Antibacterial activity of Gyt1 Aa from Bacillus thuringiensis subsp. israelensis

摘要

Cyt1 Aa is a δ-endotoxin protein that is produced by Bacillus thuringiensis subsp. israelensis It is a membrane pore-forming toxin that is lethal to insect larvae and is broadly cytolytic to vertebrate as well as invertebrate cells. Cyt1Aa is produced as a protoxin of 27 kDa. Proteolytic activation results in a reduction of the molecular mass to approximately 23-24 kDa and a threefold increase in activity. In this research, Cyt1Aa crystals were purified from B. thuringiensis IPS78/11 harbouring the expression vector pHT-cyAp20. The activity of the activated form of Cyt1Aa (23-24 kDa) was examined on a pathogenic strain of the Gram-negative Escherichia coli and the Gram-positive species Staphylococcus aureus. The Cyt1Aa minimal inhibitory concentration for E. coli and S. aureus was 1.25 and 5 μg ml~(-1), respectively. Cyt1 Aa was found to be bactericidal for E. coli, whereas it was bacteriostatic for S. aureus. Furthermore, Cyt1Aa increased the lethal effect when acting in combination with antibiotics. The association of Cyt1 Aa with cells of these two bacteria was demonstrated by Western blot analysis using antibodies against the whole δ-endotoxin crystal. Scanning electron microscopy displayed damage to Cyt1Aa-treated cells. Ion imbalance due to damage of the cell walls and membranes was confirmed by X-ray microanalysis. These experiments show that Cyt1 Aa has an antibacterial effect on pathogenic species and demonstrate, apparently for the first time, that exogenous Cyt1 Aa has a bactericidal effect upon Gram-negative bacteria.