Glycosyl conjugation to G-quadruplex ligands as a strategy to improve their antitumor and antiparasitic efficacy.

Congresos y Conferencias: Comunicación de Congreso - Conferencia invitada

Caracterización bioquímica y explotación farmacológica del mecanismo de rescate de hemo en Leishmania spp. y Trypanosoma brucei, patógenos auxótrofos para este metabolito esencial

Los protozoos de la familia Trypanosomatidae incluyen parásitos responsables de importantes enfermedades en los seres humanos y en los animales. Estos organismos son Trypanosoma brucei (subespecies gambiense y rhodesiense), Trypanosoma cruzi, y Leishmania spp. que son patógenos para los humanos causando, respectivamente, la tripanosomiasis humana africana o enfermedad del sueño, la tripanosomiasis americana o enfermedad de Chagas y la leishmaniasis (en sus distintas manifestaciones clínicas: visceral, cutánea y mucocutánea). Otros tripanosomátidos (T. b. brucei, T. congolense y T. vivax) son el agente etiológico del Nagana en el ganado, ocasionando una grave enfermedad que supone un obstáculo importante en el desarrollo económico de las áreas rurales afectadas. El control de estas enfermedades se basa en un arsenal farmacológico muy tóxico, anticuado y poco eficaz frente al que se han desarrollado resistencias clínicas. Debido a ello es urgente encontrar nuevos tratamientos. Una estrategia atractiva para conseguir este objetivo consiste en aprovechar diferencias bioquímicas entre estos organismos patógenos y el hospedador humano. El metabolismo del grupo hemo, un metabolito esencial en todos los organismos aeróbicos, constituye una de estas diferencias. Los tripanosomátidos son auxótrofos para el hemo y necesitan tomarlo del hospedador. El objetivo fundamental de esta Tesis Doctoral es el estudio del mecanismo de captación de hemo por protozoos parásitos tripanosomátidos, auxótrofos para este metabolito esencial, con el fin de identificar posibles nuevas dianas de acción terapéutica. En este trabajo se ha descrito que en Leishmania la entrada de hemo al parásito depende de un transportador específico y se ha identificado una proteína localizada en la membrana plasmática (LmFLVCRb) que realiza esta función al menos en las formas promastigotas de Leishmania. Por otra parte, los parásitos tripanosomátidos auxótrofos para el hemo, como Leishmania spp. y Trypanosoma brucei, pueden tomar el hemo a partir de la hemoglobina endocitada a través de los transportadores HRG (LmHR1 y TbHRG) localizados en los compartimentos endolisosomales. Como estas proteínas son esenciales y presentan un bajo grado de similitud con su ortólogo humano, pueden suponer una nueva diana de actuación para la lucha contra estas enfermedades.Tesis Univ. Granada

New Insights on Heme Uptake in Leishmania spp.

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

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

Abstract Background 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. Methods 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. Results 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. Conclusions 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. Keywords Heme trafficking and metabolism Iron/sulfur clusters Trypanosomatid parasites Mitochondrial ABC transporter LeishmaniaThis work was supported by grants from the Spanish Ministerio de Economía y Competitividad SAF2011-28215 (JMPV) and Junta de Andalucia BIO1786 (JMPV) and by FEDER funds from the EU to JMPV. MMG was recipient of a FPI fellowship from the Spanish Ministerio de Economía y Competitividad; MCD was recipient of a FPU fellowship from the Spanish Ministerio de Educación, Cultura y Deporte; SMC was recipient of a JAE-DOC from the Spanish CSIC (Ministerio de Economía y Competitividad), cofounded by the Fondo Social Europeo.Peer reviewe

A new ATP-binding cassette protein is involved in intracellular haem trafficking in Leishmania.

The characterization of LABCG5, a new intracellular ATP-binding cassette protein in Leishmania donovani, is described. Unlike other ABCG half-transporters, LABCG5 is not involved in either drug resistance or phospholipid efflux. However, we provide evidence suggesting that this protein is involved in intracellular haem trafficking. Thus, downregulation of LABCG5 function produced upon overexpression of an inactive version of the protein caused a dramatic growth arrest unless a haemin supplement was added or the mutated gene was eliminated. Supplementation with haemoglobin, an upstream metabolite normally sufficient to meet parasite haem requirements, was unable to rescue the growth defect phenotype. Haemoglobin endocytosis was not hampered in dominant-negative parasites and neither was haem uptake, a process that we show here to be dependent on a specific transporter. In contrast, LABCG5 function was required for the correct intracellular trafficking of haemoglobin-bound porphyrins to the mitochondria, not affecting the routing of free haem. Finally, LABCG5 binds haem through hydrophobic and electrostatic interactions. Altogether, these data suggest that LABCG5 is involved in the salvage of the haem released after the breakdown of internalized haemoglobin. As Leishmania is auxotrophic for haem, the pharmacological targeting of this route could represent a novel approach to control fatal visceral leishmaniasis.Peer Reviewe

Heme synthesis through the life cycle of the heme auxotrophic parasite Leishmania major

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 γ-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-/- 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.-Orrego, L. M., Cabello-Donayre, M., Vargas, P., Martínez-García, M., Sánchez, C., Pineda-Molina, E., Jiménez, M., Molina, R., Pérez-Victoria, J. M. Heme synthesis through the life cycle of the heme auxotrophic parasite Leishmania major

Leishmania heme uptake involves LmFLVCRb, a novel porphyrin transporter essential for the parasite

Leishmaniasis comprises a group of neglected diseases caused by the protozoan parasite Leishmania spp. As is the case for other trypanosomatid parasites, Leishmania is auxotrophic for heme and must scavenge this essential compound from its human host. In mammals, the SLC transporter FLVCR2 mediates heme import across the plasma membrane. Herein we identify and characterize Leishmania major FLVCRb (LmFLVCRb), the first member of the FLVCR family studied in a non-metazoan organism. This protein localizes to the plasma membrane of the parasite and is able to bind heme. LmFLVCRb levels in Leishmania, which are modulated by overexpression thereof or the abrogation of an LmFLVCRb allele, correlate with the ability of the parasite to take up porphyrins. Moreover, injection of LmFLVCRb cRNA to Xenopus laevis oocytes provides these cells with the ability to take up heme. This process is temperature dependent, requires monovalent ions and is inhibited at basic pH, characteristics shared by the uptake of heme by Leishmania parasites. Interestingly, LmFLVCRb is essential as CRISPR/Cas9-mediated knockout parasites were only obtained in the presence of an episomal copy of the gene. In addition, deletion of just one of the alleles of the LmFLVCRb gene markedly impairs parasite replication as intracellular amastigotes as well as its virulence in an in vivo model of cutaneous leishmaniasis. Collectively, these results show that Leishmania parasites can rescue heme through plasma membrane transporter LFLVCRb, which could constitute a novel target for therapeutic intervention against Leishmania and probably other trypanosomatid parasites in which FLVCR genes are also present

Trypanosomatid parasites rescue heme from endocytosed hemoglobin through lysosomal HRG transporters

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 down-regulation 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.We thank Ivan Hapala (IABG-SAS, Slovakia), Stephen M.Beverley (Washington University School of Medicine, USA)and Olivier Cagnac (EEZ-CSIC, Spain) for kindly providing,respectively, thehem1Dyeast strain, theLeishmaniaandthe yeast vectors used throughout this research work. Wethank Ignacio Perez-Victoria for the preparation of ApoHb.We are grateful to Jean Mathieu Bart (IPBLN-CSIC, Spain)for helpful discussions. This work was supported by Span-ish grants BIO1786 (JMPV) from the Junta de Andalucıa and SAF2011-28215 (JMPV) and BFU2014-55193-P (AE)from the Ministerio de Economıa y Competitividad and by FEDER funds from the EU to JMPV and AE. MCD was astudent of the PhD program “Biochemistry and MolecularBiology” of the University of Granada (Spain). MCD was recipient of a FPU fellowship from the Spanish Ministerio de Educacion, Cultura y Deporte; SMC was recipient of a JAE-DOC from the Spanish CSIC (Ministerio de Economıa y Competitividad), cofounded by the Fondo Social Europeo,LMOZ was recipient of a Colciencias fellowship from the Colombian Ministerio de Ciencia, Tecnologıa e Innovacion;MMG was recipient of a FPI fellowship from the Spanish Ministerio de Economıa y Competitividad. The authorsdeclare that they have no conflict of interestPeer reviewe