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ABCI3 Is a New Mitochondrial ABC Transporter from Leishmania major Involved in Susceptibility to Antimonials and Infectivity.

ABSTRACT: We have identified and characterized ABCI3 as a new mitochondrial ABC transporter from Leishmania major Localization studies using confocal microscopy, a surface biotinylation assay, and trypsin digestion after digitonin permeabilization suggested that ABCI3 presents a dual localization in both mitochondria and the plasma membrane. From studies using parasites with a single knockout of ABCI3 (ABCI3^+/-), we provide evidence that ABCI3 is directly involved in susceptibility to the trivalent form of antimony (SbIII) and metal ions. Attempts to obtain parasites with a double knockout of ABCI3 were unsuccessful, suggesting that ABCI3 could be an essential gene in L. majorABCI3^+/- promastigotes were 5-fold more resistant to Sb^III than the wild type, while ABCI3^+/- amastigotes were approximately 2-fold more resistant to pentavalent antimony (Sb^V). This resistance phenotype was associated with decreased Sb^III accumulation due to decreased Sb^III uptake. ABCI3^+/- parasites presented higher ATP levels and generated less mitochondrial superoxide after Sb^III incubation. Finally, we observed that ABCI3^+/- parasites showed a slightly higher infection capacity than wild-type and add-back ABCI3^+/-::3×FABCI3 parasites; however, after 72 h the number of ABCI3^+/- intracellular parasites per macrophage increased significantly. Our results show that ABCI3 is responsible for Sb^III transport inside mitochondria, where it contributes to enhancement of the general toxic effects caused by Sb^III To our knowledge, ABCI3 is the first ABC transporter which is involved in susceptibility toward antimony, conferring Sb^III resistance to parasites when it is partially deleted.

SUBMITTER: Arcari T

PROVIDER: S-EPMC5700302 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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ABCI3 Is a New Mitochondrial ABC Transporter from Leishmania major Involved in Susceptibility to Antimonials and Infectivity.

Arcari Talia T Manzano José Ignacio JI Gamarro Francisco F

Antimicrobial agents and chemotherapy 20171122 12

We have identified and characterized ABCI3 as a new mitochondrial ABC transporter from Leishmania major Localization studies using confocal microscopy, a surface biotinylation assay, and trypsin digestion after digitonin permeabilization suggested that ABCI3 presents a dual localization in both mitochondria and the plasma membrane. From studies using parasites with a single knockout of ABCI3 (ABCI3+/-), we provide evidence that ABCI3 is directly involved in suscep ...[more]

PMID: 28971869

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Project description:Mitochondria play essential biological functions including the synthesis and trafficking of porphyrins and iron/sulfur clusters (ISC), processes that in mammals involve the mitochondrial ATP-Binding Cassette (ABC) transporters ABCB6 and ABCB7, respectively. The mitochondrion of pathogenic protozoan parasites such as Leishmania is a promising goal for new therapeutic approaches. Leishmania infects human macrophages producing the neglected tropical disease known as leishmaniasis. Like most trypanosomatid parasites, Leishmania is auxotrophous for heme and must acquire porphyrins from the host.LmABCB3, a new Leishmania major protein with significant sequence similarity to human ABCB6/ABCB7, was identified and characterized using bioinformatic tools. Fluorescent microscopy was used to determine its cellular localization, and its level of expression was modulated by molecular genetic techniques. Intracellular in vitro assays were used to demonstrate its role in amastigotes replication, and an in vivo mouse model was used to analyze its role in virulence. Functional characterization of LmABCB3 was carried out in Leishmania promastigotes and Saccharomyces cerevisiae. Structural analysis of LmABCB3 was performed using molecular modeling software.LmABCB3 is an atypical ABC half-transporter that has a unique N-terminal extension not found in any other known ABC protein. This extension is required to target LmABCB3 to the mitochondrion and includes a potential metal-binding domain. We have shown that LmABCB3 interacts with porphyrins and is required for the mitochondrial synthesis of heme from a host precursor. We also present data supporting a role for LmABCB3 in the biogenesis of cytosolic ISC, essential cofactors for cell viability in all three kingdoms of life. LmABCB3 fully complemented the severe growth defect shown in yeast lacking ATM1, an orthologue of human ABCB7 involved in exporting from the mitochondria a gluthatione-containing compound required for the generation of cytosolic ISC. Indeed, docking analyzes performed with a LmABCB3 structural model using trypanothione, the main thiol in this parasite, as a ligand showed how both, LmABCB3 and yeast ATM1, contain a similar thiol-binding pocket. Additionally, we show solid evidence suggesting that LmABCB3 is an essential gene as dominant negative inhibition of LmABCB3 is lethal for the parasite. Moreover, the abrogation of only one allele of the gene did not impede promastigote growth in axenic culture but prevented the replication of intracellular amastigotes and the virulence of the parasites in a mouse model of cutaneous leishmaniasis.Altogether our results present the previously undescribed LmABCB3 as an unusual mitochondrial ABC transporter essential for Leishmania survival through its role in the generation of heme and cytosolic ISC. Hence, LmABCB3 could represent a novel target to combat leishmaniasis.

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Dataset Information

ABCI3 Is a New Mitochondrial ABC Transporter from Leishmania major Involved in Susceptibility to Antimonials and Infectivity.

Publications

ABCI3 Is a New Mitochondrial ABC Transporter from Leishmania major Involved in Susceptibility to Antimonials and Infectivity.

Similar Datasets

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