Bactericidal activity of Lys49 and Asp49 myotoxic phospholipases A2 from Bothrops asper snake venom--synthetic Lys49 myotoxin II-(115-129)-peptide identifies its bactericidal region.
Mammalian group-II phospholipases A2 (PLA2) of inflammatory fluids display bactericidal properties, which are dependent on their enzymatic activity. This study shows that myotoxins II (Lys49) and III (Asp49), two group-II PLA2 isoforms from the venom of Bothrops asper, are lethal to a broad spectrum of bacteria. Since the catalytically inactive Lys49 myotoxin II isoform has similar bactericidal effects to its catalytically active Asp49 counterpart, a bactericidal mechanism that is independent of an intrinsic PLA2 activity is demonstrated. Moreover, a synthetic 13-residue peptide of myotoxin II, comprising residues 115-129 (common numbering system) near the C-terminal loop, reproduced the bactericidal effect of the intact protein. Following exposure to the peptide or the protein, accelerated uptake of the hydrophobic probe N-phenyl-N-naphthylamine was observed in susceptible but not in resistant bacteria, indicating that the lethal effect was initiated on the bacterial membrane. The outer membrane, isolated lipopolysaccharide (LPS), and lipid A of susceptible bacteria showed higher binding to the myotoxin II-(115-129)-peptide than the corresponding moieties of resistant strains. Bacterial LPS chimeras indicated that LPS is a relevant target for myotoxin II-(115-129)-peptide. When heterologous LPS of the resistant strain was present in the context of susceptible bacteria, the chimera became resistant, and vice versa. Myotoxin II represents a group-II PLA2 with a direct bactericidal effect that is independent of an intrinsic enzymatic activity, but adscribed to the presence of a short cluster of basic/hydrophobic amino acids near its C-terminal loop.
Páramo
L
L
Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica.
Lomonte
B
B
Pizarro-Cerdá
J
J
Bengoechea
J A
JA
Gorvel
J P
JP
Moreno
E
E
D000818:Animals; D017837:Bothrops; D002002:Brucella / Q000187:drug effects* / Q000648:ultrastructure; D003435:Crotalid Venoms / Q000201:enzymology*; D004168:Diphtheria Toxoid / Q000737:chemistry; D004925:Escherichia / Q000187:drug effects / Q000648:ultrastructure; D005470:Fluorometry; D054501:Group II Phospholipases A2; D007527:Isoenzymes / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D008854:Microscopy, Electron; D009498:Neurotoxins / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010741:Phospholipases A / Q000494:pharmacology*; D054467:Phospholipases A2; D030162:Reptilian Proteins; D012475:Salmonella / Q000187:drug effects; D014734:Vibrio cholerae / Q000187:drug effects / Q000648:ultrastructure
D003435:Crotalid Venoms; D004168:Diphtheria Toxoid; D007527:Isoenzymes; D009498:Neurotoxins; D010446:Peptide Fragments; D030162:Reptilian Proteins; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; C061056:myotoxin II, Bothrops asper