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.
Leishmania spp. infection outcomes are dependent on both host and parasite factors. Manipulation of host signaling pathways involved in the generation of immune responses is thought to be one of the most common mechanisms used by parasites for persistence within the host. Considering the diversity of pathologies caused by different Leishmania spp., it is plausible that significant differences may exist in the mechanisms of host cell manipulation by each parasite species, which may have implications when developing new vaccine or treatment strategies. Here we show that in L. braziliensis-infection in BALB/c mice, a model of resistance, activation of ERK1/2 coincides with the peak of inflammatory responses and resolution of tissue parasitism. In contrast, in the susceptibility model of L. amazonensis-infection, an early silent phase of infection is observed, detected solely by quantification of parasite loads. At this early stage, only basal levels of P-ERK1/2 are observed. Later, after a brief shutdown of ERK1/2 phosphorylation, disease progression is observed and is associated with increased inflammation, lesion size and tissue parasitism. Moreover, the short-term down-regulation of ERK1/2 activation affected significantly downstream inflammatory pathways and adaptive T cell responses. Administration of U0126, a MEK/ERK inhibitor, confirmed this phenomenon, since bigger lesions and higher parasite loads were seen in infected mice that received U0126. To investigate how kinetics of ERK1/2 activation could affect the disease progression, U0126 was administered to L. amazonensis-infected animals earlier than the P-ERK1/2 switch off time-point. This intervention resulted in anticipation of the same effects on inflammatory responses and susceptibility phenotype seen in the natural course of infection. Additionally, in vitro inhibition of ERK1/2 affected the phagocytosis of L. amazonensis by BMDMs. Collectively, our findings reveal distinct temporal patterns of activation of inflammatory responses in L. braziliensis and L. amazonensis in the same animal background and a pivotal role for a brief and specific shutdown of ERK1/2 activation at late stages of L. amazonensis infection. Since activation of inflammatory responses is a crucial aspect for the control of infectious processes, these findings may be important for the search of new and specific strategies of vaccines and treatment for tegumentary leishmaniasis.
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