Abstract
Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.
Highlights
Leishmania amazonensis, a trypanosomatid protozoan, is the main causative agent of Tegumentary leishmaniasis in the Americas
Leishmania amazonensis promastigotes were cultured in M199 medium supplemented with 10% fetal bovine serum at 28 °C, and exponentially growing cells (Figure 1A) were used for all experiments described in this article
We examined 1,186 DAPI-stained wild-type L. amazonensis promastigotes in exponential growth and observed that most of them (973 cells) contained one kinetoplast and one nucleus (1K1N)
Summary
Leishmania amazonensis, a trypanosomatid protozoan, is the main causative agent of Tegumentary leishmaniasis in the Americas. Leishmaniasis is a spectrum of diseases with different clinical forms that affects approximately 350 million people around the globe. Recent data indicated that the disease is endemic in 98 countries, with more than 1.6 million new cases per year and over 20,000 deaths annually [1,2]. There are no effective vaccines for leishmaniasis, and the few available drugs are expensive and toxic to the host. Leishmania spp. belongs to the Trypanosomatidae family, which includes digenetic parasites with complex life cycles and different developmental forms in vertebrate and invertebrate hosts. This peculiarity is central to successful parasite adaptation and the movement of these parasites between
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