Iron is an essential element for the survival of microorganisms in vitro and in vivo, acting as a cofactor of several enzymes and playing a critical role in host-parasite relationships. Leishmania (Viannia) braziliensis is a parasite that is widespread in the new world and considered the major etiological agent of American tegumentary leishmaniasis. Although iron depletion leads to promastigote and amastigote growth inhibition, little is known about the role of iron in the biology of Leishmania. Furthermore, there are no reports regarding the importance of iron for L. (V.) braziliensis. In this study, the effect of iron on the growth, ultrastructure and protein expression of L. (V.) braziliensis was analyzed by the use of the chelator 2,2-dipyridyl. Treatment with 2,2-dipyridyl affected parasites' growth in a dose- and time-dependent manner. Multiplication of the parasites was recovered after reinoculation in fresh culture medium. Ultrastructural analysis of treated promastigotes revealed marked mitochondrial swelling with loss of cristae and matrix and the presence of concentric membranar structures inside the organelle. Iron depletion also induced Golgi disruption and intense cytoplasmic vacuolization. Fluorescence-activated cell sorting analysis of tetramethylrhodamine ester-stained parasites showed that 2,2-dipyridyl collapsed the mitochondrial membrane potential. The incubation of parasites with propidium iodide demonstrated that disruption of mitochondrial membrane potential was not associated with plasma membrane permeabilization. TUNEL assays indicated no DNA fragmentation in chelator-treated promastigotes. In addition, two-dimensional electrophoresis showed that treatment with the iron chelator induced up- or down-regulation of proteins involved in metabolism of nucleic acids and coordination of post-translational modifications, without altering their mRNA levels. Iron chelation leads to a multifactorial response that results in cellular collapse, starting with the interruption of cell proliferation and culminating in marked mitochondrial impairment in some parasites and their subsequent cell death, whereas others may survive and resume proliferating. American tegumentary leishmaniasis (ATL) is a neglected disease that is widely distributed in the Americas. The protozoan parasite Leishmania (Viannia) braziliensis is one of the main causative agents of ATL, being responsible for the development of different clinical manifestations of the disease, which ranges from self-healing cutaneous lesions to disseminated and mucocutaneous forms. Because iron is essential for the survival and growth of Leishmania, as it is required for colonization of macrophages and development of lesions in mice, several chelating compounds have been tested for their effects on the growth of these parasites. In the present work, treatment of L. (V.) braziliensis with the iron chelator 2,2-dipyridyl inhibited the growth of promastigote forms in a dose- and time-dependent manner. However, multiplication of the parasites was recovered after reinoculation in fresh culture medium. The iron chelator also induced mitochondrial dysfunction and altered expression of proteins involved in metabolism of nucleic acids and coordination of post-translational modifications. The events described above ultimately caused the death of some parasites, most likely due to mitochondrial dysfunction, whereas others adapted and survived, suggesting a plasticity or resilience of the mitochondrion in this parasite.

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 O₂ 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 provide rich substrate for explore new molecules for diagnosing purposes. Data are available via ProteomeXchange with identifier PXD017696. Leishmania braziliensis, L. panamensis, and L. guyanensis are responsible for most of the cases of tegumentary leishmaniasis (TL) in the Americas. These species are associated with a variety of clinical manifestations of TL ranging from self-healing localized cutaneous lesions to disseminated and mucocutaneous presentations that may result in severe oropharyngeal mutilation. Here, we report a comprehensive quantitative comparison of the proteome of those species. Assessment of absolute titers of ~7000 proteins revealed a very clear differentiation among them. Significant differences in energy metabolism, membrane proteins, transporters, and lipids are contributing for species-specific traits and provide rich substrate for exploring new molecules for diagnosing purposes.

Background: Aedes albopictus, a ubiquitous mosquito, is one of the main vectors of dengue and yellow fever, representing an important threat to public health worldwide. Peptidases play key roles in processes such as digestion, oogenesis, and metamorphosis of insects. However, most of the information on the proteolytic enzymes of mosquitoes is derived from insects in the adult stages and is often directed towards the understanding of blood digestion. The aim of this study was to investigate the expression of active peptidases from the preimaginal stages of Ae. albopictus. Methods: Ae. albopictus eggs, larvae, and pupae were analyzed using zymography with susbtrate-SDS-PAGE. The pH, temperature and peptidase inhibitor sensitivity was evaluated. In addition, the proteolytic activities of larval instars were assayed using the fluorogenic substrate Z-Phe-Arg-AMC. Results: The proteolytic profile of the larval stage was composed of 8 bands ranging from 17 to 130 kDa. These enzymes displayed activity in a broad range of pH values, from 5.5 to 10.0. The enzymatic profile of the eggs was similar to that of the larvae, although the proteolytic bands of the eggs showed lower intensities. The pupal stage showed a complex proteolytic pattern, with at least 6 bands with apparent molecular masses ranging from 30 to 150 kDa and optimal activity at pH 7.5. Peptidases from larval instars were active from 10°C to 60°C, with optimal activity at temperatures between 37°C and 50°C. The proteolytic profile of both the larval and pupal stages was inhibited by phenyl-methyl sulfonyl-fluoride (PMSF) and Nα-Tosyl L-lysine chloromethyl ketone hydrochloride (TLCK), indicating that the main peptidases expressed during these developmental stages are trypsin-like serine peptidases. Conclusion: The preimaginal stages of Ae. albopictus exhibited a complex profile of trypsin-like serine peptidase activities. A comparative analysis of the active peptidase profiles revealed differential expression of trypsin-like isoforms among the preimaginal stages, suggesting that some of these enzymes are stage specific. Additionally, a comparison of the peptidase expression between larvae from eggs collected in the natural environment and larvae obtained from the eggs of female mosquitoes maintained in colonies for a long period of time demonstrated that the proteolytic profile is invariable under such conditions.

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.

Chagas disease, the parasitic infection caused by Trypanosoma cruzi, afflicts about 6 million people in Latin America. Here, we investigated the hypothesis that T. cruzi may fuel heart parasitism by activating B1R, a G protein-coupled (brady) kinin receptor whose expression is upregulated in inflamed tissues. Studies in WT and B1R^−/− mice showed that T. cruzi DNA levels (15 days post infection—dpi) were sharply reduced in the transgenic heart. FACS analysis revealed that frequencies of proinflammatory neutrophils and monocytes were diminished in B1R^−/− hearts whereas CK-MB activity (60 dpi) was exclusively detected in B1R^+/+ sera. Since chronic myocarditis and heart fibrosis (90 dpi) were markedly attenuated in the transgenic mice, we sought to determine whether a pharmacological blockade of the des-Arg⁹-bradykinin (DABK)/B1R pathway might alleviate chagasic cardiomyopathy. Using C57BL/6 mice acutely infected by a myotropic T. cruzi strain (Colombian), we found that daily treatment (15–60 dpi) with R-954 (B1R antagonist) reduced heart parasitism and blunted cardiac injury. Extending R-954 treatment to the chronic phase (120–160 dpi), we verified that B1R targeting (i) decreased mortality indexes, (ii) mitigated chronic myocarditis, and (iii) ameliorated heart conduction disturbances. Collectively, our study suggests that a pharmacological blockade of the proinflammatory KKS/DABK/B1R pathway is cardioprotective in acute and chronic Chagas disease.