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Luciferase leishmania

Estefania Calvo-Alvarez, Christelle Cren-Travaillé, Aline Crouzols, Brice Rotureau
Trypanosomiases and leishmaniases, caused by a group of related protist parasites, are Neglected Tropical Diseases currently threatening >500 million people worldwide. Reporter proteins have revolutionised the research on infectious diseases and have opened up new advances in the understanding of trypanosomatid-borne diseases in terms of both biology, pathogenesis and drug development. Here, we describe the generation and some applications of a new chimeric triple reporter fusion protein combining the red-shifted firefly luciferase PpyREH9 and the tdTomato red fluorescent protein, fused by the TY1 tag...
January 25, 2018: Infection, Genetics and Evolution
Betania Barros Cota, Luiza Guimarães Tunes, Daniela Nabak Bueno Maia, Jonas Pereira Ramos, Djalma Menezes de Oliveira, Markus Kohlhoff, Tânia Maria de Almeida Alves, Elaine Maria Souza-Fagundes, Fernanda Fraga Campos, Carlos Leomar Zani
BACKGROUND In a screen of extracts from plants and fungi to detect antileishmanial activity, we found that the ethyl acetate extract of the fungus Nectria pseudotrichia, isolated from the tree Caesalpinia echinata (Brazilwood), is a promising source of bioactive compounds. OBJECTIVES The aims of this study were to isolate and determine the chemical structures of the compounds responsible for the antileishmanial activity of the organic extract from N. pseudotrichia. METHODS Compounds were isolated by chromatographic fractionation using semi-preparative high-performance liquid chromatography, and their chemical structures were determined by analytical and spectral data and by comparison with published data...
February 2018: Memórias do Instituto Oswaldo Cruz
Pinar Avci, Mahdi Karimi, Magesh Sadasivam, Wanessa C Antunes-Melo, Elisa Carrasco, Michael R Hamblin
Traditional methods of localizing and quantifying the presence of pathogenic microorganisms in living experimental animal models of infections have mostly relied on sacrificing the animals, dissociating the tissue and counting the number of colony forming units. However, the discovery of several varieties of the light producing enzyme, luciferase, and the genetic engineering of bacteria, fungi, parasites and mice to make them emit light, either after administration of the luciferase substrate, or in the case of the bacterial lux operon without any exogenous substrate, has provided a new alternative...
January 1, 2018: Virulence
Tom Beneke, Ross Madden, Laura Makin, Jessica Valli, Jack Sunter, Eva Gluenz
Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit...
May 2017: Royal Society Open Science
Joana Tavares, David Mendes Costa, Ana Rafaela Teixeira, Anabela Cordeiro-da-Silva, Rogerio Amino
Hematogenous dissemination followed by tissue tropism is a characteristic of the infectious process of many pathogens including those transmitted by blood-feeding vectors. After entering into the blood circulation, these pathogens must arrest in the target organ before they infect a specific tissue. Here, we describe a non-invasive method to visualize and quantify the homing of pathogens to the host tissues. By using in vivo bioluminescence imaging we quantify the accumulation of luciferase-expressing parasites in the host organs during the first minutes following their intravascular inoculation in mice...
May 15, 2017: Methods: a Companion to Methods in Enzymology
Cristiana T Trinconi, Juliana Q Reimão, Vivian I Bonano, Caroline R Espada, Danilo C Miguel, Jenicer K U Yokoyama-Yasunaka, Silvia R B Uliana
The aims of the present work were to test the effect of tamoxifen administered topically and the therapeutic efficacy of tamoxifen and pentavalent antimonial combinations in an experimental model of cutaneous leishmaniasis. BALB/c mice infected with a luciferase expressing line of Leishmania amazonensis were treated with topical tamoxifen in two different formulations (ethanol or oil-free cream) as monotherapy or in co-administration with pentavalent antimonial. Treatment efficacy was evaluated by lesion size and parasite burden, quantified through luminescence, at the end of treatment and 4 weeks later...
March 9, 2017: Parasitology
Leticia Pérez-Díaz, Tais Caroline Silva, Santuza M R Teixeira
Amastins are surface glycoproteins, first identified in amastigotes of T. cruzi but later found to be expressed in several Leishmania species, as well as in T. cruzi epimastigotes. Amastins are encoded by a diverse gene family that can be grouped into four subfamilies named α, β, γ, and δ amastins. Differential expression of amastin genes results from regulatory mechanisms involving changes in mRNA stability and/or translational control. Although distinct regulatory elements were identified in the 3' UTR of T...
January 2017: Molecular and Biochemical Parasitology
Eline Rouault, Hervé Lecoeur, Asma Ben Meriem, Paola Minoprio, Sophie Goyard, Thierry Lang
Characterizing the clinical, immunological and parasitological features associated with visceral leishmaniasis is complex. It involves recording in real time and integrating quantitative multi-parametric data sets from parasite infected host tissues. Although several models have been used, hamsters are considered the bona fide experimental model for Leishmania donovani studies. To study visceral leishmaniasis in hamsters we generated virulent transgenic L. donovani that stably express a reporter luciferase protein...
February 2017: Parasitology International
Adriano C Coelho, Jordana C Oliveira, Caroline R Espada, Juliana Q Reimão, Cristiana T Trinconi, Silvia R B Uliana
BACKGROUND: Leishmania braziliensis is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying L. braziliensis pathogenesis or response to chemotherapy in vivo due to the natural resistance of most mouse strains to infection with these parasites. The aim of this work was to develop an experimental set up that could be used to assess drug efficacy against L. braziliensis. The model was tested using miltefosine...
May 2016: PLoS Neglected Tropical Diseases
Tahereh Taheri, Negar Seyed, Sima Rafati
Transfection technology is an important tool in the investigation of gene function and the modulation of gene expression, thereby contributing to the advancement of basic cellular research, drug discovery, and target validation. Creation of the mutant cells through gene disruption and exogenous protein expression with noticeable phenotype like reporter genes are among other key applications. In this chapter, protocols for generating recombinant Leishmania expressing EGFP or EGFP-Luciferase and their applications are given in detail...
2016: Methods in Molecular Biology
Cristiana T Trinconi, Juliana Q Reimão, Adriano C Coelho, Silvia R B Uliana
OBJECTIVES: The objective of this study was to characterize in vitro interactions and evaluate the antileishmanial activity of tamoxifen and miltefosine combinations. METHODS: Interactions between drugs were evaluated in vitro against Leishmania amazonensis promastigotes and intracellular amastigotes by a modified isobologram method. Four different drug ratios were used to calculate the FIC index (FICI) and the mean sum of FICI. Treatment of L. amazonensis-infected BALB/c mice was initiated 4 weeks post-infection...
May 2016: Journal of Antimicrobial Chemotherapy
Samira Seif, Fereshteh Kazemi, Elham Gholami, Negar Seyed, Yasaman Taslimi, Sima Habibzadeh, Bahareh Azarian, Shahram Jamshidi, Mehrdad Hashemi, Sima Rafati, Tahereh Taheri
Optical reporter genes such as green fluorescent protein (GFP) and luciferase are efficiently and widely used in monitoring and studying the protective/therapeutic potential of candidate agents in leishmaniasis. But several observations and controversial reports have generated a main concern, whether enhanced GFP (EGFP) affects immune response. To address this issue, we studied the immunogenicity of EGFP in vivo by two lines of stably transfected parasites (Leishmania major (EGFP) or L. major (EGFP-LUC)) in BALB/c model and/or as a recombinant protein (rEGFP) produced in vitro by bacteria in parallel...
May 2016: Applied Microbiology and Biotechnology
Mozna Khraiwesh, Susan Leed, Norma Roncal, Jacob Johnson, Richard Sciotti, Philip Smith, Lisa Read, Robert Paris, Thomas Hudson, Mark Hickman, Max Grogl
Leishmaniasis is a complex tropical disease caused by kinetoplastid parasitic protozoa of the genus Leishmania and is transmitted by the sand fly insect vector. Cutaneous leishmaniasis (CL) is the most common form of this disease, and CL infections often result in serious skin lesions and scars. CL remains a public health problem in many endemic countries worldwide because of the absence of effective, safe, and cost-effective drugs for treatment. One of the strategies we chose to use to find novel chemical entities worthy of further development as antileishmanials involved screening synthetic and natural products libraries...
February 2016: American Journal of Tropical Medicine and Hygiene
Somayeh Sadeghi, Negar Seyed, Mohammad-Hossein Etemadzadeh, Saeid Abediankenari, Sima Rafati, Tahereh Taheri
Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain...
August 2015: Korean Journal of Parasitology
Raquel García-Hernández, Verónica Gómez-Pérez, Santiago Castanys, Francisco Gamarro
Drug resistance represents one of the main problems for the use of chemotherapy to treat leishmaniasis. Additionally, it could provide some advantages to Leishmania parasites, such as a higher capacity to survive in stress conditions. In this work, in mixed populations of Leishmania donovani parasites, we have analyzed whether experimentally resistant lines to one or two combined anti-leishmanial drugs better support the stress conditions than a susceptible line expressing luciferase (Luc line). In the absence of stress, none of the Leishmania lines showed growth advantage relative to the other when mixed at a 1:1 parasite ratio...
April 2015: PLoS Neglected Tropical Diseases
Juliana Q Reimão, Jordana C Oliveira, Cristiana T Trinconi, Paulo C Cotrim, Adriano C Coelho, Silvia R B Uliana
BACKGROUND: The only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease...
February 2015: PLoS Neglected Tropical Diseases
Filipa Teixeira, Helena Castro, Tânia Cruz, Eric Tse, Philipp Koldewey, Daniel R Southworth, Ana M Tomás, Ursula Jakob
Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown...
February 17, 2015: Proceedings of the National Academy of Sciences of the United States of America
Tahereh Taheri, Hana Saberi Nik, Negar Seyed, Fatemeh Doustdari, Mohammad-Hossein Etemadzadeh, Fatemeh Torkashvand, Sima Rafati
Because of the lack of an accurate and sensitive tool to evaluate the parasitemia level, treatment or prevention of leishmaniasis remains an important challenge worldwide. To monitor and track leishmanial infection by two parameters in real time, we generated stably transgenic Leishmania that express a bi-reporter protein as fused EGFP and firefly luciferase. Using two reporter genes (egfp-luc) simultaneously increases the experimental sensitivity for detection/diagnosis, and in vitro quantification of parasites as well as real-time infection in mice...
March 2015: Experimental Parasitology
Sushmita Das, Ayan Kumar Ghosh, Shikha Singh, Bhaskar Saha, Ashish Ganguly, Pradeep Das
Regulation of macrophage PCD plays an important role in pathogenesis of leishmaniasis. However, the precise involvement of any parasite molecule in this process remains uncertain. In the current study, in silico wide analysis demonstrated that genes in the Leishmania donovani genome are highly enriched for CpG motifs, with sequence frequency of 8.7%. Here, we show that unmethylated species-specific CpG motifs in LdDNA significantly (P = 0.01) delay macrophage PCD by endosomal interaction with TLR9 via the adaptor protein MyD88...
February 2015: Journal of Leukocyte Biology
Suman Gupta, Vanessa Yardley, Preeti Vishwakarma, Rahul Shivahare, Bhawna Sharma, Delphine Launay, Denis Martin, Sunil K Puri
OBJECTIVES: The objective of this study was to identify a nitroimidazo-oxazole lead molecule for the treatment of visceral leishmaniasis (VL). METHODS: A library of 72 nitroimidazo-oxazoles was evaluated in vitro for their antileishmanial activity against luciferase-transfected DD8 amastigotes of Leishmania donovani. On the basis of their in vitro potency and pharmacokinetic properties, the promising compounds were tested in acute BALB/c mouse and chronic hamster models of VL via oral administration and efficacy was evaluated by microscopic counting of amastigotes after Giemsa staining...
February 2015: Journal of Antimicrobial Chemotherapy
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