NULL MUTANTS FOR THE LMCPA CYSTEINE PROTEINASE GENE IN LEISHMANIA-MEXICANA

The parasitic protozoon Leishmania mexicana possesses an abundance of developmentally regulated cathepsin L-like cysteine proteinases expres sed at highest levels in amastigotes. We recently characterised Imcpa, a single-copy gene encoding one such proteinase, LmCPa, which differs from other homologues by possessing a 3-amino-acid insertion at the a mino terminal of the predicted mature proteinase. To investigate the r ole of LmCPa in L. mexicana, we used gene targeting of promastigotes w ith hygromycin- and phleomycin-resistance markers to generate null mut ants by disrupting sequentially both alleles of lmcpa. The promastigot e null mutants did not differ significantly from wild-type L. mexicana in growth rate or morphology and could differentiate to metacyclics a nd the amastigote-like form, both of which could infect the J774G8 mac rophage-like cell line. The null mutant amastigote-like form obtained from the J774G8 cells could also establish rump lesions in CBA mice. B y these criteria, therefore, LmCPa appears to be non-essential althoug h there is the possibility that LmCPa could be required during develop ment in the sandfly, a stage not analysed here. The apparent redundanc y of LmCPa in amastigotes may be due to the presence of other cysteine proteinases and has implications for the choice of candidate targets for rationally designed anti-leishmanial drugs.