Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

Abstract

The differentiation of proliferating epimastigote forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast -an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.

Colchicine treatment reversibly blocks cytokinesis but not mitosis in Trypanosoma cruzi epimastigotes

“…It is noteworthy that, although this atypical organelle disposition is similar to the subcellular localization of those found in the infective, non-replicating forms, the morphology of the kinetoplast is elongated and the nucleus is rounded, as seen in nontreated epimastigote forms (Elias et al 2007). In contrast, in the infective trypomastigote stage, the kinetoplast exhibits a rounded shape, while the nucleus appears elongated (Ferreira et al 2008). In this regard, we suggest that the kinetoplast movement is due to an alteration of the microtubules present in the structures connected directly or indirectly to it, like the flagellar attachment zone or basal body, disrupting the organization or the strength of these components, rather than promoting the differentiation, as postulated by Filho et al (1978).…”

“…Morphological and biochemical changes take place during differentiation from the insect non-infective epimastigote to the mammalian infective trypomastigote stages (Goldenberg and Avila 2011). Microscopy allows observing that the kinetoplast and flagellum are repositioned during in vitro metacyclogenesis (Ferreira et al 2008), demonstrating that there is an active movement of these organelles. In other trypanosomatids such as T. brucei, basal body repositioning is necessary to drive cytokinesis during the cell cycle (Lacomble et al 2010).…”

Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum

“…The mechanisms involved in adhesion to a substrate in T. brucei (Ersfeld & Gull 2001) T. cruzi (Ferreira et al 2008) and T. rangeli (Meirelles et al 2005) require the crucial and active participation of the flagella of these parasites. During metacyclogenesis, the flagellar membrane remains connected to the epithelial surface (Ersfeld & Gull 2001) representing an important interface between the parasite and its host that is essential for the fulfilment of its biological cycle.…”

Stress Induces Changes in the Phosphorylation of Trypanosoma cruzi RNA Polymerase II, Affecting Its Association with Chromatin and RNA Processing

“…As in T. brucei, the T. cruzi RNA (TcRNA) Pol II Rbp1 appears as a doublet in SDS-PAGE gels (27), suggesting that it is also phosphorylated. The fast-migrating form in most organisms is known as subunit IIa, which has a hypophosphorylated CTD, whereas the slow-migrating form is known as subunit IIo, which has a hyperphosphorylated CTD (28)(29)(30).…”