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

Abstract

BackgroundMitochondria 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.MethodsLmABCB3, 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.ResultsLmABCB3 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.ConclusionsAltogether 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1284-5) contains supplementary ma...

Trypanosomatid parasites rescue heme from endocytosed hemoglobin through lysosomal HRG transporters

“…However, at its location, LABCG5 could take up the heme exported by LHR1 from the endo‐lysosomes where Hb is digested (Campos‐Salinas et al ., ), although this possibility still requires investigation. Finally, the mitochondrial ABC transporter LABCB3, which is involved in the maturation of cytosolic iron/sulfur clusters, also promotes mitochondrial heme biogenesis from host precursors (Martinez‐Garcia et al ., ).…”

“…Briefly, parasites (grown in HMI‐9 supplemented with 10% hiFBS as a source of Hp‐Hb) were washed and lysed by freezing and thawing (three cycles). The insoluble material was removed by centrifugation at 14,000 g for 10 min and cytosolic heme were measured with the Hemin Assay Kit (Sigma) and normalized for the amount of protein as described in (Martinez‐Garcia et al ., ).…”

“…Targeted gene replacement of the L. major LmHR1 gene was performed as we have previously described (Martinez‐Garcia et al ., ) for the case of the L. major LmABCB3 gene. Briefly, a targeting DNA fragment was constructed in which the hyg gene (flanked by untranslated regions of the L. major DHFR‐TS gene (248 bp of the 5′‐UTR and 869 bp of the 3′‐UTR), conferring resistance to hygromycin B, was flanked by LmHR1 upstream (primers pairs P15 and P16) and downstream (P17 and P18) regions (Fig.…”

“…This pathway is an attractive therapeutic target: not only Leishmania requires degradation of endocytosed Hb for their growth (Patel et al ., ; Campos‐Salinas et al ., ; Guha et al ., ), but also the Hb receptor‐encoding DNA is a promising vaccine candidate for human use to prevent leishmaniasis (Guha et al ., ). The essential mitochondrial ABC transporter LmABCB3, responsible for cytosolic iron/sulfur clusters biogenesis, also plays a role in mitochondrial heme metabolism (Martinez‐Garcia et al ., ). Regarding free hemin, it is imported by the heme transporter LHR1, described in L. amazonensis (Huynh et al ., ).…”

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

“…Also, the exact role of mammalian ABCB10 in heme formation and protection of mitochondria against oxidative stress, including its substrate, remain unclear (47) (48). Recently, L. major ABCB3 (LmABCB3), another mitochondrial ABCB transporter, was described and was shown to play an essential role in mitochondrial heme and cytosolic iron sulfur cluster biogenesis (49). Although ABCI3 does not belong to this family of mitochondrial ABC transporters, we decided to study if ABCI3 could play a relevant role in porphyrin transport in L. major.…”