Automated Nuclear Analysis of Leishmania major Telomeric Clusters Reveals Changes in Their Organization during the Parasite's Life Cycle

Abstract

Parasite virulence genes are usually associated with telomeres. The clustering of the telomeres, together with their particular spatial distribution in the nucleus of human parasites such as Plasmodium falciparum and Trypanosoma brucei, has been suggested to play a role in facilitating ectopic recombination and in the emergence of new antigenic variants. Leishmania parasites, as well as other trypanosomes, have unusual gene expression characteristics, such as polycistronic and constitutive transcription of protein-coding genes. Leishmania subtelomeric regions are even more unique because unlike these regions in other trypanosomes they are devoid of virulence genes. Given these peculiarities of Leishmania, we sought to investigate how telomeres are organized in the nucleus of Leishmania major parasites at both the human and insect stages of their life cycle. We developed a new automated and precise method for identifying telomere position in the three-dimensional space of the nucleus, and we found that the telomeres are organized in clusters present in similar numbers in both the human and insect stages. While the number of clusters remained the same, their distribution differed between the two stages. The telomeric clusters were found more concentrated near the center of the nucleus in the human stage than in the insect stage suggesting reorganization during the parasite's differentiation process between the two hosts. These data provide the first 3D analysis of Leishmania telomere organization. The possible biological implications of these findings are discussed.

Cell Biology of the Trypanosome Genome

“…MCs and telomeres, which are found at the periphery in procyclic cells, are spread throughout the nucleus in bloodstream-form cells (27,149). Despite lacking homologous pol I expression sites of pathogenicity-associated genes, a similar repositioning of telo- on May 10, 2018 by guest http://mmbr.asm.org/ meres was observed in L. major (42). Here, clusters of telomeres were found to be more centrally distributed in the nuclei of the intracellular stage in humans, compared to the midgut stage in the insect vector.…”

The natural absence of RPA1N domain did not impair Leishmania amazonensis RPA-1 participation in DNA damage response and telomere protection

“…The occurrence of such breaks leads to the arrest of cell division at one of several points, although the level of damage that can be tolerated by different cell types varies markedly (Featherstone and Jackson, 1999). Single DSBs in the core of T. brucei chromosomes have been shown to trigger a robust DNA damage response and 7 parasites/lane treated with phleomycin (40 μg mL − 1 ) for 1, 3 and 6 h compared with non-treated controls (c).…”

“…We have previously shown that LaRPA-1 is copurified with LaRbp38 and DNA polymerase alpha in telomerase-positive extracts (Fernandez et al 2004). In addition, it co-immunoprecipitates telomeric DNA in vivo and co-localizes with telomeric DNA (Siqueira- .…”

Transcription Sites Are Developmentally Regulated during the Asexual Cycle of Plasmodium falciparum

“…To ensure that at the end of long acquisition periods the Z slices were correctly assembled in 3D, fluorescent microspheres were used as references by a customized algorithm that removes intrinsic microscope stage motion and chromatic aberrations, which are negligible in most applications but became important in the case of imaging the miniscule P. falciparum nucleus [29]. After corrected 3D images were assembled, the nuclei were segmented based on a semi-automatic process (modified from [30]). A user-selected region of interest (ROI), which contains a single nucleus, was submitted to automatic thresholding by the K-means clustering algorithm.…”