15 research outputs found
Automated Nuclear Analysis of Leishmania major Telomeric Clusters Reveals Changes in Their Organization during the Parasite's Life Cycle
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
Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection
The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy
An image-based drug susceptibility assay targeting the placental sequestration of Plasmodium falciparum-infected erythrocytes.
Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high-throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence-specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria
Automated detection and quantification of parasitized erythrocytes binding to plated BeWo cells.
<p>(<b>A</b>) Representative fluorescence image field showing BeWo cells in red (Syto 60-stained) and bound erythrocytes in green (anti-glycorphorin A-FITC conjugates labeled). Areas in black represent regions with no attached BeWo cells. (<b>B</b>) Image segmentation of stained BeWo cell areas (green) and erythrocyte-bound regions (yellow, the red arrow). Non-overlapping erythrocytes are indicated in red (the white arrow). Following the segmentation, binding ratios are calculated as the mean proportion of yellow areas to the green areas per microtiter well (four image fields×2000×2000 pixels). (<b>C</b>) Plot of binding ratios as a function of increasing parasitaemia, showing a strong correlation (R<sup>2</sup> = 0.79 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041765#pone.0041765-Cohen1" target="_blank">[21]</a>) between the two parameters. (<b>D</b>) Representative image fields showing an increased proportion of bound erythrocytes with increasing parasitaemia (compare the number of green spots in the image fields denoted 0.38% and 3%). The numbers below each image field represents the culture parasitaemia used in the binding.</p
Specific involvement of CSA receptors in binding of the parasitized erythrocytes to plated BeWo cells.
<p>(<b>A</b>) Significantly increased binding (Student t-test, <i>P = 0.004</i>) of CSA-panned erythrocytes (bar labeled “CSA”) to BeWo cells when analyzed in parallel and at equal parasitaemia with CD36-panned cells (bar labeled “CD36”). (<b>B</b>) Representative image fields showing a near-complete inhibition of binding between BeWo-panned erythrocytes and the plated BeWo cells in the presence of soluble CSA (1 mg/ml). Control wells were treated with DMSO at a 0.5% final concentration.</p
Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity
Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host. (c) 2010 Elsevier Ltd. All rights reserved.FAPESP Sao Paulo Research Foundation[2007/06443-0]FAPESP Sao Paulo Research Foundation[2003/02366-0]CNPq National Counsel of Technological and Scientific Development[151286/2007-8]Canadian Bureau for International Educatio
Image-based Plasmodium cytoadherence assay design.
<p>Panned <i>P. falciparum</i> FCR3 parasites (∼6-hpi) are drug-treated for 24 hours in a 384-well plate. Next, the cultures are mixed and 5 µl transferred into corresponding wells of a second plate with BeWo cells at >80% confluency. Meanwhile, the remainder 45 µl culture is further cultivated for 24 hours to complete one cell division cycle and then analyzed by the pLDH viability assay. Following a one hour binding reaction, unbound cells are washed, followed by a 15 min fixation with 4% paraformaldehyde and staining with anti-glycophorin A antibodies (bound erythrocytes) and Syto 60 (BeWo cells and parasitized erythrocytes). Stained cells are then imaged and analyzed using customized image-mining algorithms that were developed in this study.</p
Transcription Sites Are Developmentally Regulated during the Asexual Cycle of <em>Plasmodium falciparum</em>
<div><p>Increasing evidence shows that the spatial organization of transcription is an important epigenetic factor in eukaryotic gene regulation. The malaria parasite <em>Plasmodium falciparum</em> shows a remarkably complex pattern of gene expression during the erythrocytic cycle, paradoxically contrasting with the relatively low number of putative transcription factors encoded by its genome. The spatial organization of nuclear subcompartments has been correlated with the regulation of virulence genes. Here, we investigate the nuclear architecture of transcription during the asexual cycle of malaria parasites. As in mammals, transcription is organized into discrete nucleoplasmic sites in <em>P. falciparum</em>, but in a strikingly lower number of foci. An automated analysis of 3D images shows that the number and intensity of transcription sites vary significantly between rings and trophozoites, although the nuclear volume remains constant. Transcription sites are spatially reorganized during the asexual cycle, with a higher proportion of foci located in the outermost nuclear region in rings, whereas in trophozoites, foci are evenly distributed throughout the nucleoplasm. As in higher eukaryotes, transcription sites are predominantly found in areas of low chromatin density. Immunofluorescence analysis shows that transcription sites form an exclusive nuclear compartment, different from the compartments defined by the silenced or active chromatin markers. In conclusion, these data suggest that transcription is spatially contained in discrete foci that are developmentally regulated during the asexual cycle of malaria parasites and located in areas of low chromatin density.</p> </div
Determination of drug effects on BeWo cell binding of parasitized erythrocytes.
<p>Dose-response experiments were done in parallel using the developed assay (solid points) and pLDH growth assay (open rectangles) to assess the effects of CSA (<b>A</b>), artemisinin (<b>B</b>), and brefeldin A (<b>C</b>) on the binding to BeWo cells (image-based assay) and viability (pLDH assay). Data show a specific inhibition of cytoadherence but not parasite growth by CSA unlike artemisinin and BFA, which inhibited both binding and parasite growth with increasing drug concentrations.</p
Transcription occurs in discrete foci in the nucleus of asexual forms of malaria parasites.
<p>Synchronized populations of <i>P. falciparum</i> were labeled with BrUTP, and nascent RNA (BrRNA, in green) was detected by indirect immunofluorescence, showing that transcription sites are organized in foci distributed throughout the nucleus in early rings (2 hpi), rings (10 hpi), throphozoites (22 hpi) and schizonts (34 hpi). DNA was stained with DAPI and artificially colored in red for enhanced contrast. Representative images are shown. Bars, 1 µm.</p