title: Permeabilization of E. coli K12 inner and outer membranes by bothropstoxin-I, A LYS49 phospholipase A2 from Bothrops jararacussu.
abstract: Although lacking catalytic activity, the Lys49-PLA(2)s damage artificial membranes by a Ca(2+)-independent mechanism, and demonstrate a potent bactericidal effect. The relationship between the membrane-damaging activity and bactericidal effect of bothropstoxin-I (BthTx-I), a Lys49-PLA(2) from the venom of Bothrops jararacussu, was evaluated for the wild-type protein and a series of site-directed mutants in the active site and C-terminal regions of the protein. The membrane permeabilization effect against the inner and outer membranes of Escherichia coli K12 was evaluated by fluorescence changes of Sytox Green and N-phenyl-N-naphthylamine, respectively. With the exception of H48Q, all mutants reduced the bactericidal activity, which correlated with a reduction of the permeabilization effect both against the inner bacterial membrane. No significant differences in the permeabilization of the bacterial outer membrane were observed between the native, wild-type recombinant and mutant proteins. These results suggest different permeabilization mechanisms against the inner and outer bacterial membranes. Furthermore, the structural determinants of bacterial inner membrane damage identified in this study correlate with those previously observed for artificial membrane permeabilization, suggesting that a common mechanism of membrane damage underlies the two effects.
authors.affiliation: Department of Chemistry, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Monte Alegre, CEP 14040-901, Ribeirão Preto, SP, Brazil.
authors.forename: Elisangela Aparecida
authors.initials: EA
authors.lastname: Aragão
authors.forename: Lucimara
authors.initials: L
authors.lastname: Chioato
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
mesh_terms: D000818:Animals; D017837:Bothrops / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects*; D004926:Escherichia coli / Q000187:drug effects*; D009154:Mutation; D010539:Permeability; D054467:Phospholipases A2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030162:Reptilian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D030162:Reptilian Proteins; D054467:Phospholipases A2