Leishmania species are responsible for a broad spectrum of diseases, denominated Leishmaniasis, affecting over 12 million people worldwide. During the last decade, there have been impressive efforts for sequencing the genome of most of the pathogenic Leishmania spp. as well as hundreds of strains, but large-scale proteomics analyses did not follow these achievements and the Leishmania proteome remained mostly uncharacterized. Here, we report a comprehensive comparative study of the proteomes of strains representing L. braziliensis, L. panamensis and L. guyanensis species. Proteins extracted by SDS-mediated lysis were processed following the multi-enzyme digestion-filter aided sample preparation (FASP) procedure and analysed by high accuracy mass spectrometry. "Total Protein Approach" and "Proteomic Ruler" were applied for absolute quantification of proteins. Principal component analysis demonstrated very high reproducibility among biological replicates and a very clear differentiation of the three species. Our dataset comprises near 7000 proteins, representing the most complete Leishmania proteome yet known, and provides a comprehensive quantitative picture of the proteomes of the three species in terms of protein concentration and copy numbers. Analysis of the abundance of proteins from the major energy metabolic processes allow us to highlight remarkably differences among the species and suggest that these parasites depend on distinct energy substrates to obtain ATP. Whereas L. braziliensis relies the more on glycolysis, L. panamensis and L. guyanensis seem to depend mainly on mitochondrial respiration. These results were confirmed by biochemical assays showing opposite profiles for glucose uptake and O 2 consumption in these species. In addition, we provide quantitative data about different membrane proteins, transporters, and lipids, all of which contribute for significant species-specific differences and PLOS NEGLECTED TROPICAL DISEASES

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In American Tegumentary Leishmaniasis production of cytokines, reactive oxygen species and nitric oxide (NO) by host macrophages normally lead to parasite death. However, some Leishmania braziliensis strains exhibit natural NO resistance. NO-resistant strains cause more lesions and are frequently more resistant to antimonial treatment than NO-susceptible ones, suggesting that NO-resistant parasites are endowed with specific mechanisms of survival and persistence. To tests this, we analyzed the effect of pro- and antioxidant molecules on the infectivity in vitro of L. braziliensis strains exhibiting polar phenotypes of resistance or susceptibility to NO. In addition, we conducted a comprehensive quantitative mass spectrometry-based proteomics analysis of those parasites. NO-resistant parasites were more infective to peritoneal macrophages, even in the presence of high levels of reactive species. Principal component analysis of protein concentration values clearly differentiated NO-resistant from NO-susceptible parasites, suggesting that there are natural intrinsic differences at molecular level among those strains. Upon NO exposure, NO-resistant parasites rapidly modulated their proteome, increasing their total protein content and glutathione (GSH) metabolism. Furthermore, NO-resistant parasites showed increased glucose analogue uptake, and increased abundance of phosphotransferase and G6PDH after nitrosative challenge, which can contribute to NADPH pool maintenance and fuel the reducing conditions for the recovery of GSH upon NO exposure. Thus, increased glucose consumption and GSH-mediated redox capability may explain the natural resistance of L. braziliensis against NO.

Visceral leishmaniasis (VL) is a neglected disease caused by Leishmania parasites. Although significant morbidity and mortality in tropical and subtropical regions of the world are associated with VL, the low investment for developing new treatment measures is chronic. Moreover, resistance and treatment failure are increasing for the main medications, but the emergence of resistance phenotypes is poorly understood at the protein level. Here, we analyzed the development of resistance to miltefosine upon experimental selection in a L. infantum strain. Time to miltefosine resistance emergence was ~six months and label-free quantitative mass-spectrometry-based proteomics analyses revealed that this process involves a remodeling of components of the membrane and mitochondrion, with significant increase in oxidative phosphorylation complexes, particularly on complex IV and ATP synthase, accompanied by increased energy metabolism mainly dependent on β-oxidation of fatty acids. Proteins canonically involved in ROS detoxification did not contribute to the resistant process whereas sterol biosynthesis enzymes could have a role in this development. Furthermore, changes in the abundance of proteins known to be involved in miltefosine resistance such as ABC transporters and phospholipid transport ATPase were detected. Together, our data show a more complete picture of the elements that make up the miltefosine resistance phenotype in L. infantum.

Malnutrition is a risk factor for developing visceral leishmaniasis and its severe forms. Our group demonstrated that malnourished animals infected with Leishmania infantum had severe atrophies in lymphoid organs and T cell subpopulations as well as altered levels of thymic and splenic chemotactic factors, all of which resulted in dysfunctional lymphoid microenvironments that promoted parasite proliferation. Here, we hypothesize that malnutrition preceding parasite infection leads to structural and immunological changes in the gut mucosae, resulting in a failure in the immune response sensed in the intestine. To evaluate this, we analyzed the immunopathological events resulting from protein malnutrition in the guts of BALB/c mice infected with L. infantum. We observed lymphocytic/lymphoplasmacytic inflammatory infiltrates and lymphoid hyperplasia in the duodenum of well-nourished-infected mice; such alterations were worsened when malnutrition preceded infection. Parasite infection induced a significant increase of duodenal immunoglobulin A (IgA) of well-nourished animals, but those levels were significantly decreased in malnourished-infected mice. In addition, increased levels of Th17-related cytokines in duodenums of malnourished animals supported local inflammation. Together, our results suggest that the gut plays a potential role in responses to L. infantum infection—and that such responses are impaired in malnourished individuals.