Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in Leishmania mexicana

Abstract

SummaryIn the past, ultrastructural investigations of Leishmania mexicana amastigotes revealed structures that were tentatively identified as autophagosomes. This study has now provided definitive data that autophagy occurs in the parasite during differentiation both to metacyclic promastigotes and to amastigotes, autophagosomes being particularly numerous during metacyclic to amastigote form transformation. Moreover, the results demonstrate that inhibiting two major lysosomal cysteine peptidases (CPA and CPB) or removing their genes not only interferes with the autophagy pathway but also prevents metacyclogenesis and transformation to amastigotes, thus adding support to the hypothesis that autophagy is required for cell differentiation. The study suggests that L. mexicana CPA and CPB perform similar roles to the aspartic peptidase PEP4 and the serine peptidase PRB1 in Saccharomyces cerevisiae. The results also provide an explanation for why L. mexicana CPA/ CPB-deficient mutants transform to amastigotes very poorly and lack virulence in macrophages and mice.

Autophagy Is Involved in Nutritional Stress Response and Differentiation in Trypanosoma cruzi

“…Moreover, CPA and CPB from Leishmania mexicana were recently found to be essential for both autophagosome degradation and differentiation of that parasite. In addition, LmAtg4.2 was suggested to be involved in autophagosome formation, thus being upstream of CPA and CPB (39,46).…”

Glycosome turnover inLeishmania majoris mediated by autophagy

“…Further characterization showed that Leishmania expressing GFP-ATG8 form GFP-labeled puncta corresponding to autophagosomes and that autophagy is induced by starvation, as in yeast and mammals, as well as increasing during differentiation. 20,21 During procyclic to metacyclic promastigote differentiation, GFP-ATG8 appears in tubular structures resembling the MVT-lysosome and the PEconjugated form of ATG8 is more abundant as shown by western blotting. Leishmania with defects in the autophagy pathway, either overexpressing a dominant-negative VPS4, important in endosomal sorting pathways, 21 or lacking the major lysosomal peptidases CPA and CPB, 20 are unable to fully differentiate, in addition to being unable to survive prolonged starvation.…”

“…20,21 During procyclic to metacyclic promastigote differentiation, GFP-ATG8 appears in tubular structures resembling the MVT-lysosome and the PEconjugated form of ATG8 is more abundant as shown by western blotting. Leishmania with defects in the autophagy pathway, either overexpressing a dominant-negative VPS4, important in endosomal sorting pathways, 21 or lacking the major lysosomal peptidases CPA and CPB, 20 are unable to fully differentiate, in addition to being unable to survive prolonged starvation. These data show that autophagy is important for successful parasite differentiation, presumably because it is required for protein turnover during the cellular remodeling that occurs throughout the differentiation process.…”

“…As ATG8 remains associated with the autophagosome, GFP-ATG8 is a common molecular marker for tracking autophagosomes by fluorescence microscopy, from their formation to their delivery to the lysosome. Genes encoding proteins with similarity to the core ATG proteins in yeast and mammals have been described in Leishmania species, 19,20 suggesting that these parasites possess a functional autophagy pathway. Further characterization showed that Leishmania expressing GFP-ATG8 form GFP-labeled puncta corresponding to autophagosomes and that autophagy is induced by starvation, as in yeast and mammals, as well as increasing during differentiation.…”

Biological Roles of Peptidases in Trypanosomatids~!2009-11-26~!2010-02-15~!2010-03-18~!

“…Also shown was that the absence of the cpb genes resulted in a shift in the immune response from predominantly Th2 immune response (wild-type) to the Th1 response, normally observed when the CPB isoenzymes are present [133]. It has also been suggested that in L. mexicana these enzymes are vital for autophagy and differentiation of the parasite [136]. Recently, our group described that drug-induced Leishmania autophagy is accompanied by enhanced cpb expression [137].…”