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Characterization of unusual families of ATG8-like proteins and ATG12 in the protozoan parasite Leishmania major.

ABSTRACT: 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.

SUBMITTER: Williams RA

PROVIDER: S-EPMC2642932 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Characterization of unusual families of ATG8-like proteins and ATG12 in the protozoan parasite Leishmania major.

Williams Roderick A M RA Woods Kerry L KL Juliano Luiz L Mottram Jeremy C JC Coombs Graham H GH

Autophagy 20090204 2

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 ...[more]

PMID: 19066473

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Project description:The importance of autophagy in parasites with a digenetic life cycle like Leishmania spp. is significant. The parasite survives as promastigotes in the insect gut and as immotile amastigotes in mammals. This study demonstrates increased autophagy in Leishmania parasite during progression of in vitro life cycle and upon exposure to stress stimuli like starvation, oxidative stress, and drugs. Autophagy inhibition during stress exposure increased cell death, indicating the importance of autophagy in cellular defense against adverse conditions. Atg8 protein, a homolog of mammalian autophagy protein LC3 is expressed in Leishmania parasite but its function remains unknown. Overexpression of Atg8 (Atg8-OE) rendered the parasites resistant to stress and capable of infecting macrophages in substantial numbers; however, disruption of the Atg8 gene (?Atg8) resulting in suppression of Atg8 protein expression, increased susceptibility to stress and reduced the capability to cause infection. A critical event in the Leishmania parasite lifecycle is the differentiation of promastigote forms to the disease causing amastigote forms. The failure of ?Atg8 parasites lacking Atg8 protein to differentiate into amastigotes, unlike the Atg8-OE and vector-transfected parasites, clearly indicated Atg8 involvement in a crucial event. The inability of ?Atg8 parasites to infect macrophages in vitro was verified in an in vivo mouse model of leishmaniases where infection could not be induced by the ?Atg8 parasites. Autophagy is known to be involved in the remodeling of damaged organelles. The accumulation of Atg8 around damaged mitochondria suggested increase of autophagy in the vicinity of the organelle. This buildup was prevented when mitochondria generated reactive oxygen species that were quenched, suggesting them as possible signaling molecules for sensing mitochondrial instability. In summary, our study provides new evidences for a crucial role of Atg8 protein in sustaining Leishmania parasite survival during life cycle and stress exposure, differentiation to amastigotes, and their infective abilities.

Dataset Information

Characterization of unusual families of ATG8-like proteins and ATG12 in the protozoan parasite Leishmania major.

Publications

Characterization of unusual families of ATG8-like proteins and ATG12 in the protozoan parasite Leishmania major.

Similar Datasets

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