Examinando por Autor "Cribb, Pamela"

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    Construction of three new Gateway® expression plasmids for Trypanosoma cruzi
    (Instituto Oswaldo Cruz, 2014-12) Alonso, Victoria Lucía; Ritagliati, Carla; Cribb, Pamela; Serra, Esteban Carlos
    We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).
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    In vitro drug screening against all life cycle stages of Trypanosoma cruzi using parasites expressing β-galactosidase
    (MyJove Corporation, 2021-11) Alonso, Victoria Lucía; Manarin, Romina; Perdomo, Virginia Gabriela; Gulin, Julián Ernesto Nicolás; Serra, Esteban Carlos; Cribb, Pamela
    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.
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    Light modulates important pathogenic determinants and virulence in ESKAPE pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus
    (American Society for Microbiology, 2021-02-08) Tuttobene, Marisel Romina; Pérez, J. F.; Pavesi, Estefanía S.; Pérez Mora, Bárbara; Biancotti, Daiana; Cribb, Pamela; Altilio, Matías; Müller, Gabriela Leticia; Gramajo, Hugo Cesar; Tamagno, G.; Ramírez, María Soledad; Diacovich, Lautaro; Mussi, María Alejandra; https://orcid.org/0000-0002-9904-7890; https://orcid.org/0000-0002-3339-0100; https://orcid.org/0000-0002-4168-3624; Rabinovich, Gabriel: provide HaCaT cells; Voyich, Jovanka: provide the hla and complemented hla mutant
    Light sensing has been extensively characterized in the human pathogen Acinetobacter baumannii at environmental temperatures. However, the influence of light on the physiology and pathogenicity of human bacterial pathogens at temperatures found in warm-blooded hosts is still poorly understand. In this work, we show that Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa (ESKAPE) priority pathogens, which have been recognized by the WHO and the CDC as critical, can also sense and respond to light at temperatures found in human hosts. Most interestingly, in these pathogens, light modulates important pathogenicity determinants as well as virulence in an epithelial infection model, which could have implications in human infections. In fact, we found that alpha-toxin-dependent hemolysis, motility, and growth under iron-deprived conditions are modulated by light in S. aureus. Light also regulates persistence, metabolism, and the ability to kill competitors in some of these microorganisms. Finally, light exerts a profound effect on the virulence of these pathogens in an epithelial infection model, although the response is not the same in the different species; virulence was enhanced by light in A. baumannii and S. aureus, while in A. nosocomialis and P. aeruginosa it was reduced. Neither the BlsA photoreceptor nor the type VI secretion system (T6SS) is involved in virulence modulation by light in A. baumannii. Overall, this fundamental knowledge highlights the potential use of light to control pathogen virulence, either directly or by manipulating the light regulatory switch toward the lowest virulence/persistence configuration. IMPORTANCE: Pathogenic bacteria are microorganisms capable of producing disease. Dangerous bacterial pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, are responsible for serious intrahospital and community infections in humans. Therapeutics is often complicated due to resistance to multiple antibiotics, rendering them ineffective. In this work, we show that these pathogens sense natural light and respond to it by modulating aspects related to their ability to cause disease; in the presence of light, some of them become more aggressive, while others show an opposite response. Overall, we provide new understanding on the behavior of these pathogens, which could contribute to the control of infections caused by them. Since the response is distributed in diverse pathogens, this notion could prove a general concept.
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    Microtubules regulate brush border formation
    (Wiley, 2017-11-06) Tonucci, Facundo Mauro; Ferretti, Anabela Cecilia; Almada, Evangelina; Cribb, Pamela; Vena, Rodrigo; Hidalgo, Florencia; Favre, Cristian; Tyska, Matt J.; Kaverina, Irina; Larocca, María Cecilia; https://orcid.org/0000-0002-6967-5431
    Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity.
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    Optimization and biological validation of an in vitro assay using the transfected Dm28c/pLacZ Trypanosoma cruzi strain
    (Oxford University Press, 2021-07-13) Gulin, Julián Ernesto Nicolás; Rocco, Daniela Marisa; Alonso, Victoria Lucía; Cribb, Pamela; Altcheh, Jaime; García-Bournissen, Facundo
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    Overexpression of bromodomain factor 3 in Trypanosoma cruzi (TcBDF3) affects differentiation of the parasite and protects it against bromodomain inhibitors
    (Wiley, 2016-06-06) Alonso, Victoria Lucía; Ritagliati, Carla; Cribb, Pamela; Cricco, Julia Alejandra; Serra, Esteban Carlos; Glaxo Smith Kline (GSK): provide I-BET151
    The bromodomain is the only protein domain known to bind acetylated lysine. In the last few years many bromodomain inhibitors have been developed in order to treat diseases such as cancer caused by aberrant acetylation of lysine residues. We have previously characterized Trypanosoma cruzi bromodomain factor 3 (TcBDF3), a bromodomain with an atypical localization that binds acetylated α-tubulin. In the present work we show that parasites overexpressing TcBDF3 exhibit altered differentiation patterns and are less susceptible to treatment with bromodomain inhibitors. We also demonstrate that recombinant TcBDF3 is able to bind to these inhibitors in vitro in a concentration-dependant manner. In parallel, the overexpression of a mutated version of TcBDF3 negatively affects growth of epimastigotes. Recent results, including the ones presented here, suggest that bromodomain inhibitors can be conceived as a new type of anti-parasitic drug against trypanosomiasis.
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    Overexpression of cytoplasmic TcSIR2RP1 and mitochondrial TcSIR2RP3 impacts on Trypanosoma cruzi growth and cell invasion
    (Public Library of Science (PLOS), 2015-04-15) Ritagliati, Carla; Alonso, Victoria Lucía; Manarin, Romina; Cribb, Pamela; Serra, Esteban Carlos
    Abstract 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.
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    Overexpression of Trypanosoma cruzi high mobility group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity
    (Springer Nature, 2019-01-17) Tavernelli, Luis Emilio; Motta, María Cristina M.; Silva Gonçalves, Camila; Santos da Silva, Marcelo; Elías, María Carolina; Alonso, Victoria Lucía; Serra, Esteban Carlos; Cribb, Pamela; Dr. De Gaudenzi, Javier: provide the pTcINDEX-eGFP strain
    Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.
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    Quorum and light signals modulate acetoin/butanediol catabolism in Acinetobacter spp
    (Frontiers Media, 2019-06-20) Tuttobene, Marisel Romina; Fernández-García, Laura; Blasco, Lucía; Cribb, Pamela; Ambroa, Anton; Müller, Gabriela Leticia; Fernández-Cuenca, Felipe; Bleriot, Inés; Rodríguez, Ramiro Esteban; Barbosa, Beatriz G. V.; Lopez-Rojas, Rafael; Trastoy, Rocío; López, María; Bou, Germán; Tomás, María; Mussi, María Alejandra
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    Trypanosoma cruzi High Mobility Group B (TcHMGB) can act as an inflammatory mediator on mammalian cells
    (Public Library of Science (PLOS), 2017-02-08) Cribb, Pamela; Perdomo, Virginia Gabriela; Alonso, Victoria Lucía; Manarin, Romina; Barrios-Payán, Jorge; Marquina-Castillo, Brenda; Tavernelli, Luis Emilio; Hernández-Pando, Rogelio