Expression of the major surface glycoprotein of Leishmania, gp63, in wild‐type and sinefungin‐resistant promastigotes

In this study, we have surveyed gp63 expression in sinefungin‐(SF)‐resistant and wild‐type Leishmania promastigotes. Documentation of gp63 expression in Leishmania promastigotes was carried out by Western blotting, purification of the protein and assessment of gp63 protease activity. We demonstrated a 3–4‐fold and 1.5–2‐fold increase of gp63 protein in SF‐resistant Leishmania donovani and Leishmania tropica promastigotes compared to wild‐type, respectively. Northern blot analysis showed that the increase in the amount of gp63 protein in SF‐resistant compared to wild‐type parasites was concomitant with an increase in gp63 mRNA. No extrachromosomal DNA was identified by alkaline lysis of isolated DNA samples and Southern blot analysis. Treatment of SF‐resistant and wild‐type L. donovani promastigotes with cycloheximide resulted in an increase of the steady state levels of gp63 mRNA in the SF‐resistant parasites to approximately fivefold that of the wild type. After treating parasites with actinomycin D, estimated gp63 mRNA t1/2 in the wild type was 40 min and increased to 83 min in SF‐resistant promastigotes. Therefore, the overexpression of gp63 may be mediated, at least in part, by post‐transcriptional stabilization of a gp63 transcript by a protein factor. Down regulation of the latter factor may account for the observed increase in gp63 expression in SF‐resistant promastigotes. Attempts to correlate gp63 expression with promastigote virulence suggested that the observed increase in gp63 expression did not result in a significant change in the virulence of SF‐resistant compared to wild‐type L. donovani promastigotes.