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

Abstract

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; β-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.

Extracellular Vesicles Released by Leishmania (Leishmania) amazonensis Promastigotes with Distinct Virulence Profile Differently Modulate the Macrophage Functions

“…Several studies have shown that the long-term culture of L. infantum, L. major, and L. amazonensis alters the expression of virulence factors, changes the parasite metabolism, and impairs their ability to infect mice [28,[31][32][33][34]. Although these studies are still in their infancy, they shed light on the importance of evaluating the loss of infectivity of Leishmania after a long period of culture when in vitro.…”

“…This hypothesis is highly speculative; however, studies on parasites that lost one of these two virulence factors showed a decrease in their ability to infect macrophages and mice [37][38][39]. In addition, [34] showed that the L. amazonensis cultured for a long-term duration was less infective in vitro compared to the parasites recently recovered from an in vivo infection. Therefore, it is important to highlight the importance of carrying out studies with virulent parasites since their passage for long periods in culture can alter the phenotypic and infective characteristics of the parasites.…”

Papel de miRNAs na regulação da via de produção de poliaminas/NO em macrófagos derivados de THP-1 infectados com Leishmania amazonensis

“…A metabolômica foi utilizada para caracterizar o metabolismo do parasita (Akpunarlieva et al, 2017;Castilho-Martins et al, 2015;Crepaldi et al, 2019;Saunders et al, 2014;Westrop et al, 2015) e se mostrou uma ferramenta útil, uma vez que os mecanismos de controle pós-transcricionais e pós-traducionais dificultam o acesso ao fenótipo do parasita utilizando apenas transcriptômica e proteômica (Scheltema et al 2010;Kloehn et al 2015;Atan et al 2018).…”