Translation initiation in eukaryotes requires eIF4E, the cap binding protein, which mediates its function through an interaction with the scaffolding protein eIF4G, as part of the eIF4F complex. In trypanosomatids, four eIF4E homologues have been described but the specific function of each is not well characterized. Here, we report a study of these proteins in Trypanosoma brucei (TbEIF4E1 through 4). At the sequence level, they can be assigned to two groups: TbEIF4E1 and 2, similar in size to metazoan eIF4E1; and TbEIF4E3 and 4, with long N-terminal extensions. All are constitutively expressed, but whilst TbEIF4E1 and 2 localize to both the nucleus and cytoplasm, TbEIF4E3 and 4 are strictly cytoplasmic and are also more abundant. After knockdown through RNAi, TbEIF4E3 was the only homologue confirmed to be essential for viability of the insect procyclic form. In contrast, TbEIF4E1, 3 and 4 were all essential for the mammalian bloodstream form. Simultaneous RNAi knockdown of TbEIF4E1 and 2 caused cessation of growth and death in procyclics, but with a delayed impact on translation, whilst knockdown of TbEIF4E3 alone or a combined TbEIF4E1 and 4 knockdown led to substantial translation inhibition which preceded cellular death by several days, at least. Only TbEIF4E3 and 4 were found to interact with T. brucei eIF4G homologues; TbEIF4E3 bound both TbEIF4G3 and 4 whilst TbEIF4E4 bound only to TbEIF4G3. These results are consistent with TbEIF4E3 and 4 having distinct but relevant roles in initiation of protein synthesis.

Trypanosomatid protozoans are reliant on posttranscriptional processes to control gene expression. Regulation occurs at the levels of mRNA processing, stability, and translation, events that may require the participation of the poly(A) binding protein (PABP). Here, we have undertaken a functional study of the three distinct Leishmania major PABP (LmPABP) homologues: the previously described LmPABP1; LmPABP2, orthologous to the PABP described from Trypanosoma species; and LmPABP3, unique to Leishmania. Sequence identity between the three PABPs is no greater than 40%. In assays measuring binding to A-rich sequences, LmPABP1 binding was poly(A) sensitive but heparin insensitive; LmPABP2 binding was heparin sensitive and less sensitive to poly(A), compatible with unique substitutions observed in residues implicated in poly(A) binding; and LmPABP3 displayed intermediate properties. All three homologues are simultaneously expressed as abundant cytoplasmic proteins in L. major promastigotes, but only LmPABP1 is present as multiple isoforms. Upon transcription inhibition, LmPABP2 and -3 migrated to the nucleus, while LmPABP1 remained predominantly cytoplasmic. Immunoprecipitation assays showed an association between LmPABP2 and -3. Although the three proteins bound to a Leishmania homologue of the translation initiation factor eukaryotic initiation factor 4G (eIF4G) (LmEIF4G3) in vitro, LmPABP1 was the only one to copurify with native LmEIF4G3 from cytoplasmic extracts. Functionality was tested using RNA interference (RNAi) in Trypanosoma brucei, where both orthologues to LmPABP1 and -2 are required for cellular viability. Our results indicate that these homologues have evolved divergent functions, some of which may be unique to the trypanosomatids, and reinforces a role for LmPABP1 in translation through its interaction with the eIF4G homologue.

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The current therapy for leishmaniasis, which affects annually about 2 million people, is far from satisfactory. All available drugs require parenteral administration and are potentially toxic. Plant essential oils have been traditionally used in folk medicine and appear as valuable alternative source for chemotherapeutic compounds. In this study, we demonstrated the effect of essential oils from Cymbopogon citratus, Lippia sidoides, and Ocimum gratissimum on growth and ultrastructure of Leishmania chagasi promastigote forms. Steam distillation was used to isolate the essential oils, and their constituents were characterized by gas chromatography coupled to mass spectrometry and nuclear magnetic resonance. All essential oils showed in vitro inhibitory action on L. chagasi promastigotes growth in a dose-dependent way, with IC(50)/72 h of 45, 89, and 75 microg/mL for C. citratus, L. sidoides, and O. gratissimum, respectively. Drastic morphological alterations were observed in all essential oil-treated parasites, including cell swelling, accumulation of lipid droplets in the cytoplasm, and increase of acidocalcisome volume. Furthermore, aberrant-shaped cells with multi-septate body were observed by scanning electron microscopy, suggesting an additional effect on cytokinesis. Taken together, our data show that these essential oils affect the parasite viability being the C. citratus essential oil the most effective against L. chagasi.

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Leishmaniasis is a neglected disease present in more than 88 countries. The currently adopted chemotherapy faces challenges related to side effects and the development of resistance. Photodynamic therapy (PDT) is emerging as a therapeutic modality for cutaneous leishmaniasis. Zn(ii) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (ZnTE-2-PyP4+, ZnP) is a cationic, water-soluble, zinc porphyrin-based photosensitizer whose photodynamic effect on Leishmania braziliensis was analyzed by evaluating the number of visibly undamaged and motile cells, cell membrane integrity, mitochondrial membrane potential, and ultrastructural damage. Treatment of parasites with ZnP and light induced damage in up to 90% of L. braziliensis promastigote cells. Propidium iodide labeling suggested the loss of plasma membrane integrity. In samples treated with ZnP and light, a hyperpolarization of the mitochondrial membrane potential was also observed. Ultrastructural evaluation of promastigotes after photodynamic treatment indicated a loss of cytoplasmic material and the presence of vacuoles. Scanning electron microscopy showed wrinkling of the plasma membrane and a reduced cell volume. Additionally, the number of amastigotes per macrophage was reduced by about 40% after photodynamic application. The treatment showed no considerable toxicity against mammalian cells. Therefore, the results indicated that PDT associated with ZnTE-2-PyP4+ represents a promising alternative to cutaneous leishmaniasis treatment.