Proteomic identification of Bacillus thuringiensis subsp. israelensis toxin Cry4Ba binding proteins in midgut membranes from Aedes (Stegomyia) aegypti Linnaeus (Diptera, Culicidae) larvae.
ABSTRACT Novel Bacillus thuringiensis subsp. israelensis (Bti) Cry4Ba toxin-binding proteins have been identified in gut brush border membranes of the Aedes (Stegomyia) aegypti mosquito larvae by combining 2-dimensional gel electrophoresis (2DE) and ligand blotting followed by protein identification using mass spectrometry and database searching. Three alkaline phosphatase isoforms and aminopeptidase were identified. Other Cry4Ba binding proteins identified include the putative lipid raft proteins flotillin and prohibitin, V-ATPase B subunit and actin. These identified proteins might play important roles in mediating the toxicity of Cry4Ba due to their location in the gut brush border membrane. Cadherin-type protein was not identified, although previously, we identified a midgut cadherin AgCad1 as a putative Cry4Ba receptor in Anopheles gambiae mosquito larvae [Hua, G., Zhang, R., Abdullah, M.A., Adang, M.J., 2008. Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity. Biochemistry 47, 5101-5110]. Other identified proteins in this study that might have lesser roles include mitochondrial proteins such as ATP synthase subunits, mitochondrial processing peptidase and porin; which are likely contaminants from mitochondria and are not brush border membrane components. Trypsin-like serine protease was also identified as a protein that binds Cry4Ba. Identification of these toxin-binding proteins will lead to a better understanding of the mode of action of this toxin in mosquito.
Article: Larval midgut modifications associated with Bti resistance in the yellow fever mosquito using proteomic and transcriptomic approaches.[show abstract] [hide abstract]
ABSTRACT: Bacillus thuringiensis var. israelensis (Bti) is a natural larval mosquito pathogen producing pore-forming toxins targeting the midgut of Diptera larvae. It is used worldwide for mosquito control. Resistance mechanisms of an Aedes aegypti laboratory strain selected for 30 generations with field-collected leaf litter containing Bti toxins were investigated in larval midguts at two levels: 1. gene transcription using DNA microarray and RT-qPCR and 2. differential expression of brush border membrane proteins using DIGE (Differential In Gel Electrophoresis). Several Bti Cry toxin receptors including alkaline phosphatases and N-aminopeptidases and toxin-binding V-ATPases exhibited altered expression levels in the resistant strain. The under-expression of putative Bti-receptors is consistent with Bt-resistance mechanisms previously described in Lepidoptera. Four soluble metalloproteinases were found under-transcribed together with a drastic decrease of metalloproteinases activity in the resistant strain, suggesting a role in resistance by decreasing the amount of activated Cry toxins in the larval midgut. By combining transcriptomic and proteomic approaches, we detected expression changes at nearly each step of the ingestion-to-infection process, providing a short list of genes and proteins potentially involved in Bti-resistance whose implication needs to be validated. Collectively, these results open the way to further functional analyses to better characterize Bti-resistance mechanisms in mosquitoes.BMC Genomics 06/2012; 13:248. · 4.07 Impact Factor
Article: Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis.[show abstract] [hide abstract]
ABSTRACT: Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes.PLoS ONE 01/2012; 7(5):e37034. · 4.09 Impact Factor