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

Additional file 5: Figure S3. of LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis

The ScATM1 residues that interact with glutathione are conserved in LmABCB3. The alignment of the indicated amino acids of S. cerevisiae ScATM1, H. sapiens HsABCB7 and L. major LmABCB3 (ClustalW software) shows that LmABCB3 share the ScATM1 residues forming hydrogenen bonds with GSH (highlighted in yellow). The E433 residue of HsABCB7 mutated to lysine in XLSA/A patients is indicated by a red arrow. Other ScATM1 residues surrounding bound GSH are highlighted in green. Identical (*), strongly similar (:) and weakly similar (.) amino acids are coloured in red, green and blue, respectively. (TIF 379 kb

Additional file 2: Figure S1. of LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis

The UNE domain is exclusive of Leishmania ABCB3. Schematic representation of putative mitochondrial ABCB transporters showing the unique N-terminal extension (UNE), the Transmembrane Domain (TMD), the Nucleotide Binding Domain (NBD) and the theoretical molecular weight. Lm: L. major; Li: L. infantum; Lmx: L. mexicana; Lb: L. braziliensis; Tb: T. brucei; Tc: T. cruzi; Hs: H. sapiens; Sc: S. cereviciae. (TIF 1523 kb

Additional file 4: Figure S2. of LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis

Expression of LmABCB3-GFP and LmABCB3_∆UNE-GFP. Western blot analysis of total protein from LmABCB3-GFP (lane 1) or LmABCB3_∆UNE-GFP (lane 2) and GFP (lane 3) expressing L. major parasites. Immunodetection were performed with antibody anti-GFP incubation at a 1:5000 dilution. The molecular mass standards (kDa) from Bio-Rad are indicated on the left. (TIF 2482 kb