An alpha-amylase is a novel receptor for Bacillus thuringiensis ssp. israelensis Cry4Ba and Cry11Aa toxins in the malaria vector mosquito Anopheles albimanus (Diptera: Culicidae)

Abstract

Bacillus thuringiensis ssp. israelensis (Bti) produces four Cry toxins (Cry4Aa, Cry4Ba, Cry10Aa and Cry11Aa), and two Cyt proteins (Cyt1Aa and Cyt2Ba), toxic to mosquito-larvae of the genus Aedes, Anopheles and Culex, important human disease vectors that transmit dengue virus, malaria and filarial parasites respectively. Previous work showed that Bti is highly toxic to Anopheles albimanus, the main vector for transmission of malaria in Mexico. In this work, we analysed the toxicity of isolated Cry proteins of Bti and identified an An. albimanus midgut protein as a putative Cry4Ba and Cry11Aa receptor molecule. Biossays showed that Cry4Ba and Cry11Aa of Bti are toxic to An. albimanus larvae. Ligand blot assays indicated that a 70 kDa glycosylphosphatidylinositol-anchored protein present in midgut brush border membrane vesicles of An. albimanus interacts with Cry4Ba and Cry11Aa toxins. This protein was identified as an alpha-amylase by mass spectrometry and enzymatic activity assays. The cDNA that codes for the alpha-amylase was cloned by means of 5'- and 3'-RACE experiments. Recombinant alpha-amylase expressed in Escherichia coli specifically binds Cry4Ba and Cry11Aa toxins.

Fig. 1

A 70 kDa GPI-anchored protein that interacts with Cry4Ba and Cry11Aa toxins is an α-amylase. A. Cry4Ba and Cry11Aa toxins bind aGPI-anchored protein. BBMVs isolated fromAn. albimanus midgut larvae were treated withphospholipase C to release the GPI-anchoredprotein fraction, which was subjected toSDS-PAGE and electroblotted. Proteins thatbound biotin-labelled Cry4Ba and Cry11Aatoxins were detected by a ligand blot assay. B. The main component of the BBMVGPI-anchored protein fraction is a 70 kDaα-amylase. BBMV total proteins and BBMVproteins anchored by GPI were run in aSDS-PAGE and stained with Coomasie blue.Arrow shows the protein band that wassequenced by LC-MS/MS.

Fig. 2

Cry4Ba toxin binds an acidic, 70 kDa GPI-anchored protein. Colloidal Coomasie blue stained 2D gel (A) and ligand blot using biotin-labelled Cry4Ba toxin (B). Arrow shows the spot that was subjected to LC-MS/MS sequencing.

Fig. 3

A Cry4Ba binding protein shows α-glucosidase activity. In-gel α-glucosidase assay of the BBMV GPI-anchored protein fraction and the protein bound by an Agarose-Cry4Ba column.

Fig. 4

Nucleotide sequence of aamy1 gene and its deduced amino acid sequence. The initiation and stop codons are indicated by a star () and an asterisk (*) respectively. Peptide sequences obtained from LC-MS/MS identification are underlined. The N-terminal hydrophobic domain that corresponds to the putative signal peptide is shown in lower case. The ω-site for GPI modification and the amino acid putatively implicated in the cleavage site is shown as a pentagon. Asparagines predicted to be N-glycosylated are encircled. Seven conserved regions (I to VII) characteristic of the α-amylase family proteins are boxed. The catalytically important residues, the histidines responsible for substrate binding and the amino acids involved in binding of calcium ions are referred by diamonds, squares or a double underline respectively.The GenBank accession number of aamy1 gene is GQ344953.

Fig. 4

Nucleotide sequence of aamy1 gene and its deduced amino acid sequence. The initiation and stop codons are indicated by a star () and an asterisk (*) respectively. Peptide sequences obtained from LC-MS/MS identification are underlined. The N-terminal hydrophobic domain that corresponds to the putative signal peptide is shown in lower case. The ω-site for GPI modification and the amino acid putatively implicated in the cleavage site is shown as a pentagon. Asparagines predicted to be N-glycosylated are encircled. Seven conserved regions (I to VII) characteristic of the α-amylase family proteins are boxed. The catalytically important residues, the histidines responsible for substrate binding and the amino acids involved in binding of calcium ions are referred by diamonds, squares or a double underline respectively.The GenBank accession number of aamy1 gene is GQ344953.

Fig. 5

Specific binding of Cry4Ba toxin to heterologous expressed Aamy1 protein. A. Ligand blot analysis of cell extracts ofE. coli expressing Aamy1 protein usingbiotin-labelled Cry4Ba, Cry11Aa or Cry3Aatoxins. As a negative control, cell extractsprepared from E. coli cells transformed withan empty plasmid, in the absence of anybiotin-labelled Cry toxin but incubated withStreptavidin-peroxidase (lane marked with adash). B. Homologous competition of Cry4Ba bindingto heterologous expressed Aamy1 protein.Ligand blot analysis of cell extracts of E. coliexpressing Aamy1 protein usingbiotin-labelled Cry4Ba toxin in the absence orthe presence of an excess (50×) of unlabelledCry4Ba protein.