Bactericidal Effect Of Ly49-Phospholipase A2 (Lys49-PLA2): A Role Of The C-Terminal Region Of Bothropstoxin-I In The Damage Of Bactericidal And Artificial Membranes.

¹Aragão, E.A.; ²Chioato, L. and ¹Ward, R.J.

1 Depto. de Química, FFCLRP-USP, Ribeirão Preto; 2 Depto. de Bioquímica e Imunologia, FMRP-USP, Ribeirão Preto.

The interaction of the catalytically inactive Lys49-PLA2s with liposome membranes provokes the rapid Ca²⁺-independent release of liposome contents. The Lys-PLA2s also demonstrate bactericidal activity, and this work aims to evaluate a possible correlation between the bactericidal and artificial membrane damaging activities. We have suggested that a transition between “open” and “closed” conformations, and consequent changes in the position of the C-terminal region of the homodimeric bothropstoxin-I (BthTx-I), a Lys49-PLA2 isolated from the venom of Bothropsjararacussu, may be responsible for membrane damage. A series of 9 charge elimination or aromatic substitution site-directed mutants in the C-terminal loop of BthTx-I were expressed in E.coli, and purified by cation exchange chromatography. The membrane damaging activities as measured by release of liposome entrapped calcein, and the bactericidal activity against E. coli K12 of all 9 mutants were compared. At concentrations where 100% inhibition of colony forming units (CFU) was observed for native and wild type recombinant proteins, the K122A, F125W, K127A and K129A mutants showed significantly reduced CFU inhibitory activity. These same 4 mutants also demonstrated reduced damaging activity against liposome membranes, supporting the suggestion that the bactericidal and artificial membrane damaging activities are correlated.

Support: FAPESP, CNPQ and PRP-USP.

Correspondence to: li_usp@yahoo.com.br