Leishmania (Sauroleishmania) tarentolae versus pathogenic species: comparative evaluation of protease activity, glycoconjugates, resistance to complement and metabolome composition.

BACKGROUND: Leishmania tarentolae is a non-pathogenic species found in lizards representing an important model for Leishmania biology. However, several aspects of this Sauroleishmania remain unknown to explain its low level of virulence. OBJECTIVES: We reported several aspects of L. tarentolae biology including glycoconjugates, proteolytic activities and metabolome composition in comparison to pathogenic species (Leishmania amazonensis, Leishmania braziliensis, Leishmania infantum and Leishmania major). METHODS: Parasites were cultured for extraction and purification of lipophosphoglycan (LPG), immunofluorescence probing with anti-gp63 and resistance against complement. Parasite extracts were also tested for proteases activity and metabolome composition. FINDINGS: Leishmania tarentolae does not express LPG on its surface. It expresses gp63 at lower levels compared to pathogenic species and, is highly sensitive to complement-mediated lysis. This species also lacks intracellular/extracellular activities of proteolytic enzymes. It has metabolic differences with pathogenic species, exhibiting a lower abundance of metabolites including ABC transporters, biosynthesis of unsaturated fatty acids and steroids, TCA cycle, glycine/serine/threonine metabolism, glyoxylate/dicarboxylate metabolism and pentose-phosphate pathways. MAIN CONCLUSIONS: The non-pathogenic phenotype of L. tarentolae is associated with alterations in several biochemical and molecular features. This reinforces the need of comparative studies between pathogenic and non-pathogenic species to elucidate the molecular mechanisms of virulence during host-parasite interactions.

Leishmania (Sauroleishmania) tarentolae versus pathogenic species: comparative evaluation of protease activity, glycoconjugates, resistance to complement and metabolome composition

BACKGROUND Leishmania tarentolae is a non-pathogenic species found in lizards representing an important model for Leishmania biology. However, several aspects of this Sauroleishmania remain unknown to explain its low level of virulence. OBJECTIVES We reported several aspects of L. tarentolae biology including glycoconjugates, proteolytic activities and metabolome composition in comparison to pathogenic species (Leishmania amazonensis, Leishmania braziliensis, Leishmania infantum and Leishmania major). METHODS Parasites were cultured for extraction and purification of lipophosphoglycan (LPG), immunofluorescence probing with anti-gp63 and resistance against complement. Parasite extracts were also tested for proteases activity and metabolome composition. FINDINGS Leishmania tarentolae does not express LPG on its surface. It expresses gp63 at lower levels compared to pathogenic species and, is highly sensitive to complement-mediated lysis. This species also lacks intracellular/extracellular activities of proteolytic enzymes. It has metabolic differences with pathogenic species, exhibiting a lower abundance of metabolites including ABC transporters, biosynthesis of unsaturated fatty acids and steroids, TCA cycle, glycine/serine/threonine metabolism, glyoxylate/dicarboxylate metabolism and pentose-phosphate pathways. MAIN CONCLUSIONS The non-pathogenic phenotype of L. tarentolae is associated with alterations in several biochemical and molecular features. This reinforces the need of comparative studies between pathogenic and non-pathogenic species to elucidate the molecular mechanisms of virulence during host-parasite interactions.

Mapping Alterations Induced by Long-Term Axenic Cultivation of Leishmania amazonensis Promastigotes With a Multiplatform Metabolomic Fingerprint Approach.

Leishmaniases are widespread neglected diseases with an incidence of 1.6 million new cases and 40 thousand deaths per year. Leishmania parasites may show distinct, species-specific patterns of virulence that lead to different clinical manifestations. It is well known that successive in vitro passages (SIVP) lead to the attenuation of virulence, but neither the metabolism nor the pathways involved in these processes are well understood. Herein, promastigotes of a virulent L. amazonensis strain recently isolated from mice was compared to SIVP derived and attenuated promastigotes, submitted to 10, 40, and 60 axenic passages and named R10, R40, and R60, respectively. In vitro assays and in vivo tests were performed to characterize and confirmed the attenuation profiles. A metabolomic fingerprint comparison of R0, R10, and R60 was performed by means of capillary electrophoresis, liquid and gas chromatography coupled to mass spectrometry. To validate the metabolomic data, qPCR for selected loci, flow cytometry to measure aPS exposure, sensitivity to antimony tartrate and ROS production assays were conducted. The 65 identified metabolites were clustered in biochemical categories and mapped in eight metabolic pathways: ABC transporters; fatty acid biosynthesis; glycine, serine and threonine metabolism; ß-alanine metabolism; glutathione metabolism; oxidative phosphorylation; glycerophospholipid metabolism and lysine degradation. The obtained metabolomic data correlated with previous proteomic findings of the SVIP parasites and the gene expression of 13 selected targets. Late SIVP cultures were more sensitive to SbIII produced more ROS and exposed less phosphatidylserine in their surface. The correspondent pathways were connected to build a biochemical map of the most significant alterations involved with the process of attenuation of L. amazonensis. Overall, the reported data pointed out to a very dynamic and continuous metabolic reprogramming process, accompanied by changes in energetic, lipid and redox metabolisms, membrane remodeling and reshaping of parasite-host cells interactions, causing impacts in chemotaxis, host inflammatory responses and infectivity at the early stages of infection.