Complement resistance of Leishmania amazonensis promastigotes is independent of parasite proteases and lysis of sensitive forms is not due to natural antibodies in normal human serum

Leishmania amazonensis promastigotes cultivated in vitro differentiate from complement-sensitive to complement-resistant forms. In order to determine the possible involvement of parasite proteases in this process, L. amazonensis promastigotes were collected daily and their proteolytic enzyme patterns analyzed using polacrylamide gels copolymerized with gelatin. Although promastigotes at different growth stages showed differences in protease patterns, these changes did not correlate with their susceptibility to complement. The major protease of promastigotes, gp63, was expressed at the same level throughout culture, regardless of the complement resistance of the promastigotes. Furthermore, inhibitors specific for the classes of proteases found in L. amazonensis promastigotes did not interfere with the complement-mediated killing of promastigotes. We also investigated the binding of natural antibodies to promastigotes. at different stages of growth using ELISA. Although complement-sensitive promastigotes bound significantly more antibodies from fresh normal human serum than complement-resistant promastigotes, equivalent amounts of C3 were detected on their surfaces following complement activation. Moreover, serum depleted of anti-Leishmania antibodies was an efficient in killing promastigotes and the intact serum. These data suggest that the resistance of L. amazonensis to complement killing involves strategies other than that of the regulated expression of endogenous proteases capable of inactivating complement components, or the differential ability to bind natural antibodies that might interfere with complement deposition on the parasite surface.