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LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis.

ABSTRACT: 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.

SUBMITTER: Martinez-Garcia M

PROVIDER: S-EPMC4700571 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Project description:Iron-sulfur clusters are required in a variety of biological processes. Biogenesis of iron-sulfur clusters includes assembly of iron-sulfur clusters on scaffold complexes and transfer of iron-sulfur clusters to recipient apoproteins by iron-sulfur carriers, such as nitrogen-fixation-subunit-U (NFU)-type proteins. Arabidopsis thaliana has three plastid-targeted NFUs: NFU1, NFU2, and NFU3. We previously discovered that nfu2 -/- nfu3 -/- mutants are embryo lethal. The lack of viable nfu2 -/- nfu3 -/- mutants posed a serious challenge. To overcome this problem, we characterized nfu2-1 -/- nfu3-2+/- and nfu2-1+/- nfu3-2 -/- sesquimutants. Simultaneous loss-of-function mutations in NFU2 and NFU3 have an additive effect on the declines of 4Fe-4S-containing PSI core subunits. Consequently, the sesquimutants had much lower PSI and PSII activities, much less chlorophyll, and much smaller plant sizes, than nfu2-1 and nfu3-2 single mutants. These observations are consistent with proposed roles of NFU3 and NFU2 in the biogenesis of chloroplastic 4Fe-4S. By performing spectroscopic and in vitro reconstitution experiments, we found that NFU1 may act as a carrier for chloroplastic 4Fe-4S and 3Fe-4S clusters. In line with this hypothesis, loss-of-function mutations in NFU1 resulted in significant declines in 4Fe-4S- and 3Fe-4S-containing chloroplastic proteins. The declines of PSI activity and 4Fe-4S-containing PSI core subunits in nfu1 mutants indicate that PSI is the main target of NFU1 action. The reductions in 4Fe-4S-containing PSI core proteins and PSI activity in nfu3-2, nfu2-1, and nfu1 single mutants suggest that all three plastid-targeted NFU proteins contribute to the biogenesis of chloroplastic 4Fe-4S clusters. Although different insertion sites of T-DNA lines may cause variations in phenotypic results, mutation severity could be an indicator of the relative importance of the gene product. Our results are consistent with the hypothesis that NFU3 contributes more than NFU2 and NFU2 contributes more than NFU1 to the production of 4Fe-4S-containing PSI core subunits.

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LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis.

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