title: Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis.
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.
authors.affiliation: Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
authors.forename: Angeles
authors.initials: A
authors.lastname: Cancino-Rodezno
authors.forename: Luis
authors.initials: L
authors.lastname: Lozano
authors.forename: Cris
authors.initials: C
authors.lastname: Oppert
authors.forename: Julieta I
authors.initials: JI
authors.lastname: Castro
authors.forename: Humberto
authors.initials: H
authors.lastname: Lanz-Mendoza
authors.forename: Sergio
authors.initials: S
authors.lastname: Encarnación
authors.forename: Amy E
authors.initials: AE
authors.lastname: Evans
authors.forename: Sarjeet S
authors.initials: SS
authors.lastname: Gill
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
authors.forename: Juan L
authors.initials: JL
authors.lastname: Jurat-Fuentes
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
mesh_terms: D000330:Aedes / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism*; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D007313:Insecta / Q000235:genetics / Q000378:metabolism; D007814:Larva / Q000235:genetics* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis