Evaluation of NanoLuc, RedLuc and Luc2 as bioluminescent reporters in a cutaneous leishmaniasis model

New drugs for the treatment of human leishmaniasis are urgently needed, considering the limitations of current available options. However, pre-clinical evaluation of drug candidates for leishmaniasis is challenging. The use of luciferase-expressing parasites for parasite load detection is a potentially powerful tool to accelerate the drug discovery process. We have previously described the use of Leishmania amazonensis mutants expressing firefly luciferase (Luc2) for drug testing. Here, we describe three new mutant L. amazonensis lines that express different variants of luciferases: NanoLuc, NanoLuc-PEST and RedLuc. These mutants were evaluated in drug screening protocols. NanoLuc-parasites, in spite of high bioluminescence intensity in vitro, were shown to be inadequate in discriminating between live and dead parasites. Bioluminescence detection from intracellular amastigotes expressing NanoLuc-PEST, RedLuc or Luc2 proved more reliable than microscopy to determine parasite killing. Increased sensitivity was observed in vivo with RedLuc-expressing parasites as compared to NanoLuc-expressing L. amazonensis. Our data indicates that NanoLuc is not suitable for in vivo parasite burden determination. Additionally, RedLuc and the conventional luciferase Luc2 demonstrated equivalent sensitivity in an in vivo model of cutaneous leishmaniasis

First occurrence of an autochthonous canine case of Leishmania (Leishmania) infantum chagasi in the municipality of Campinas, State of São Paulo, Brazil

An autochthonous case of visceral leishmaniasis is reported in a dog (Canis familiaris) as an apparently natural infection in a non-endemic area. DNA obtained from spleen and liver samples produced the expected fragment in a Leishmania-specific rDNA-based nested-PCR assay. The PCR product, a 490 bp fragment, was sequenced and the nucleotide sequence was identical to that of Leishmania (Leishmania) infantum chagasi. These results are surprising since no autochthonous human or canine cases of visceral leishmaniasis have ever been reported in this municipality. This case suggests that natural transmission of this disease is occurring in this area

Leishmania: Overexpression and comparative structural analysis of the stage-regulated meta 1 gene

The meta I gene of Leishmania major is upregulated in metacyclic promastigotes and encodes an 11.5-kDa protein with no significant similarities to other proteins in the existing databases. in this paper, we characterize the homologous meta 1 genes in L. amazonensis and L. donovani. Proteins encoded by this gene in all three species present a high degree of identity. the meta 1 gene cannot be replaced by gene targeting in L. major, suggesting an essential role for the protein, at least in promastigotes. Overexpression of the meta 1 protein in L. amazonensis generates parasites that are more virulent than wild-type organisms in vivo. (C) 1999 Academic Press.Univ São Paulo, Inst Ciencias Biomed, Dept Parasitol, BR-05508900 São Paulo, BrazilUniv London Imperial Coll Sci Technol & Med, Dept Biochem, London SW7 2AZ, EnglandUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Microscopia Elect, São Paulo, BrazilCent Drug Res Inst, Div Biochem, Lucknow 226001, Uttar Pradesh, IndiaUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Microscopia Elect, São Paulo, BrazilWeb of Scienc

Leishmanicidal activity of primary S-nitrosothiols against Leishmania major and Leishmania amazonensis: Implications for the treatment of cutaneous leishmaniasis

Nitric oxide (NO) is considered a key molecule in the defense against intracellular pathogens, particularly Leishmania. The expression of inducible nitric oxide synthase and consequent production of NO by infected macrophages has been shown to correlate with leishmaniasis resistance in the murine model as well as in human patients. Nitric oxide donors have been used successfully in the treatment of cutaneous leishmaniasis in humans, although their mechanisms of action are not fully understood. In the present work, the dose-dependent cytotoxic effects of the NO-donors S-nitroso-N-acetyl-L-Cysteine (SNAC) and S-nitrosoglutathione (GSNO) against Leishmania were evaluated. GSNO inhibited the growth of Leishmania major and Leishmania amazonensis with in vitro 50% inhibitory concentrations (IC50) of 68.8 +/- 22.86 and 68.9 +/- 7.9 mu mol L-1, respectively. The IC50 for SNAC against L. major and L. amazonensis were, respectively, 54.6 +/- 8.3 and 181.6 +/- 12.5 mu mol L-1. The leishmanicidal activity of GSNO, but not of SNAC, was reversed by ascorbic acid (AA) and dithiothreitol (DTT), suggesting that the mechanism of action of GSNO is related to the transnitrosation of parasite proteins. These results demonstrate that SNAC and GSNO have leishmanicidal activity, and are thus potential therapeutic agents against cutaneous leishmaniasis. (c) 2006 Elsevier Inc. All rights reserved.15320921