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 51-kDa protein has been shown in the past to be much more susceptible than the 42-kDa protein to proteolysis by larval gut enzymes (1,4,7). As indicated by immunoblots, the reverse is the case with respect to breakdown products of these proteins in cell extracts of B. subtilis (this study ; 7, 8).…”

Section: Resultsmentioning

confidence: 54%

“…lanes j and k with m and n) and may involve changes in the properties of the modified proteins. resistant to further proteolysis (1,4,6,7). Evidence has been provided indicating that the latter process is accompanied by an activation of the toxin and that the 39-kDa derivative lacks 10 amino acids at the N terminus and 17 amino acids at the C terminus; both deletions correspond to potential chymotrypsin sites (6,8,9).…”

Section: Resultsmentioning

confidence: 99%

“…Following ingestion, the 51-and 42-kDa proteins are degraded to proteins of about 43 and 39 kDa, respectively (1,7). Evidence has been presented indicating that this processing leads to an activation of the toxin (6,9).…”

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confidence: 99%

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Deletion analysis of the 51-kilodalton protein of the Bacillus sphaericus 2362 binary mosquitocidal toxin: construction of derivatives equivalent to the larva-processed toxin

Clark

Baumann

1990

J Bacteriol

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Bacillus sphaericus 2362 produces a binary toxin consisting of 51-and 42-kDa proteins, both of which are required for toxicity to mosquito larvae. Upon ingestion by larvae, these proteins are processed to 43 and 39 kDa, respectively. Using site-directed mutagenesis, we have obtained N-and C-terminal deletions of the 51-kDa protein and expressed them in B. subtilis by using the subtilisin promoter. Removal of 21 amino acids from the N terminus and 53 amino acids from the C terminus resulted in a protein with the same electrophoretic properties as the 43-kDa degradation product which accumulates in the guts of mosquito larvae. This protein was toxic only in the presence of the 42-kDa protein. A deletion of 32 amino acids at the N terminus combined with a 53-amino-acid deletion at the C terminus resulted in a protein which retained toxicity. Toxicity was lost upon a further deletion of amino acids at potential chymotrypsin sites (41 at the N terminus, 61 at the C terminus). Comparison of the processing of the 51-and the 42-kDa proteins indicated that in spite of their sequence similarity proteolysis occurred at different sites.

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Section: Resultsmentioning

confidence: 54%

Section: Resultsmentioning

confidence: 99%

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Deletion analysis of the 51-kilodalton protein of the Bacillus sphaericus 2362 binary mosquitocidal toxin: construction of derivatives equivalent to the larva-processed toxin

Clark

Baumann

1990

J Bacteriol

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“…The presence of trypsinlike proteases in mosquito larvae has been demonstrated (18). Toxin processing by trypsin and chymotrypsinlike enzymes in mosquito gut extracts has been demonstrated for the 51.4-and 41.9-kDa toxins from the highly toxic B. sphaericus strains (1,8,9,12,13) and for the mosquitocidal toxins of B. thuringiensis (28a). Given the highly specific cleavage of the 97-kDa toxin by gut extracts, we suggest a similar processing of the Mtx2l protein by a trypsinlike enzyme.…”

Section: Discussionmentioning

confidence: 99%

Proteolytic processing of the mosquitocidal toxin from Bacillus sphaericus SSII-1

1992

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The 97-kDa protein Mtx2l, derived from the 100-kDa mosquitocidal protein (Mtx) from BaciUus sphaericus SSII-1 by the deletion of the putative signal sequence, was expressed as a fusion protein with glutathione S-transferase in Escherichia coli, and the fusion protein was purified by affinity chromatography. The fusion protein bound to glutathione agarose was cleaved with thrombin to release the Mtx21 protein. The 97-kDa Mtx21 protein was found to be toxic to Cukx quinquefasciatus larvae with a 50% lethal concentration of 15 ng/ml. Treating Mtx21 with crude mosquito larval gut extracts gave rise to two major peptides of 70 and 27 kDa. Treating the 97-kDa Mtx21 protein with trysin also gave rise to a similar proteolytic cleavage pattern. N-terminal sequencing showed that the 27-kDa peptide was derived from the N-terminal region of the 97-kDa protein and that the 70-kDa protein was from the C-terminal region of the 97-kDa protein. The 27-kDa peptide has all the previously identified regions of homology with the catalytic peptides of the ADP-ribosyltransferase toxins, such as pertussis toxin S1 peptide, while the 70-kDa peptide has three internal regions of homology.Bacillus sphaericus is an aerobic, gram-positive, sporeforming bacterium which is widespread in soil and aquatic environments. Some strains of B. sphaericus have been found to be pathogenic to mosquito larvae (14), and the pathogenicity of these bacteria has been attributed to the expression of toxic proteins (26,29,31,32 The high-toxicity strains have been shown to express a pair of proteins with relative mobilities corresponding to molecular masses of 63 and 43 kDa (5). Later studies in which the genes encoding these proteins were cloned and sequenced showed the molecular masses to be 51.4 and 41.9 kDa (2, 19). Both these proteins are required for toxicity to mosquito larvae (7, 13) and are expressed predominantly during sporulation (27). B. sphaericus SSII-1 is a lowtoxicity strain isolated from mosquito larva cadavers collected in India (29). The genes encoding the 51.4-and 41.9-kDa binary toxin have been shown to be absent from B.sphaericus . In order to study the nature of toxicity of B. sphaericus SSII-1, we have cloned a gene encoding a 100-kDa protein and have designated it the mix (mosquitocidal toxin) gene (31). The N-terminal region of the Mtx protein was found to have significant homology with the catalytic subunits of ADP-ribosyltransferase toxins, such as the pertussis toxin S1 subunit (28), and had no significant homology with the pair of toxins from the high-toxicity B. sphaericus strains (31). The aim of the present study was to purify the Mtx protein and to understand the processing of this protein in mosquito larvae to gain further insight into its mode of action. MATERIALS AND METHODSMaterials and strains. pGEX-1 and pUC18 were obtained from Pharmacia. Glutathione agarose, thrombin, and trypsin were purchased from Sigma USA. Escherichia coli (DH5) * Corresponding author. cells harboring plasmids were grown in Luria broth (24) supplem...

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“…When assayed against mosquito larvae, both proteins were found to be necessary for toxicity, indicating that the B. sphaericus larvicide may act as a binary toxin (Broadwell et al, 1990a, 6;Davidson et al, 1990;Baumann et al, 1991). Following ingestion of the toxin by larvae, the 51 and 42 kDa proteins are processed to lower molecular mass forms of approximately 44 and 39 kDa respectively (Broadwell & Baumann, 1987;Davidson et al, 1987aDavidson et al, , 1990Aly et al, 1989;Broadwell et al, 1990a). Studies using binary toxin purified from the spores of B. sphaericus, a mixture of the 5 1 and 42 kDa proteins, have shown that toxin binding occurs in the gastric caecum and posterior midgut of Culex larvae (Davidson, 1988).…”

Section: Introductionmentioning

confidence: 99%

Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains

Oei

Hindley²,

Berry³

1992

Journal of General Microbiology

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Highly larvicidal strains of Bacillus sphaericus produce a binary toxin composed of 51 and 42 kDa proteins which binds to sharply delineated regions of the gastric caecum and posterior midgut of susceptible larvae of the mosquito Culex quinquefmciafus. To investigate the role of the individual subunits and the organization of functional binding regions within the toxin, plasmids were constructed for the expression in Escherichia culi of the toxin proteins and their NH2-and COOH-terminal deletion derivatives as fusions with glutathione S-transferase (GST). Toxin proteins were purified by af6nity chromatography followed by cleavage from the GST carrier with thrombin. The LCs0 values for the purified toxin proteins and their deletion derivatives were determined. The binding patterns of fluorescently labelled toxin suggested that the 51 kDa protein is the primary binding component of the toxin and mediates the regional binding and internalization of the 42kDa protein. Examination of the toxin deletion derivatives revealed that the NH2-terminal region of the 51 kDa protein was required for binding to the larval gut, whilst the COOH-terminal region was responsible for interacting with the 42 kDa protein. Toxicity was strongly correlated with the subsequent internalization of the toxin, probably by endocytosis.

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Ingestion, dissolution, and proteolysis of the Bacillus sphaericus toxin by mosquito larvae

Cited by 33 publications

References 14 publications

Deletion analysis of the 51-kilodalton protein of the Bacillus sphaericus 2362 binary mosquitocidal toxin: construction of derivatives equivalent to the larva-processed toxin

Deletion analysis of the 51-kilodalton protein of the Bacillus sphaericus 2362 binary mosquitocidal toxin: construction of derivatives equivalent to the larva-processed toxin

Proteolytic processing of the mosquitocidal toxin from Bacillus sphaericus SSII-1

Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains

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