Construction of three new Gateway® expression plasmids for Trypanosoma cruzi

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).Fil: 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; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Microbiología; ArgentinaFil: 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: Cribb, Pamela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Microbiología; Argentina. 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; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

BlsA integrates light and temperature signals into iron metabolism through fur in the human pathogen Acinetobacter baumannii

Light modulates global features of the important human pathogen Acinetobacter baumannii lifestyle including metabolism, tolerance to antibiotics and virulence, most of which depend on the short BLUF-type photoreceptor BlsA. In this work, we show that the ability to circumvent iron deficiency is also modulated by light at moderate temperatures, and disclose the mechanism of signal transduction by showing that BlsA antagonizes the functioning of the ferric uptake regulator (Fur) in a temperature-dependent manner. In fact, we show that BlsA interacts with Fur in the dark at 23 °C, while the interaction is significantly weakened under blue light. Moreover, under iron deprived conditions, expression of Fur-regulated Acinetobactin siderophore genes is only induced in the dark in a BlsA-dependent manner. Finally, growth under iron deficiency is supported in the dark rather than under blue light at moderate temperatures through BlsA. The data is consistent with a model in which BlsA might sequester the repressor from the corresponding operator-promoters, allowing Acinetobactin gene expression. The photoregulation of iron metabolism is lost at higher temperatures such as 30 °C, consistent with fading of the BlsA-Fur interaction at this condition. Overall, we provide new understanding on the functioning of the widespread Fur regulator as well as short-BLUFs.Fil: Tuttobene, Marisel R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; 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: Mussi, María Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentin

SHORT COMMUNICATION - One-tube Nested Polymerase Chain Reaction for Detection of Chlamydia trachomatis

Here we present a one-tube nested PCR test, which allows the detection of minimal quantities of Chlamydia trachomatis   in human fluids. This assay includes the use of an internal control to avoid false negative results due to the presence of inhibitors. The results obtained show that this assay is robust enough to be used for clinical diagnosis

Optimization and biological validation of an in vitro assay using the transfected Dm28c/pLacZ Trypanosoma cruzi strain

There is an urgent need to develop safer and more effective drugs for Chagas disease, as the current treatment relies on benznidazole (BZ) and nifurtimox (NFX). Using the Trypanosoma cruzi Dm28c strain genetically engineered to express the Escherichia coli b-galactosidase gene, lacZ, we have adapted and validated an easy, quick and reliable in vitro assay suitable for high-throughput screening for candidate compounds with anti-T. cruzi activity. In vitro studies were conducted to determine trypomastigotes sensitivity to BZ and NFX from Dm28c/pLacZ strain by comparing the conventional labour-intensive microscopy counting method with the colourimetric assay. Drug concentrations producing the lysis of 50% of trypomastigotes (lytic concentration 50%) were 41.36 and 17.99 mM for BZ and NFX, respectively, when measured by microscopy and 44.74 and 38.94 mM, for the colourimetric method, respectively. The optimal conditions for the amastigote development inhibitory assay were established considering the parasite–host relationship (i.e. multiplicity of infection) and interaction time, the time for colourimetric readout and the incubation time with the b-galactosidase substrate. The drug concentrations resulting in 50% amastigote development inhibition obtained with the colourimetric assay were 2.31 mM for BZ and 0.97 mM for NFX, similar to the reported values for the Dm28c wild strain (2.80 and 1.5 mM, respectively). In summary, a colourimetric assay using the Dm28c/pLacZ strain of T. cruzi has been set up, obtaining biologically meaningful sensibility values with the reference compounds on both trypomastigotes and amastigotes forms. This development could be applied to high-throughput screening programmes aiming to identify compounds with anti-T. cruzi in vitro activity.Fil: Gulin, Julián Ernesto Nicolás. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP-CONICET); Argentina.Fil: Gulin, Julián Ernesto Nicolás. Hospital de Niños “Dr. Ricardo Gutiérrez”. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Gulin, Julián Ernesto Nicolás. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas (INBIOMED-CONICET); Argentina.Fil: Rocco, Daniela Marisa. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP-CONICET); Argentina.Fil: Rocco, Daniela Marisa. Hospital de Niños “Dr. Ricardo Gutiérrez”. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: Alonso, Victoria Lucía. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Cribb, Pamela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Altcheh, Jaime. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP-CONICET); Argentina.Fil: Altcheh, Jaime. Hospital de Niños “Dr. Ricardo Gutiérrez”. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: García-Bournissen, Facundo. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas (IMIPP-CONICET); Argentina.Fil: García-Bournissen, Facundo. Hospital de Niños “Dr. Ricardo Gutiérrez”. Servicio de Parasitología y Enfermedad de Chagas; Argentina.Fil: García-Bournissen, Facundo. University of Western Ontario. Schulich School of Medicine & Dentistry. Department of Paediatrics. Division of Paediatric Clinical Pharmacology; Canada