Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei

TbPARG in Trypanosoma brucei. A) TbPARG localization in untreated (control) and in procyclic cultures exposed to 500 μM H2O2 for 10 min. IFI was carried out as reported in our previous work [33]. TbPARG was identified with our home-made antibody against TcPARG [33]; and PAR was identified with a commercial antibody against PAR (BD). White bar represents 50 μm. B) Western blot analysis of 40 μg protein per lane revealed with a commercial anti-PARG antibody (Antibody Verify) in T. brucei procyclic (PC) and bloodstream (BST) forms. The arrow indicates the band with the expected molecular weight (approximately 60 kDa). The membrane stained with Red Ponceau was used as a loading control. (TIF 4272 kb

Trypanosoma cruzi PARP is enriched in the nucleolus and is present in a thread connecting nuclei during mitosis.

Poly (ADP-ribose) polymerase (PARP) is responsible for the synthesis of ADP-ribose polymers, which are involved in a wide range of cellular processes such as preservation of genome integrity, DNA damage signaling and repair, molecular switches between distinct cell death pathways, and cell cycle progression. Previously, we demonstrated that the only PARP present in T. cruzi migrates to the nucleus upon genotoxic stimulus. In this work, we identify the N-terminal domain as being sufficient for TcPARP nuclear localization and describe for the first time that TcPARP is enriched in the parasite's nucleolus. We also describe that TcPARP is present in a thread-like structure that connects two dividing nuclei and co-localizes with nucleolar material and microtubules. Furthermore, ADP-ribose polymers could also be detected in this thread during mitosis. These findings represent a first approach to new potential TcPARP functions inside the nucleus and will help understand its role well beyond the largely described DNA damage response protein in trypanosomatids

VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt

Poly-ADP-ribose (PAR) is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs) and degraded by poly-ADP-ribose-glycohydrolase (PARG). Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair). Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt). In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO). PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications

An AMP-activated protein kinase complex with two distinctive alpha subunits is involved in nutritional stress responses in Trypanosoma cruzi.

Trypanosoma cruzi, the etiological agent of Chagas disease, has a digenetic life cycle. In its passage from the insect vector to the mammalian host, and vice versa, it must be prepared to cope with abrupt changes in environmental conditions, such as carbon source, pH, temperature and osmolarity, in order to survive. Sensing and signaling pathways that allow the parasite to adapt, have unique characteristics with respect to their hosts and other free-living organisms. Many of the canonical proteins involved in these transduction pathways have not yet been found in the genomes of these parasites because they present divergences either at the functional, structural and/or protein sequence level. All of this makes these pathways promising targets for therapeutic drugs. The AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by environmental stresses such as osmotic stress, hypoxia, ischaemia and exercise that results in reduction of ATP and increase of AMP levels. Thus, AMPK is regarded as a fuel gauge, functioning both as a nutrient and an energy sensor, to maintain energy homeostasis and, eventually, to protect cells from death by nutrient starvation. In the present study we report the characterization of AMPK complexes for the first time in T. cruzi and propose the function of TcAMPK as a novel regulator of nutritional stress in epimastigote forms. We show that there is phosphotransferase activity specific for SAMS peptide in epimastigotes extracts, which is inhibited by Compound C and is modulated by carbon source availability. In addition, TcAMPKα2 subunit has an unprecedented functional substitution (Ser x Thr) at the activation loop and its overexpression in epimastigotes led to higher autophagic activity during prolonged nutritional stress. Moreover, the over-expression of the catalytic subunits resulted in antagonistic phenotypes associated with proliferation. Together, these results point to a role of TcAMPK in autophagy and nutrient sensing, key processes for the survival of trypanosomatids and for its life cycle progression

Identification of a Wee1-like kinase gene essential for procyclic Trypanosoma brucei survival.

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M

Role of TcPARG in <i>Trypanosoma cruzi</i> epimastigotes proliferation and cell cycle progression.

<p>A) Effect of the PARG inhibitors ADP-HPD and DEA on <i>T. cruzi</i> growth and survival was determined by incubating epimastigotes at an initial density of 10⁷ parasites/ml in the continuous presence of inhibitors at 1 µM. The number of epimastigotes was determined daily by counting formaldehyde-fixed parasites in a Neubauer chamber. All data points were determined in triplicates and shown as means with standard deviation. The significance of the results versus the control at day 4 was analyzed with t test and indicated in the figure (* p0.05). B) Effect of ADP-HPD at 1 µM concentration on cell cycle progression of epimastigotes was determined by adding the inhibitor at the indicated concentration to the culture media of hydroxyurea synchronized parasites after digitonin permeabilization. Samples were drawn every 2 hours for 14 hours and DNA content was determined by propidium iodide staining followed by flow cytometry analysis. The percentage of epimastigotes in each cell cycle phase was determined by setting gates according to the DNA content in the 0 hs of the control sample and maintained for all other samples. The data were analyzed using the Cyflogic software.</p

Effect of PARG inhibitors on <i>T. cruzi</i> infection on Vero or A549 host cells.

<p><i>T. cruzi</i> trypomastigotes were purified from the supernatant of previously infected cells and preincubated for 30 min in the respective culture medium in the absence (Control) or presence of 1 µM PARG inhibitor (DEA). Twenty-four hours Vero, A549 wild type or shPARG (hPARG silenced) cell monolayers were infected with 50 trypomastigotes/cell. The infection process was followed by microscopic direct visualization. At the indicated days (A and C) or at day 6 post-infection (B and D), percentage of infected cells and number of amastigotes intracellular were determined on May-Grünwald Giemsa stained samples. Amastigotes and cells were counted using the ImageJ software in at least 7 fields. The number of trypomastigotes/ml in the supernatant of infected cell cultures was determined by counting unfixed trypomastigotes in a Neubauer chamber at the indicated days (E) or at day 9 post-infection (F). All points were determined in triplicates and shown as means with standard deviation. Significance of the result versus the Control (***p0.001; two way ANOVA) or Wild Type Control (***p0.001; **, p0.01; two way ANOVA) is indicated.</p

Expression of TcPARG throughout the <i>Trypanosoma cruzi</i> life-cycle.

<p>(A) Microarray expression data for TcPARG over the course of <i>T. cruzi</i> life-cycle. TcPARG mRNA relative abundance was evaluated by using the transcriptome analysis of different <i>T. cruzi</i> stages available at Gene Expression Omnibus database (<a href="http://www.ncbi.nlm.nih.gov/geo" target="_blank">www.ncbi.nlm.nih.gov/geo</a>, DataSets: GSE14641). Shown are mean microarray log₂ ratios (stage/reference) for TS significantly regulated in <i>Trypanosoma cruzi</i> amastigotes (AMA), trypomastigotes (TRYP), epimastigotes (EPI), and metacyclic trypomastigotes (META). (B) Western blot analysis of the three life-cycle stages of <i>T. cruzi</i>. Protein extracts (35 µg) of amastigote, epimastigote or trypomastigote stages of <i>T. cruzi</i> were solved in a 10% polyacrylamide gel, transfer to a nitrocellulose membrane and revealed with an anti-TcPARG (1:10000) specific antiserum. β-tubulin was used as loading control.</p