Cellular remodeling during differentiation is essential for lifecycle progression of many unicellular eukaryotic pathogens such as Leishmania, but the mechanisms involved are largely uncharacterized. The role of endosomal sorting in differentiation was analyzed in Leishmania major by overexpression of a dominant-negative ATPase, VPS4. VPS4 E235Q accumulated in vesicles from the endocytic pathway, and the mutant L. major was deficient in endosome sorting. Mutant parasites failed to differentiate to the obligate infective metacyclic promastigote form. Furthermore, the autophagy pathway, monitored via the expression of autophagosome marker GFP-ATG8, and shown to normally peak during initiation of metacyclogenesis, was disrupted in the mutants. The defect in late endosome-autophagosome function in the VPS4 E235Q parasites made them less able to withstand starvation than wild-type L. major. In addition, a L. major ATG4-deficient mutant was found also to be defective in the ability to differentiate. This finding, that transformation to the infective metacyclic form is dependent on late endosome function and, more directly, autophagy, makes L. major a good model for studying the roles of these processes in differentiation.

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.

Leishmania major possesses, apparently uniquely, four families of ATG8-like genes, designated ATG8, ATG8A, ATG8B and ATG8C, and 25 genes in total. L. major ATG8 and examples from the ATG8A, ATG8B and ATG8C families are able to complement a Saccharomyces cerevisiae ATG8-deficient strain, indicating functional conservation. Whereas ATG8 has been shown to form putative autophagosomes during differentiation and starvation of L. major, ATG8A primarily form puncta in response to starvation-suggesting a role for ATG8A in starvation-induced autophagy. Recombinant ATG8A was processed at the scissile glycine by recombinant ATG4.2 but not ATG4.1 cysteine peptidases of L. major and, consistent with this, ATG4.2-deficient L. major mutants were unable to process ATG8A and were less able to withstand starvation than wild-type cells. GFP-ATG8-containing puncta were less abundant in ATG4.2 overexpression lines, in which unlipidated ATG8 predominated, which is consistent with ATG4.2 being an ATG8-deconjugating enzyme as well as an ATG8A-processing enzyme. In contrast, recombinant ATG8, ATG8B and ATG8C were all processed by ATG4.1, but not by ATG4.2. ATG8B and ATG8C both have a distinct subcellular location close to the flagellar pocket, but the occurrence of the GFP-labeled puncta suggest that they do not have a role in autophagy. L. major genes encoding possible ATG5, ATG10 and ATG12 homologues were found to complement their respective S. cerevisiae mutants, and ATG12 localized in part to ATG8-containing puncta, suggestive of a functional ATG5-ATG12 conjugation pathway in the parasite. L. major ATG12 is unusual as it requires C-terminal processing by an as yet unidentified peptidase.