First body of evidence suggesting a role of a tankyrase-binding motif (TBM) of vinculin (VCL) in epithelial cells

Background: Adherens junctions (AJ) are involved in cancer, infections and neurodegeneration. Still, their composition has not been completely disclosed. Poly(ADP-ribose) polymerases (PARPs) catalyze the synthesis of poly(ADP-ribose) (PAR) as a posttranslational modification. Four PARPs synthesize PAR, namely PARP-1/2 and Tankyrase-1/2 (TNKS). In the epithelial belt, AJ are accompanied by a PAR belt and a subcortical F-actin ring. F-actin depolymerization alters the AJ and PAR belts while PARP inhibitors prevent the assembly of the AJ belt and cortical actin. We wondered which PARP synthesizes the belt and which is the PARylation target protein. Vinculin (VCL) participates in the anchorage of F-actin to the AJ, regulating its functions, and colocalized with the PAR belt. TNKS has been formerly involved in the assembly of epithelial cell junctions. Hypothesis: TNKS poly(ADP-ribosylates) (PARylates) epithelial belt VCL, affecting its functions in AJ, including cell shape maintenance. Materials and Methods: Tankyrase-binding motif (TBM) sequences in hVCL gene were identified and VCL sequences from various vertebrates, Drosophila melanogaster and Caenorhabditis elegans were aligned and compared. Plasma membrane-associated PAR was tested by immunocytofluorescence (ICF) and subcellular fractionation in Vero cells while TNKS role in this structure and cell junction assembly was evaluated using specific inhibitors. The identity of the PARylated proteins was tested by affinity precipitation with PAR-binding reagent followed by western blots. Finally, MCF-7 human breast cancer epithelial cells were subjected to transfection with Tol2-plasmids, carrying a dicistronic expression sequence including Gallus gallus wt VCL (Tol-2-GgVCL), or the same VCL gene with a point mutation in TBM-II (Tol2-GgVCL/*TBM) under the control of a β-actin promoter, plus green fluorescent protein following an internal ribosome entry site (IRES-GFP) to allow the identification of transfected cells without modifying the transfected protein of interest. Results and discussion: In this work, some of the hypothesis predictions have been tested. We have demonstrated that: (1) VCL TBMs were conserved in vertebrate evolution while absent in C. elegans; (2) TNKS inhibitors disrupted the PAR belt synthesis, while PAR and an endogenous TNKS pool were associated to the plasma membrane; (3) a VCL pool was covalently PARylated; (4) transfection of MCF-7 cells leading to overexpression of Gg-VCL/*TBM induced mesenchymal-like cell shape changes. This last point deserves further investigation, bypassing the limits of our transient transfection and overexpression system. In fact, a 5th testable prediction would be that a single point mutation in VCL TBM-II under endogenous expression control would induce an epithelial to mesenchymal transition (EMT). To check this, a CRISPR/Cas9 substitution approach followed by migration, invasion, gene expression and chemo-resistance assays should be performed.Fil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Valsecchi, Wanda Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Fernandez Villamil, Silvia Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Lafon Hughes, Laura I.. Universidad de la Republica. Centro Universitario del Litoral Norte.; Urugua

PARP Inhibitor Olaparib Causes No Potentiation of the Bleomycin Effect in VERO Cells, Even in the Presence of Pooled ATM, DNA-PK, and LigIV Inhibitors

Poly(ADP-ribosyl)polymerase (PARP) synthesizes poly(ADP-ribose) (PAR), which is anchored to proteins. PAR facilitates multiprotein complexes’ assembly. Nuclear PAR affects chromatin’s structure and functions, including transcriptional regulation. In response to stress, particularly genotoxic stress, PARP activation facilitates DNA damage repair. The PARP inhibitor Olaparib (OLA) displays synthetic lethality with mutated homologous recombination proteins (BRCA-1/2), base excision repair proteins (XRCC1, Polβ), and canonical nonhomologous end joining (LigIV). However, the limits of synthetic lethality are not clear. On one hand, it is unknown whether any limiting factor of homologous recombination can be a synthetic PARP lethality partner. On the other hand, some BRCA-mutated patients are not responsive to OLA for still unknown reasons. In an effort to help delineate the boundaries of synthetic lethality, we have induced DNA damage in VERO cells with the radiomimetic chemotherapeutic agent bleomycin (BLEO). A VERO subpopulation was resistant to BLEO, BLEO + OLA, and BLEO + OLA + ATM inhibitor KU55933 + DNA-PK inhibitor KU-0060648 + LigIV inhibitor SCR7 pyrazine. Regarding the mechanism(s) behind the resistance and lack of synthetic lethality, some hypotheses have been discarded and alternative hypotheses are suggested.Fil: Perini, Valentina. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Schacke, Michelle. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Liddle, Pablo. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Keszenman, Deborah J.. Universidad de la Republica. Centro Universitario del Litoral Norte.; UruguayFil: Lafon Hughes, Laura. Instituto de Investigaciones Biológicas "Clemente Estable"; Urugua

Host Cell Poly(ADP-ribose) glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Trypanosoma cruzi, etiological agent of Chagas´ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADPribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stagedependant manner. Indirect immunofluorescense assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl)pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 μM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 μM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas´ disease.Fil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Schlesinger, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Kevorkian, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Flawia, Mirtha Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Fernandez Villamil, Silvia Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentin

A versatile CRISPR/Cas9 editing approach in Trypanosoma cruzi

Development of CRISPR/Cas9 as a tool for genomic edition brought a new perspective to the study of Trypanosoma cruzi, an organism usually reluctant to other gene editing technologies. Most often, epimastigotes are co-transfected with a single plasmid bearing both the gene for Cas9-GFP expression and a sequence to be translated into a single guide RNA (sgRNA), jointly with a lineal donor DNA encompassing a selection marker flanked by sequences homologous to the target gene. Here, we tested an alternative approach for the generation of Phosphodiesterase (PDE) knockout parasites. We obtained epimastigotes from Tul II strain stably expressing Cas9-GFP in the nucleus in all parasite stages, with no detrimental effects on epimastigote growth or differentiation nor on trypomastigote infection capability. These Cas9-GFP epimastigotes were co-transfected with the sgRNA + DNA donor pair, according to the intended gene target. sgRNA were obtained by in vitro transcription using a template DNA bearing the specific + scaffold sequence under a T7 promoter. To obtain the donor DNA we designed a "pre-donor" formed by a sequence including several restriction enzyme recognition sites flanked by 30-bp arms homologous to the sequence adjacent sgRNA annealing target. This "pre-donor" allowed to easily generate a variety of donor DNAs by cloning alternative selection markers. DNA extracts (boiling-preps) from 4-day post-transfection cultures were evaluated by PCR using "mixed" primer pairs: while one of the primers annealed to the target gene, the second primer annealed to a sequence in the donor DNA, allowing assessment of its correct insertion in the gene of interest. Advantages of this take on CRISPR/Cas9 edition include its versatility for choosing and switching between alternative selection markers and a quick and affordable generation of the components of the system and analysis of the transfected cultures, while possibly facilitating complementation assays on the KO lines.Fil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Prego, Alejo Facundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Llanos, Manuel. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Alberca, Lucas Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; ArgentinaFil: Bellera, Carolina Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; ArgentinaFil: Gavernet, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; ArgentinaFil: Talevi, Alan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencas Exactas. Laboratorio de Investigación y Desarrollo de Bioactivos; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaXI Congreso de la Sociedad Argentina de ProtozoologíaMendozaArgentinaSociedad Argentina de Protozoologí

Inhibition of Poly(ADP-Ribose)Glycohydrolase activity affects lysosomal function and hampers T. cruzi infection in Vero cells

Chagas disease is a potentially life-threatening protozoan infection but, despite its high incidence and large economic costs associated to it, effective pharmacological treatments are lacking. The search for new anti-chagasic drugs has focused on potential targets in the parasite itself but heterogeneity among different strains of Trypanosoma cruzi - the etiological agent- has hampered these efforts. Like other protozoa, T. cruzi invades the host cell and this complex interplay can determine the outcome of the infection: the parasite must manipulate host cell signaling pathways to achieve its purpose. Therefore, targeting the host signals that promote T. cruzi infection can be therapeutically valuable. Poly(ADP-ribose) (PAR) ?crucial for DNA damage response among other processes- participates in host cell response to the parasitic infection: Poly(ADP-ribose)polymerase-1 inhibitors decrease T. cruzi infection while Poly(ADP-ribose)glycohydrolase (PARG) inhibition or silencing almost completely abrogates it, raising interest in PAR signaling and the role of PARG in the host-parasite interaction.PAR levels raised early after infection (15 min) and remained elevated during the complete cell infection cycle, as determined by ELISA using a PAR-detecting reagent. PARG inhibition by DEA 1 μM or silencing by shRNA caused reduced T. cruzi cell invasion, indicating that PARG might be important during this initial step. T. cruzi can invade the host cell by lysosome-independent, lysosome-dependent and autophagic pathways but they must all culminate in the fusion of the trypomastigote-bearing parasitophorous vacuole (TcPV) to lysosomes. Absence of PARG activity did not hamper the formation of TcPV with early endosomal characteristics as shown by staining against EEA1 (early endosomal antigen) or the use of a FYVE-eGFP probe to detect PIP3-rich vacuoles, nor did it affect infection levels when cells were subjected to nutritional stress, suggesting PARG is not participating in the initial stages of the lysosome-independent and autophagic pathways. However, PARG activity seems crucial for lysosomal function: PARG-inhibited or silenced Vero cells showed reduced DQ-BSA Red and Lysotracker DND-99 staining, indicating proteolytic activity and pH are altered. A drastic reduction in LAMP-1 signal was also detected. PARG inhibition and silencing also appears to affect the reorganization of host cell cytoskeleton during T. cruzi invasion. These results indicate that PARG activity is important for the maintenance of lysosomal activity, which is crucial for the initial steps of T. cruzi infection.Fil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Fernandez Villamil, Silvia Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaMolecular Parasitology Meeting XXXIRockvilleEstados UnidosGenetic Society of Americ

Poly(ADP-ribose) metabolism in human parasitic protozoa

Poly(ADP-ribosyl)ation reactions constitute a post-translational protein modification synthesized in higher eukaryotes by a family of poly(ADP-ribose)polymerases (PARP) and catabolized mainly by poly(ADP-ribose) glycohydrolase (PARG). The best understood role of PARP is the maintenance of genomic integrity via the promotion of DNA repair that leads to cell survival when low levels of genotoxic stress occur. The participation of PARP in unleashing cell death at higher levels of damage has also been broadly studied. The biology of poly(ADP-ribosyl)ation in protozoan parasites, however, still remains a mystery. This review will examine the presence of the key enzyme involved in ADP-ribose polymer (PAR) metabolism in protozoan parasites associated with human diseases. Theoretical and experimental data obtained up to date have revealed the presence of PAR metabolism only in the trypanosomatids Trypanosoma cruzi and T. brucei, the apicomplexan Toxoplasma gondii and Entamoeba histolytica. T. cruzi and T. brucei, as opposed to humans and other organisms, have only one PARP and one PARG with subcellular localizations that are distinct from the ones described for their mammalian counterparts. The topics discussed in this review describe the first studies on PAR metabolism in trypanosomatids, specially the role of PAR on DNA damage response, cell cycle progression and cell death after genotoxic stimuli. The results described show differences in some aspects of PAR metabolism in trypanosomatids in comparison to other eukaryotes. New questions about the function of this metabolic pathway in the parasites under study are open and we hope it encourages the research community to explore this signaling pathway as a new possible target of clinical relevance in these and other disease-causing parasites.Fil: Fernandez Villamil, Silvia Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Role of TcPARP during the metacyclogenesis of Trypanosoma cruzi

Poly(ADP-ribosyl)ation is a post-translational modification catalyzed by poly(ADP- ribose)polymerases (PARPs). As a reversible modification, it is regulated by a delicate balance ofsynthesis and degradation, and is involved in various nuclear processes related to DNAmetabolism. To adapt to its complex life cycle, T. cruzi undergoes several differentiationprocesses such as metacyclogenesis, where the replicative epimastigote transforms into theinfective trypomastigote. Here we evaluated the role of TcPARP during T. cruzi metacyclogenesisusing Olaparib, a specific PARP inhibitor, with two differentiation techniques: TAU medium andblood agar. Our results using two different T. cruzi strains show that, depending on thedifferentiation technique used, the inhibition of TcPARP stimulates or hinders metacyclogenesis.We also compared the formation of reactive oxygen species (ROS) and autophagosomes duringthe induction of metacyclogenesis on blood agar, in order to understand the pathways that arebeing triggered in this process. Preliminary results indicate that while ROS formation could betriggering the metacyclogenesis in blood agar, autophagy would not be involved in this process.In order to further study this process, we are generating TcPARP knockout cell lines that will helpus corroborate our results.Fil: Svagzdys, Ailin. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Fernandez Villamil, Silvia Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaParasitravaganzaSmithfieldAustraliaThe Australian Society for Parasitolog

Metabolism of Poly-ADP-ribose in Trypanosomatids

The poly-ADP-ribosylation reaction is a post-translational modification involved invarious cellular processes including DNA repair, DNA replication, gene expression,maintenance of genomic stability, cell death and cellular differentiation. Poly(ADPribose)polymerase (PARP) is an abundant nuclear protein that is potently activated by DNA strand nicks and breaks. This enzyme transfers ADP-ribose units from NAD+ to several proteins implicated in chromatin architecture and DNA metabolism, but the main acceptor protein is PARP itself. The homopolymer of ADP-ribose is mainly catabolized by poly(ADP-ribose)glycohydrolase (PARG), a mechanism through which PARP and other modified proteins may be restored to their native state. Poly-ADP-ribose metabolism has been studied in several organisms, but limited information is available about this process in trypanosomatids. In our laboratory, we have demonstrated the presence of PARP in the trypanosomatids Crithidia fasciculata, Trypanosoma cruzi and Trypanosoma brucei, all of which showed properties characterizing other representatives of the PARP group. By using the genome project of T. cruzi and T. brucei we identified TcPARP and TbPARP as well as TcPARG and TbPARG sequences and found that they presented several conserved structural characteristics and only showed slight differences when compared with PARPs and PARGs from other organisms. In addition, we characterized the purified C. fasciculata PARP and recombinant TcPARP biochemically and found that both enzymes were strongly activated by nicked DNA. We also found that histones increase PARP activity and demonstrated the covalent attachment of [32P]-ADPribose moieties to histones, which are catalyzed by PARP. In further experiments, we demonstrated that C. fasciculata PARP and recombinant TcPARP require no magnesium or any other metal ion cofactor for their activity and are inhibited by typical PARP inhibitors: nicotinamide, 3-aminobenzamide, theophylline and thymidine. TcPARP and TcPARG were localized to the cell nucleus. Our findings demonstrated that covalent poly-ADP-ribosylation of PARP itself or DNA topoisomerase I resulted in the inhibitionof their activities, which were restored after removing the ADP-ribose polymers. DNAdamaging agents, which trigger different repair mechanisms, induce poly-ADP-ribosesynthesis in the nucleus, as was established by our group in epimastigotes of T. cruzi.The role of poly-ADP-ribose metabolism in trypanosomatids remains to be furtherinvestigated, and unraveling it could be important for the development of new therapies against these parasites.Fil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Fernandez Villamil, Silvia Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Tankyrase inhibitors hinder Trypanosoma cruzi infection by altering host-cell signalling pathways

Chagas disease is a potentially life-threatening protozoan infection affecting around 8 million people, for which only chemotherapies with limited efficacy and severe adverse secondary effects are available. The etiological agent, Trypanosoma cruzi, displays varied cell invading tactics and triggers different host cell signals, including the Wnt/b-catenin pathway. Poly(ADP-ribose) (PAR) can be synthetized by certain members of the poly(ADP-ribose) polymerase (PARP) family: PARP-1/-2 and Tankyrases-1/2 (TNKS). PAR homeostasis participates in the host cell response to T. cruzi infection and TNKS are involved in Wnt signalling, among other pathways. Therefore, we hypothesized that TNKS inhibitors (TNKSi) could hamper T. cruzi infection. We showed that five TNKSi (FLALL9, MN64, XAV939, G007LK & OULL9) diminished T. cruzi infection of Vero cells. As most TNKSi did not affect the viability of axenically cultivated parasites, our results suggested that TNKSi were interfering with parasite-host cell signalling. Infection by Trypanosoma cruzi induced nuclear translocation of b-catenin, as well as up-regulation of TNF-a expression and secretion. These changes were hampered by TNKSi. Further signals should be monitored in this model and in vivo. As a TNKS inhibitor has entered cancer clinical trials with promising results, our findings encourage further studies aiming at drug repurposing strategies.Fil: Lafon Hughes, Laura. Universidad de la República; UruguayFil: Fernandez Villamil, Silvia Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

Inhibition of PARG activity affects lysosomal function and hampers T. cruzi infection in Vero cells

Chagas disease is a potentially life-threatening protozoan infection with little therapeuticalternatives. Since Trypanosoma cruzi triggers different host cell signaling pathways, targetingthem can be therapeutically valuable. Poly(ADP-ribose) (PAR) participates in host cell responseduring the infection: Poly(ADP-ribose)Polymease-1 inhibition or silencing decreases T .cruziinfection and Poly(ADP-ribose)glycohyrolase (PARG) inhibition or silencing almost completelyabrogates it. New results showed PAR raised early after infection (15 min) and remainedelevated. T. cruzi invades the host cell by lysosome-independent, lysosome-dependent orautophagic pathways. However, they must all culminate in the fusion of the trypomastigotebearingparasitophorous vacuole (TcPV) to lysosomes. PARG inhibition or silencing during theinvasion step caused a significant reduction in T. cruzi cell invasion. Absence of PARG activitydidn ́t hamper formation of TcPV with early endosomal characteristics (EEA1+ or PIP3+vacuoles), nor infection levels under nutritional stress, suggesting PARG is unimportant in earlylysosome-independent and autophagic pathways. However, PARG activity seems crucial forlysosomal function: PARG-silenced or inhibited cells reduced DQ-BSA Red and LysotrackerDND-99 staining, indicating proteolytic activity and pH are altered. LAMP-1 signal was alsodrastically reduced. PARG activity seems important for the maintenance of lysosomal activity,and, therefore, for the initial steps of T. cruzi infection.Fil: Chiatellino, Maria Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Svagzdys, Ailin. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Fernandez Villamil, Silvia Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vilchez Larrea, Salomé Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaParasitravaganzaSmithfieldAustraliaAustralian Society for Parasitolog