Generation and Characterization of a Dual-Reporter Transgenic Leishmania braziliensis Line Expressing eGFP and Luciferase

Abstract

In this study, we generated a transgenic strain of Leishmania braziliensis, an etiological agent associated with a diversity of clinical manifestations of leishmaniasis ranging from localized cutaneous to mucocutaneous to disseminated disease. Transgenic parasites expressing reporter proteins are valuable tools for studies of parasite biology, host-pathogen interactions, and anti-parasitic drug development. To this end, we constructed an L. braziliensis line stably expressing the reporters eGFP and luciferase (eGFP-LUC L. braziliensis). The integration cassette co-expressing the two reporters was targeted to the ribosomal locus (SSU) of the parasite genome. Transgenic parasites were characterized for their infectivity and stability both in vitro and in vivo. Parasite maintenance in axenic long-term culture in the absence of selective drugs did not alter expression of the two reporters or infection of BALB/c mice, indicating stability of the integrated cassette. Infectivity of eGFP-LUC, L. braziliensis, both in vivo and in vitro was similar to that obtained with the parental wild type strain. The possibility of L. braziliensis tracking and quantification using fluorescence and luminescence broadens the scope of research involving this neglected species, despite its importance in terms of public health concerning the leishmaniasis burden.

Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23

“…Studies reporting on the use of homologous recombination in L. braziliensis demonstrate the generation of stable transgenic parasite lines from integration of DNA constructs into the SSU rDNA genomic locus. These include L. braziliensis lines expressing reporter genes, e.g., luciferase or eGFP, which hold potential for parasite tracking and monitoring effects of antileishmanial compounds in vitro and in vivo [ 67 , 68 , 69 ], and over expressing parasite lines for the analysis of gene products, e.g., to assess antimony susceptibility and resistance mechanisms [ 70 , 71 , 72 ]. Moreover, circular extrachromosomal cosmids can be stably introduced into L. braziliensis to over-express stretches of genomic DNA and connect the over expression phenotypes to biological processes such as virulence [ 73 ] and antimony resistance [ 59 ].…”

Challenges and Tools for In Vitro Leishmania Exploratory Screening in the Drug Development Process: An Updated Review

“…Nevertheless, in the field of cell viability analysis, flow cytometry constitutes an interesting alternative [75,76], which is accurate and largely used to automate the reading of results. Another approach uses the detection and quantification of engineered cells expressing fluorescent gene reporters [77,78] such as green fluorescent protein (GFP) [32,[79][80][81] and bioluminescent gene reporters such as luciferase [82][83][84][85][86], or a combination of both [87][88][89][90]. These methods are proven to be more sensitive and enable faster read-outs and higher throughput [91].…”

<p>Evaluation of in vitro and in vivo Efficacy of a Novel Amphotericin B-Loaded Nanostructured Lipid Carrier in the Treatment of <em>Leishmania braziliensis</em> Infection</p>

“…modified to express green fluorescent protein (GFP) were cultured at 24°C in Schneider's Insect medium supplemented with 10% FBS, 100 U/mL penicillinstreptomycin-glutamine and 50μg/mL of Geneticin ® antibiotic (G418). 25 Leishmania braziliensis GFP was exclusively employed in uptake assessment assays.…”