Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis: Modulation of gp63 level in promastigotes and their infection ofmacrophages in vitro

The major surface glycoprotein (gp63) of Leishmania amazonensis is a metall oprotease implicated in the infection of mammalian macrophages. The express ion of gp63 and its participation in this infection were further examined b y modulating the level of this molecule in a virulent gp63-abundant wild-ty pe clone. Promastigotes were transfected with gp63 genes cloned into a Leis hmania-specific vector in two different orientations, leading to the expres sion of gp63 sense and antisense RNAs. With increasing selective pressure, cell surface gp63 was increasingly augmented in the transfectants with sens e transcripts and suppressed to a very low level in those with antisense tr anscripts. Thus, the expression of gp63 from chromosomal, repetitive genes is not stringently regulated at the protein level and can be substantially reduced by episomal antisense transcription of a single copy. The transfect ants differed significantly only in the level of gp63, thereby allowing spe cific evaluation of this molecule in leishmanial infection of macrophages i n vitro. Kinetic studies of infection in vitro indicate that gp63 plays a r ole not only in the binding of this parasite to these macrophages but also in its intramacrophage survival and replication.