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.
Highlights
Leishmaniasis is a neglected tropical disease caused by the protozoan pathogen Leishmania spp. that is transmitted by sand flies
Sequencing of the ITS-1 amplicon demonstrated high identity (97%) of eGFPLUC and Wild type (WT) L. braziliensis (Lb) (Genebank Id- FN398335.1; MHOM/BR/2002/NMT-RBO018) (Supplementary Figure 2). These results demonstrate the successful integration of this dual-reporter system containing eGFP and Luc into the Lb genome
In spite of the status of L. braziliensis as a neglected species, and considering that this species is largely responsible for most cases of cutaneous leishmaniasis (CL), we sought to generate a transgenic dual-reporter line co-expressing eGFP and luciferase from a clinical isolate
Summary
Leishmaniasis is a neglected tropical disease caused by the protozoan pathogen Leishmania spp. that is transmitted by sand flies. The two most common forms of leishmaniasis are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). In Brazil, Leishmania braziliensis is the leading cause of CL, which mostly manifests as localized lesions on the skin, but may metastasize to mucosal sites (Bittencourt and Netto, 1995). Another common manifestation is disseminated leishmaniasis (Carvalho et al, 1994), which is characterized by the presence of large numbers of papular and acneiform ulcers affecting different parts of the body. In contrast to American Cutaneous Leishmaniasis, disseminated leishmaniasis is associated with impaired
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