The Stage-regulated Expression of Leishmania mexicana CPB Cysteine Proteases Is Mediated by an Intercistronic Sequence Element*

Abstract The tandemly arranged CPB genes ofLeishmania mexicana are polycistronically transcribed and encode cysteine proteases that are differentially stage-specific;CPB1 and CPB2 are expressed predominantly in metacyclics, whereas CPB3–CPB18 are expressed mainly in amastigotes. The mechanisms responsible for this differential expression have been studied via gene analysis and re-integration of individual CPB genes, and variants thereof, into aCPB-deficient parasite mutant. Comparison of the nucleotide sequences of the repeat units of CPB1 and CPB2with CPB2.8 (typical of CPB3–CPB18) revealed two major regions of divergence as follows: one of 258 base pairs (bp) corresponding to the C-terminal extension of CPB2.8; another, designated InS, of 120 bp, with insertions totaling 57 bp, localized to the intercistronic region downstream of CPB1 andCPB2. Cell lines expressing CPB2.8 orCPB2 with the 3′-untranslated region and intercistronic sequence of CPB2.8 showed up-regulation in amastigotes. Conversely, metacyclic-specific expression occurred withCPB2 or CPB2.8 with the 3′-untranslated region and intercistronic sequence of CPB2. Moreover, the InS down-regulated expression in amastigotes of a reporter gene integrated into the CPB locus. It is proposed that the InS mediates metacyclic-specific stage-regulated expression ofCPB by affecting the maturation of polycistronic pre-mRNA. This is the first well defined cis-regulatory element implicated in post-transcriptional stage-specific gene expression in Leishmania.