Overexpression of Cytoplasmic TcSIR2RP1 and Mitochondrial TcSIR2RP3 Impacts on Trypanosoma cruzi Growth and Cell Invasion

Background: Trypanosoma cruzi is a protozoan pathogen responsible for Chagas disease. Current therapies are inadequate because of their severe host toxicity and numerous side effects. The identification of new biotargets is essential for the development of more efficient therapeutic alternatives. Inhibition of sirtuins from Trypanosoma brucei and Leishmania ssp. showed promising results, indicating that these enzymes may be considered as targets for drug discovery in parasite infection. Here, we report the first characterization of the two sirtuins present in T. cruzi. Methodology: Dm28c epimastigotes that inducibly overexpress TcSIR2RP1 and TcSIR2RP3 were constructed and used to determine their localizations and functions. These transfected lines were tested regarding their acetylation levels, proliferation and metacyclogenesis rate, viability when treated with sirtuin inhibitors and in vitro infectivity. Conclusion: TcSIR2RP1 and TcSIR2RP3 are cytosolic and mitochondrial proteins respectively. Our data suggest that sirtuin activity is important for the proliferation of T. cruzi replicative forms, for the host cell-parasite interplay, and for differentiation among life-cycle stages; but each one performs different roles in most of these processes. Our results increase the knowledge on the localization and function of these enzymes, and the overexpressing T. cruzi strains we obtained can be useful tools for experimental screening of trypanosomatid sirtuin inhibitors.Fil: Ritagliati, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Alonso, Victoria Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Manarin, Romina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cribb, Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Serra, Esteban Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Benznidazole blocks NF-kB activation but not AP-1 through inhibition of IKK

Previously, we demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and NO release in macrophages by inhibiting NF-kappaB. We now proceeded to elucidate the molecular mechanisms by which BZL exerts its inhibitory action on NF-kappaB. We demonstrated that the inhibitory effect of BZL is not extended to other macrophage responses, since it did not inhibit other typical hallmarks of macrophage activation such as phagocytosis, MHC-II molecules expression or production of reactive oxygen species (ROS) by NADPH oxidase. BZL was able to interfere specifically with the activation of NF-kappaB pathway without affecting AP-1 activation in RAW 264.7 macrophages, not only in LPS-mediated activation, but also for other stimuli, such as pro-inflammatory cytokines (IL-1beta, TNF-alpha), PMA or H(2)O(2). Also, BZL delayed the activation of p38 MAPK, but not that of ERK1/2 and JNK. Finally, treatment with BZL inhibited IkappaBalpha phosporylation and hence its degradation, whereas it did not block IkappaB kinase (IKK) alpha/beta phosphorylation. Collectively, BZL behaves as a broad range specific inhibitor of NF-kappaB activation, independently of the stimuli tested.This work was supported by ANPCyT PICTPeer Reviewe

Tumor Necrosis Factor-α Regulates Glucocorticoid Synthesis in the Adrenal Glands of Trypanosoma cruzi Acutely-Infected Mice. The Role of TNF-R1

Adrenal steroidogenesis is under a complex regulation involving extrinsic and intrinsic adrenal factors. TNF-α is an inflammatory cytokine produced in response to tissue injury and several other stimuli. We have previously demonstrated that TNF-R1 knockout (TNF-R1−/−) mice have a dysregulated synthesis of glucocorticoids (GCs) during Trypanosoma cruzi acute infection. Since TNF-α may influence GCs production, not only through the hypothalamus-pituitary axis, but also at the adrenal level, we now investigated the role of this cytokine on the adrenal GCs production. Wild type (WT) and TNF-R1−/− mice undergoing acute infection (Tc-WT and Tc-TNF-R1−/− groups), displayed adrenal hyperplasia together with increased GCs levels. Notably, systemic ACTH remained unchanged in Tc-WT and Tc-TNF-R1−/− compared with uninfected mice, suggesting some degree of ACTH-independence of GCs synthesis. TNF-α expression was increased within the adrenal gland from both infected mouse groups, with Tc-WT mice showing an augmented TNF-R1 expression. Tc-WT mice showed increased levels of P-p38 and P-ERK compared to uninfected WT animals, whereas Tc-TNF-R1−/− mice had increased p38 and JNK phosphorylation respect to Tc-WT mice. Strikingly, adrenal NF-κB and AP-1 activation during infection was blunted in Tc-TNF-R1−/− mice. The accumulation of mRNAs for steroidogenic acute regulatory protein and cytochrome P450 were significantly increased in both Tc-WT and Tc-TNF-R1−/− mice; being much more augmented in the latter group, which also had remarkably increased GCs levels. TNF-α emerges as a potent modulator of steroidogenesis in adrenocortical cells during T. cruzi infection in which MAPK pathways, NF-κB and AP-1 seem to play a role in the adrenal synthesis of pro-inflammatory cytokines and enzymes regulating GCs synthesis. These results suggest the existence of an intrinsic immune-adrenal interaction involved in the dysregulated synthesis of GCs during murine Chagas disease.Fil. Villar, Silvina R. Instituto de Inmunología, Facultad de Ciencias Médicas, Universidad Nacional de Rosario; ArgentinaFil. Ronco, María Teresa. Instituto de Fisiología Experimental (IFISE-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario; Argentin

Efficacy of novel benznidazole solutions during the experimental infection with Trypanosoma cruzi

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. About 8. million people throughout Latin America are infected causing approximately 10,000 deaths annually. Benznidazole, available as unique 100. mg tablets in many of the endemic countries, is currently the drug of choice for the specific treatment of this condition. Despite of the large number of pediatric patients infected, there are no commercial liquid dosage forms available to treat this trypanosomiasis. This work showed that novel benznidazole-water-polyethylene glycol 400 solutions are active against T. cruzi in a murine model of Chagas' disease. Present results constitute the first demonstration on the usefulness of benznidazole solutions in infected mice.Fil: Manarin, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología; ArgentinaFil: Lamas, Maria Celina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Bottasso, Emanuel. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología; ArgentinaFil: Serra, Esteban Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Revelli, Silvia Susana. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología; ArgentinaFil: Salomon, Claudio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentin

The expression of sirtuins is developmentally regulated.

<p>Equal amounts of parasite lysate from epimastigotes (E), amastigotes (A) and trypomastigotes (T) were loaded on SDS-PAGE followed by (A) Coomassie staining or (B) western blot analysis using the following antibodies: anti-<i>Tc</i>SIR2RP1, anti-<i>Tc</i>SIR2RP3 and anti-Tubulin as load control.</p

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing β-galactosidase

Alonso, V. L., Manarin, R., Perdomo, V., Gulin, E., Serra, E., Cribb, P. In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing β-galactosidase. J. Vis. Exp. (177), e63210, doi:10.3791/63210 (2021).Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi. T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission. This paper describes an assay based on measuring the activity of the cytoplasmic β-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red β-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a β-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.Fil: Alonso, Victoria Lucia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.Fil: Manarin, Romina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.Fil: Perdomo, Virginia Gabriela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.Fil: Serra, Esteban C. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.Fil: Cribb, Pamela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Parasitología; Argentina.Fil: Serra, Esteban. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Cribb, Pamela. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Gulin, Julián Ernesto Nicolás. Universidad de Buenos Aires. Instituto de Investigaciones Biomédicas (INBIOMED); Argentina

Overexpression of sirtuins changes the acetylation pattern in epimastigotes.

<p>Equal amounts of parasite lysate from each line in the absence (-, white bars) or presence (+, grey bars) of 0.25 μg/ml Tetracycline 24 hours post induction, were loaded on SDS-PAGE (A) followed by Western blot analysis using rabbit anti-Acetyl-lysine (B) and mouse anti-Tubulin and anti-Acetylated Tubulin antibodies (C). The deacetylated proteins in the anti-Acetyl-lysine western blot are depicted with black arrowsheads. The intensity of the acetylated-tubulin bands was quantified from n = 3 independent experiments and normalized to α-tubulin intensity. The bar graph represents the mean ± SEM; * p<0.05, ** p<0.005 (unpaired, two-tailed Student t test).</p

Sirtuin overexpression impacts on Vero cells infection.

<p>The infection and the post-infection incubation were performed in the absence or presence of 0.25 μg/ml Tetracycline: (-/-), Tet was never added to the medium; (+/-), trypomastigotes were pre-treated with Tet for 3 hours prior to infection, and it was added during the infection but not after; (-/+), trypomastigotes were not induced, Tet was only added for 48 hours post-infection at the amastigote stage; (+/+), trypomastigotes were pre-treated and Tet was present at all times. The conditions (-/-) and (+/+) were also tested in the presence of 75 μM Nicotinamide (+ NAM). The percentage of infected cells (A) and the number of amastigotes per cell (B) were determined by counting Giemsa-stained slides using a light microscope. Results are expressed as means ± SEM of triplicates, and represent one of three independent experiments performed. Each condition was analyzed by unpaired Mann-Whitney two-tailed Student t test with the control (-/-): * p<0.05, ** p<0.005, *** p<0.001. The same statistical analysis was used to compare uninduced and induced parasites in the presence of NAM (-/-, +NAM and +/+, +NAM): σ p<0.05.</p

Effect of the overexpression of sirtuins on the growth rate of epimastigotes.

<p>Growth curves of epimastigotes transfected with p<i>Tc</i>INDEXGW-<i>Tc</i>SIR2RP1HA and <i>Tc</i>SIR2RP3HA in the absence (closed circles, grey line) or presence (closed squares, black line) of 0.5 μg/ml Tetracycline (which was re-added every 5 days) counted daily during 15 days. Results are representative of three independent experiments.</p

<i>Tc</i>SIR2RP1 localizes in the cytoplasm and <i>Tc</i>SIR2RP3 in the mitochondrion.

<p>(A) Immunofluorescence assay of Dm28<i>c</i> wild type epimastigotes using rabbit antibodies against <i>Tc</i>SIR2RP1 and <i>Tc</i>SIR2RP3 and cytosolic and mitochondrial markers (mouse anti-TAT and MitoTracker, respectively). Cy3-conjugated anti-rabbit (red) and FITC-conjugated anti-mouse and anti-rabbit (green) were used as secondary antibodies, and DNA was counterstained with DAPI (blue). Scale bar: 5 μm. (B) Immunofluorescence assay of Dm28<i>c</i> expressing <i>Tc</i>SIR2RP1HA and <i>Tc</i>SIR2RP3HA (0.25 μg/ml Tetracycline) using rat anti-HA and cytosolic and mitochondrial markers (rabbit anti-TAT and anti-MDHm). Cy3-conjugated anti-rabbit (red) and FITC-conjugated anti-rat (green) were used as secondary antibodies, and DNA was counterstained with DAPI (blue). Scale bar: 5 μm. (C) Western blot analysis of the subcellular fractionation of the induced lines obtained by differential centrifugation. Equal amounts of each fraction were loaded on SDS-PAGE (left panel) followed by western blot with anti-TAT (cytosolic marker), anti-MDHm (mitochondrial marker), anti-BDF2 (nuclear marker) and anti-HA. N, nucleus; LG, large granules; SG, small granules; M, microsomes; S, final supernatant.</p