This study focuses on the role of the population structure of Leishmania spp. on the adaptive capacity of the parasite. Herein, we investigate the contribution of subpopulations of the L. (V.) braziliensis Thor strain (Thor03, Thor10 and Thor22) in the profile of murine macrophages infection. Infection assays were performed with binary combinations of these subpopulations at stationary phases. The initial interaction time showed major effects on the combination assays, as demonstrated by the significant increase in the infection rate at 5 h. Based on the endocytic index (EI), Thor10 (EI = 563.6) and Thor03 (EI = 497) showed a higher infection load compared to Thor22 (EI = 227.3). However, the EI decreased in Thor03 after 48 h (EI = 447) and 72 h (EI = 388.3) of infection, and showed changes in the infection level in all Thor10/Thor22 combinations. Assays with CellTrace CFSE-labelled Thor22 promastigotes indicated an increase (~1.5 fold) in infection by this subpopulation in the presence of Thor10 when compared to the infection profile of Thor03/Thor22 combinations in the same proportions. In addition, the potential of these subpopulations, alone or in binary combinations, to modulate the expression of cytokines and nitric oxide (NO) in vitro was investigated. Lower NO and tumour necrosis factor-α production levels were observed for all Thor10/Thor22 combinations at 24 h compared to these subpopulations alone. In contrast, Thor03/Thor22 combination assays increased IL-10 production at this time. Collectively, these results provide in vitro evidence on the potential of L. (V.) braziliensis population structure to play a relevant role in a host infection by this parasite.

The lack of an adequate model for Leishmania (Viannia) braziliensis infection is a limiting factor for studying American tegumentary leishmaniasis (ATL). The golden hamster (Mesocricetus auratus) is a promising model because besides being highly susceptible to dermotropic Leishmania infection, the lesions are very similar to cutaneous leishmaniasis (CL) in humans. However, different Leishmania isolates or species and/or protocols have resulted in different outcomes, whereas no study has evaluated the reproducibility of L. braziliensis infection in this model. The natural history of L. braziliensis infection in 34 hamsters was evaluated by using a single parasite isolate in 8 independent experiments under similar experimental conditions. Clinical, histological and immunological analyses were performed. The hamsters presented skin ulcers similar to those observed in ATL. The intra-experiment lesion increment tended to show an intermediary variance. Histological analysis of infected skins showed granulomatous reaction, scarce amastigotes, and Schaumann's bodies. Blood lymphocytes proliferated in response to leishmanial antigens. The severity of the infection was positively correlated to spleen weight, and the titres of anti-Leishmania IgG antibodies. Our findings indicate that the hamster is an appropriate model for immunopathogenesis studies of CL caused by L. braziliensis, supporting its use in clinical, vaccine and chemotherapy experimental protocols.

Current treatments for different clinical forms of leishmaniasis are unsatisfactory, highly toxic and associated with increasing failure rates resulting from the emergence of resistant parasites. Leishmania (Viannia) braziliensis is the main aetiological agent of different clinical forms of American tegumentary leishmaniasis, including the mucosal form for which treatment has high failure rates. The aim of this work was to investigate the activity of the Morita-Baylis-Hillman adduct, methyl 2-{2-[hydroxy(2-nitrophenyl)methyl])acryloyloxy} benzoate in vitro against isolates of L. (V.) braziliensis obtained from patients with different clinical manifestations of tegumentary leishmaniasis: localized cutaneous leishmaniasis, mucosal leishmaniasis and disseminated cutaneous leishmaniasis. The adduct effectively inhibited the growth of promastigotes of the different isolates of L. (V.) braziliensis (IC50 ⩽ 7·77 μg/ml), as well as reduced the infection rate of macrophages infected with these parasites (EC50 ⩽ 1·37 μg/ml). It is remarkable to state that the adduct was more effective against intracellular amastigotes (P ⩽ 0·0045). The anti-amastigote activity correlated with an immunomodulatory effect, since the adduct was able to decrease the production of IL-6 and IL-10 by the infected macrophages. However, its effect was independent of nitric oxide production. This work demonstrates the anti-leishmanial activity of methyl 2-{2-[hydroxy(2-nitrophenyl)methyl])acryloyloxy} benzoate and suggests its potential in the treatment of human infections caused by L. (V.) braziliensis.

Leishmaniasis is a vector-borne disease and an important public health issue. Glycosaminoglycan ligands in Leishmania parasites are potential targets for new strategies to control this disease. We report the subcellular distribution of heparin-binding proteins (HBPs) in Leishmania (Viannia) braziliensis and specific biochemical characteristics of L. (V.) braziliensis HBPs. Promastigotes were fractionated, and flagella and membrane samples were applied to HiTrap Heparin affinity chromatography columns. Heparin-bound fractions from flagella and membrane samples were designated HBP Ff and HBP Mf, respectively. Fraction HBP Ff presented a higher concentration of HBPs relative to HBP Mf, and SDS-PAGE analyses showed 2 major protein bands in both fractions (65 and 55 kDa). The 65 kDa band showed gelatinolytic activity and was sensitive to inhibition by 1,10-phenanthroline. The localization of HBPs on the promastigote surfaces was confirmed using surface plasmon resonance (SPR) biosensor analysis by binding the parasites to a heparin-coated sensor chip; that was inhibited in a dose-dependent manner by pre-incubating the parasites with variable concentrations of heparin, thus indicating distinct heparin-binding capacities for the two fractions. In conclusion, protein fractions isolated from either the flagella or membranes of L. (V.) braziliensis promastigotes have characteristics of metallo-proteinases and are able to bind to glycosaminoglycans.

Introduction. Evaluation of Leishmania drug susceptibility depends on in vitro SbV susceptibility assays, which are labour-intensive and may give a biased view of the true parasite resistance. Molecular markers are urgently needed to improve and simplify the monitoring of SbV-resistance. We analysed here the gene expression profile of 21 L. braziliensis clinical isolates in vitro defined as SbV-resistant and -sensitive, in order to identify potential resistance markers. Methods. The differential expression of 13 genes involved in SbV metabolism, oxidative stress or housekeeping functions was analysed during in vitro promastigote growth. Results. Expression profiles were up-regulated for 5 genes only, each time affecting a different set of isolates (mosaic picture of gene expression). Two genes, ODC (ornithine decarboxylase) and TRYR (trypanothione reductase), showed a significantly higher expression rate in the group of SbV-resistant compared to the group of SbV-sensitive parasites (P<0·01). However, analysis of individual isolates showed both markers to explain only partially the drug resistance. Discussion. Our results might be explained by (i) the occurrence of a pleiotropic molecular mechanism leading to the in vitro SbV resistance and/or (ii) the existence of different epi-phenotypes not revealed by the in vitro SbV susceptibility assays, but interfering with the gene expression patterns.

An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response.

Gene expression is known to vary significantly during the Leishmania life-cycle. Its monitoring might allow identification of molecular changes associated with the infective stages (metacyclics and amastigotes) and contribute to the understanding of the complex host-parasite relationships. So far, very few studies have been done on Leishmania (Viannia) braziliensis, one of the most pathogenic species. Such studies require, first of all, reference molecular markers. In the present work, we applied differential display analysis (DD analysis) in order to identify transcripts that might be (i) candidate markers of metacyclics and intracellular amastigotes of L. (V.) braziliensis or (ii) potential controls, i.e. constitutively expressed. In total, 48 DNA fragments gave reliable sequencing data, 29 of them being potential markers of infective stages and 12 potential controls. Eight sequences could be identified with reported genes. Validation of the results of DD analysis was done for 4 genes (2 differentially expressed and 2 controls) by quantitative real-time PCR. The infective insect stage-specific protein (meta 1) was more expressed in metacyclic-enriched preparations. The oligopeptidase b showed a higher expression in amastigotes. Two genes, glucose-6-phosphate dehydrogenase and a serine/threonine protein kinase, were found to be similarly expressed in the different biological samples.

Proteolytic activities of 5 strains of Leishmania (Viannia) braziliensis isolated from Brazilian and Colombian patients, presenting distinct clinical manifestations, were characterized and compared using whole-promastigote extracts and extracellular secretions. Zymographic assays concerning whole-cell extracts and supernatants resulted in the detection of high molecular weight bands, ranging from 50 to 125 kDa. Proteolytic activities from both whole-cell extracts and supernatants were optimal in a pH range 5·5 to 9·0 for all analysed strains. Such protease activities were inhibited when 10 mM 1,10-phenanthroline was assayed, strongly suggesting that the enzymes responsible for hydrolysis of the substrate belong to the metalloproteases class. Distinct profiles of metalloproteases were observed among the studied L. (V.) braziliensis strains. Differences among the microorganisms might be related to the geographical origin of the strains and/or to the clinical presentation.

Specific glycolipids (GLs) from Leishmania (Viannia) braziliensis promastigotes were isolated and purified. A monoclonal antibody directed to carbohydrate epitopes of these GLs was produced. mAb SST-1 recognizes a low molecular weight GL as established by solid-phase radioimmunoassay and HPTLC immunostaining, and does not cross-react with lipophosphoglycan isolated from L. (V.) braziliensis promastigotes. An indirect immunofluorescence study indicated that the antigenic GLs are present at the L. (V.) braziliensis promastigote surface. SST-1 reacted with promastigotes of L. (V.) naiffi and L. (V.) guyanensis, but not with species in the L. Leishmania subgenus i.e. L. (L.) amazonensis, L. (L.) chagasi, or L. (L.) major. All L. (V.) braziliensis serodemes tested were reactive with SST-1. These results indicate that SST-1 recognizes specific GLs expressed by species of the Viannia subgenus, and will be particularly useful for identification of L. (V.) braziliensis.

The major surface protease (msp or gp63) of Leishmania plays a major role in the host–parasite interaction. We analysed here the structure of the msp gene locus in Leishmania (Viannia) braziliensis and compared it to results obtained in other species. Physical mapping of cosmid contigs revealed a minimum of 37 genes per haploid genome and at least 8 different msp gene families. Within the same organism, these genes showed a nucleotide sequence varying in certain stretches from 3 to 34%, and a mosaic structure. From an evolutionary point of view, major differences were observed between subgenera Viannia and Leishmania, both in terms of msp gene number and sequence. Within subgenus Viannia, phenetic analysis revealed three clusters in which sequence variants of L. (Viannia) braziliensis and L. (Viannia) guyanensis were interspersed. Functional implications of our results were explored from predicted L. (Viannia) braziliensis protein sequences: regions encoding the msp catalytic site showed a conserved sequence, while regions encoding surface domains possibly involved in the host–parasite interaction (macrophage adhesion sites and immunodominant B-cell and T-cell epitopes) were variable. We speculate that this would be an adaptive strategy of the parasite.

The immunolocalization of Leishmania (Viannia) braziliensis stage-specific antigens recognized by mAbs was analysed by transmission electron microscopy. The antigen recognized by mAb SST-2 was present at the surface of promastigotes, including the flagellum and flagellar pocket. The reactivity of SST-2 with isolates of different serodemes showed a pronounced microheterogeneity in terms of the number of reactive bands within the low molecular weight range from 24 to 33 kDa. The 180 kDa glycoprotein recognized by mAb SST-3 was present only in the flagellar membrane. SST-3 also recognized multiple discrete bands from 160 to 200 kDa, as observed in several serodemes. In contrast, mAb SST-4, which recognizes a 98 kDa antigen, showed weak labelling on the promastigote surface by transmission electron microscopy and indirect immunofluorescence. Based on Western blotting, indirect immunofluorescence, and solid-phase radio-immunoassay, the antigens recognized by mAbs SST-2, SST-3 and SST-4 were present in all L. (V.) braziliensis analysed, from 7 different serodemes.

The present studies on infections with Leishmania (Viannia) braziliensis in rhesus macaques were made to characterize the evolution of different parasite strains and the immune responses they elicited in this experimental host. A standardized inoculum of promastigotes was injected intradermally either above the eyelid or on the forearm of each monkey. Sixteen infected monkeys developed longstanding infections which lasted until the end of the observation period (33 months). The time required for lesion development was very variable, not only for the isolates showing molecular differences but also for individual animals in groups infected with the same parasite strain. The inocula produced lesions of variable severity, ranging from localized cutaneous leishmaniasis (CL) with a tendency to spontaneous healing to non-healing disease. One infected animal developed persistent metastatic skin and mucosal lesions. Anti-Leishmania antibodies and parasite-specific T-cell responses were induced by the experimental infections. As the granulomatous inflammatory response found at the lesions in L. (V.) braziliensis-infected M. mulatta was similar to that in patients with CL, this primate model could be useful for studying the pathophysiology and immunoregulatory events associated with disease evolution, as well as for the evaluation of new drugs or candidate vaccines.