Heme requirement and intracellular trafficking in Trypanosoma cruzi epimastigotes

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Abstract

Epimastigotes multiplies in the insect midgut by taking up nutrients present in the blood meal including heme bound to hemoglobin of red blood cell. During blood meal digestion by vector proteases in the posterior midgut, hemoglobin is clipped off into amino acids, peptides, and free heme. In this paper, we compared the heme and hemoglobin uptake kinetics and followed their intracellular trafficking. Addition of heme to culture medium increased epimastigote proliferation in a dose-dependent manner, while medium supplemented with hemoglobin enhanced growth after 3-day lag phase. Medium supplemented with globin-derived peptides stimulated cell proliferation in a dose-independent way. Using Palladium mesoporphyrin IX (Pd-mP) as a fluorescent heme-analog, we observed that heme internalization proceeded much faster than that observed by hemoglobin-rhodamine. Binding experiments showed that parasites accumulated the Pd-mP into the posterior region of the cell whereas hemoglobin-rhodamine stained the anterior region. Finally, using different specific inhibitors of ABC transporters we conclude that a P-glycoprotein homologue transporter is probably involved in heme transport through the plasma membrane.

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Materials and methods

Parasites.Trypanosoma cruzi strains Y (CT-IOC-106) and Dm28c (CT-IOC-010) strains were provided by the Trypanosomatid Collection of the Oswaldo Cruz Institute, Fiocruz, Brazil. The protozoa were grown at 28 °C for 7 days in brain–heart infusion medium (BHI), and supplemented with 30 μM hemin (heme-Cl) and 10% fetal calf serum (FCS). Parasite growth was monitored by cell counting in a Neubauer chamber.

Heme, hemoglobin, and globin-derived peptides effects on T. cruzi epimastigotes growth. To

Effects of supplementary hemin on different strains of T. cruzi epimastigotes

We investigated the effects of hemin on the in vitro growth of two strains (Y, Dm28c) of T. cruzi. By varying the hemin concentration from 0 to 1 mM, we observed an increased parasite proliferation in a dose-dependent manner (Fig. 1A and B). Strain Y and clone Dm28c cultivated in BHI medium or BHI medium supplemented with 3, 30, 100, and 1000 μM of heme exhibited respective median generation times of 5.33, 4.07, 3.38, 3.06, 2.66 days (n = 4) and 5.40, 4.61, 4.40, 3.59, 3.30 days (n = 5),

Acknowledgments

We express our gratitude to Dr. Ulysses Lins and Dr. Thais Souto Padron, from Instituto de Microbiologia Prof Paulo de Góes, UFRJ, to allow the mounting of Pd-mP filter on their epifluorescence microscope Zeiss Axioplan 2. We thank Dr. E. Pays and Dr. C. Felu (Brussels) for critical reading of the manuscript.

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    This work was supported by FAPERJ, PADCT, CNPq, Howard Hughes Medical Institute (to P.L. Oliveira) and PRONEX (to P.L. Oliveira).

    1

    Present address: Lab. de Microbiologia Celular, Departamento de Micobacterioses, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Brazil.

    2

    These authors contributed equally to this work.

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