Amino acid substitutions in alphaA and alphaC of Cyt2Aa2 alter hemolytic activity and mosquito-larvicidal specificity.
Cyt2Aa2 produced by Bacillus thuringiensis subsp. darmstadiensis exhibits in vitro cytolytic activity against broad range of cells but shows specific in vivo toxicity against larvae of Dipteran insects. To investigate the role of amino acids in alphaA and alphaC of this toxin, 3 single-point mutants (A61C, S108C and V109A) were generated. All 3 mutant proteins were highly produced as inclusion bodies that could be solubilized and activated by proteinase K similar to that of the wild type. Hemolytic activity of A61C and S108C mutants was significantly reduced whereas the V109A mutant showed comparable hemolytic activity to the wild type. Interestingly, the A61C mutant exhibited high larvicidal activity to both Aedes aegypti and Culex quinquefasciatus. S108C and V109A mutants showed low activity against C. quinquefasciatus but relatively high toxicity to A. aegypti. These results demonstrated for the first time that amino acids in alphaA and alphaC are involved in the selectivity of the Cyt toxin to the targeted organism.
Promdonkoy
Boonhiang
B
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phahonyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand. boonhiang@biotec.or.th
Rungrod
Amporn
A
Promdonkoy
Patcharee
P
Pathaichindachote
Wanwarang
W
Krittanai
Chartchai
C
Panyim
Sakol
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000187:drug effects; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D009033:Culicidae / Q000187:drug effects / Q000502:physiology*; D004731:Endotoxins / Q000737:chemistry / Q000378:metabolism*; D004912:Erythrocytes / Q000187:drug effects; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism*; D006461:Hemolysis / Q000187:drug effects / Q000502:physiology*; D007814:Larva / Q000187:drug effects; D008969:Molecular Sequence Data; D009032:Mosquito Control; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D012756:Sheep; D012995:Solubility / Q000187:drug effects; D013045:Species Specificity
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D050505:Mutant Proteins; D021122:Protein Subunits; C030956:insecticidal crystal protein, Bacillus Thuringiensis