Pathogenic trypanosomatid parasites are auxotrophic for heme and they must scavenge it from their human host. Trypanosoma brucei (responsible for sleeping sickness) and Leishmania (leishmaniasis) can fulfill heme requirement by receptor-mediated endocytosis of host hemoglobin. However, the mechanism used to transfer hemoglobin-derived heme from the lysosome to the cytosol remains unknown. Here we provide strong evidence that HRG transporters mediate this essential step. In bloodstream T. brucei, TbHRG localizes to the endolysosomal compartment where endocytosed hemoglobin is known to be trafficked. TbHRG overexpression increases cytosolic heme levels whereas its downregulation is lethal for the parasites unless they express the Leishmania orthologue LmHR1. LmHR1, known to be an essential plasma membrane protein responsible for the uptake of free heme in Leishmania, is also present in its acidic compartments which colocalize with endocytosed hemoglobin. Moreover, LmHR1 levels modulated by its overexpression or the abrogation of an LmHR1 allele correlate with the mitochondrial bioavailability of heme from lysosomal hemoglobin. In addition, using heme auxotrophic yeasts we show that TbHRG and LmHR1 transport hemoglobin-derived heme from the digestive vacuole to the cytosol. Collectively, these results show that trypanosomatid parasites rescue heme from endocytosed hemoglobin through endolysosomal HRG transporters, which could constitute novel drug targets.
An understanding of the taxonomic status and vector distribution of anophelines is crucial to malaria control efforts. Previous phylogenetic analyses have supported the description of six species of the Neotropical malaria vector Anopheles (Nyssorhynchus) albitarsis s.l. (Diptera: Culicidae): Anopheles albitarsis, An. deaneorum, An. marajoara, An. oryzalimnetes, An. janconnae and An. albitarsis F. To evaluate the taxonomic status of An. albitarsis s.l. mosquitoes collected in various localities of the Colombian Caribbean region, specimens were analyzed using the complete mtDNA Cytochrome Oxidase I (COI) gene, the ribosomal DNA internal transcribed spacer 2 (ITS2) region and partial nuclear DNA White gene sequences. Phylogenetic analyses of the COI sequences detected a new lineage near An. janconnae in the Caribbean region of Colombia and determined its position relative to the other members of the complex. However, the ITS2 and White gene sequences lacked resolution to support a new lineage near An. janconnae or the An. janconnae clade. Nothing is known about the possible involvement in malaria transmission in Colombia of this new lineage, but its phylogenetic closeness to Anopheles janconnae, which has been incriminated in local malaria transmission in Brazil, is provocative.
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...
Heme is an essential molecule synthetized through a broadly conserved 8-step route that has been lost in trypanosomatid parasites. Interestingly, Leishmania reacquired by horizontal gene transfer from g-proteobacteria the genes coding for the last 3 enzymes of the pathway. Here we show that intracellular amastigotes of Leishmania major can scavenge heme precursors from the host cell to fulfill their heme requirements, demonstrating the functionality of this partial pathway. To dissect its role throughout the L. major life cycle, the significance of L. major ferrochelatase (LmFeCH), the terminal enzyme of the route, was evaluated. LmFeCH expression in a heterologous system demonstrated its activity. Knockout promastigotes lacking lmfech were not able to use the ferrochelatase substrate protoporphyrin IX as a source of heme. In vivo infection of Phlebotomus perniciosus with knockout promastigotes shows that LmFeCH is not required for their development in the sandfly. In contrast, the replication of intracellular amastigotes was hampered in vitro by the deletion of lmfech. However, LmFeCH 2/2 parasites produced disease in a cutaneous leishmaniasis murine model in a similar way as control parasites. Therefore, although L. major can synthesize de novo heme from macrophage precursors, this activity is dispensable being an unsuited target for leishmaniasis treatment.
Tritryps are protozoan parasites that belong to the Trypanosomatidae family, which encompasses the etiologic agents of leishmaniasis, African and American trypanosomiasis. These parasites undergo different stress conditions across their life cycle, such as nutritional stress, which needs to be deadened in order to guarantee the survival of the parasite inside its vector and mammal hosts respectively. Here we show that the lack of the serine threonine kinase PKB / AKT-like function, either by allosteric inhibition of its Plekstrin domain (PH) in T. cruzi, the reduction of the gene transcripts in T. brucei by RNAi assays, or by the genomic knockout of its alleles in L. major, causes the parasites to be less tolerant to nutritional stress and promotes apoptosis-like events, including DNA fragmentation, mitochondrial damage and loss of plasma membrane integrity. Additionally, we observed that the Akt-like gene knockout in L. major impairs its infective capacity. This work confirms some of the previously described functions regarding parasite survival for AKT-like kinases in the Leishmania genus. The present work also provides strong evidence of the probable function of Akt-like in T. cruzi and T. brucei survival and infectivity.Author summaryEndemic countries are called to play a paramount role in the discovery of new drug candidates through the application of new drug development strategies, among them the rational drug design method have proven to be compatible with the development of new drug for orphan and neglected diseases since it substantially reduces the costs of discovery and development, a desirable condition for public funded initiatives. Previously we have identified a new parasite protein kinase (AKT-like) as promising new target candidate by means of computational tools and probed its biological role in trypanosomatids. We show that inhibition of the AKT-like kinase in trypanosomatids by different approaches (chemical inhibition, interference RNA and gene knockout) decreases the fitness and survival of the parasites in vitro, interfering with the capacity of the cell to react and survive stress conditions similar to those experienced by the cell in the natural life cycle. Additionally our results strongly supports the potential of a new family of compounds as potential trypanocidal agents previously described by bioinformatic means. Altogether we show here that the specific inhibition of the AKT-like is a promising strategy for the further development of anti-trypanosome drugs. The downstream mechanisms of the observed cellular effects in the ATK pathways is still to be determined and may be the subject of future studies.
The protozoan parasite Leishmania, responsible for leishmaniasis, is one of the few aerobic organisms that cannot synthesize the essential molecule heme. Therefore, it has developed specialized pathways to scavenge it from its host. In recent years, some proteins involved in the import of heme, such as LHR1 and LFLVCRB, have been identified, but relevant aspects regarding the process remain unknown. Here, we characterized the kinetics of the uptake of the heme analogue Zn(II) Mesoporphyrin IX (ZnMP) in Leishmania major promastigotes as a model of a parasite causing cutaneous leishmaniasis with special focus on the force that drives the process. We found that ZnMP uptake is an active, inducible, and pH-dependent process that does not require a plasma membrane proton gradient but requires the presence of the monovalent cations Na+ and/or K+. In addition, we demonstrated that this parasite can efflux this porphyrin against a concentration gradient. We also found that ZnMP uptake differs among different dermotropic or viscerotropic Leishmania species and does not correlate with LHR1 or LFLVCRB expression levels. Finally, we showed that these transporters have only partially overlapping functions. Altogether, these findings contribute to a deeper understanding of an important process in the biology of this parasite.
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