Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening

Singh, Nasib; Gupta, Reema; Jaiswal, Anil K.; Sundar, Shyam; Dube, Anuradha

doi:10.1093/jac/dkp206

Cited by 44 publications

(38 citation statements)

References 23 publications

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“…Although there are some reporters about the toxic effect of EGFP expression in some cells (such as muscle (Wallace et al 2013), Ku80-deficient hamster cells (Koike et al 2013), retina (Rex et al 2004), mouse fibroblast, hamster kidney cell and Huh-7 cells from hepatoma cancer (Liu et al 1999), adult stem cells from rat hepatic (Taghizadeh and Sherley 2008), but recombinant parasites encoding reporters as EGFP have received great attention and are routinely used (Breton et al 2005;Costa Sdos et al 2011;Kamau et al 2001;Mehta et al 2008;Singh et al 2009;Varela M et al 2009) without adverse effects on parasite.…”

Section: Discussionmentioning

confidence: 99%

EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

Seif

Kazemi

Gholami

et al. 2015

Appl Microbiol Biotechnol

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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. Disease progression was followed by footpad swelling measurements and parasite burden in draining lymph nodes using microtitration assay and real-time PCR, and immune responses were also evaluated in spleen. EGFP-expressing parasites generated larger swellings in comparison with wild-type (L. major) while mice immunized with rEGFP and challenged with wild-type parasite were quite comparable in footpad swelling with control group without significant difference. However, both conventional and molecular approaches revealed no significant difference in parasite load between different groups. More importantly, no significant inflammatory responses were detected in groups with higher swelling size measured by interferon-γ (IFN-γ), interleukin (IL)-10, IL-5, and nitric oxide against frozen and thawed lysate of parasite as stimulator. Altogether, these results clearly revealed that EGFP protein expressed in prokaryotic and eukaryotic hosts is not an immunological reactive molecule and acts as a neutral protein without any side effects in mice. So, EGFP expressing Leishmania could be a safe and reliable substitution for wild-types that simplifies in situ follow-up and eliminates the animal scarification wherever needed during the study.

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Section: Discussionmentioning

confidence: 99%

EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

Seif

Kazemi

Gholami

et al. 2015

Appl Microbiol Biotechnol

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“…This allows for shorter acquisition time values (200-300 ms for FPs versus 5-10 min for bioluminescence), which reduces the amount of background noise [8]. However, newly developed near-infrared emitting proteins avoid any background interference derived from organs or tissues (green-orange range autofluorescence) [9].…”

Section: Fluorescent Versus Luminescent Reportersmentioning

confidence: 99%

“…Furthermore, GFP-transfected Leishmania donovani parasites obtained from meglumine antimoniate-resistant and sensitive clinical isolates were used to infect J744A.1 macrophages. Intracellular amastigotes from resistant isolates showed sensitivity to miltefosine, pentamidine, paromomycin, and amphotericin B, but retained the resistance to antimonials after genetic manipulation [9]. More recently, a novel mCherry + Leishmania major transgenic strain has arisen as a suitable tool for massive drug testing on the intracellular form (Fig.…”

Section: Use Of Fluorescent Trypanosomatids In Htsmentioning

confidence: 99%

Trypanosomatids see the light: recent advances in bioimaging research

Calvo-Álvarez

Álvarez-Velilla

Fernández-Prada

et al. 2015

Drug Discovery Today

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“…Such parasites can be used to elucidate novel drug targets as well as vaccine candidates based on whether the gene under study is essential for both the promastigote and amastigote stages of the parasite or, only the amastigote stage. In addition, genetically modified organisms can also be used in metabolic pathways studies, structure-function relationship investigates (14), screening of new drugs (15), host–parasite interaction, and post-infection analysis among others, to enhance our understanding of these lower eukaryotes. Considering the success of LAV strategies against many viral, bacterial, and protozoan diseases (although to different extents), these are now considered the gold standard for protection against intra-cellular pathogens (12).…”

Section: Genetic Modification In Leishmania: Applications and Typesmentioning

confidence: 99%

Genetically Modified Organisms and Visceral Leishmaniasis

Chhajer

Ali

2014

Front. Immunol.

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Vaccination is the most effective method of preventing infectious diseases. Since the eradication of small pox in 1976, many other potentially life compromising if not threatening diseases have been dealt with subsequently. This event was a major leap not only in the scientific world already burdened with many diseases but also in the mindset of the common man who became more receptive to novel treatment options. Among the many protozoan diseases, the leishmaniases have emerged as one of the largest parasite killers of the world, second only to malaria. There are three types of leishmaniasis namely cutaneous (CL), mucocutaneous (ML), and visceral (VL), caused by a group of more than 20 species of Leishmania parasites. Visceral leishmaniasis, also known as kala-azar is the most severe form and almost fatal if untreated. Since the first attempts at leishmanization, we have killed parasite vaccines, subunit protein, or DNA vaccines, and now we have live recombinant carrier vaccines and live attenuated parasite vaccines under various stages of development. Although some research has shown promising results, many more potential genes need to be evaluated as live attenuated vaccine candidates. This mini-review attempts to summarize the success and failures of genetically modified organisms used in vaccination against some of major parasitic diseases for their application in leishmaniasis.

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Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening

Abstract: The GFP-transfectants were found to be suitable for FACS-based ex vivo screening assays. They were also infective to hamsters up to day 60 post-infection.

Cited by 44 publications

References 23 publications

EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

Trypanosomatids see the light: recent advances in bioimaging research

Genetically Modified Organisms and Visceral Leishmaniasis

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