Leonardo Saboia-Vahia

Proteomic characterization of the released/secreted proteins of Leishmania (Viannia) braziliensis promastigotes.

Extracellular proteins secreted/released by protozoan parasites are key mediators of the host-parasite interaction. To characterise the profile of proteins secreted/released by Leishmania (Viannia) braziliensis promastigotes, a proteomic approach combining two-dimensional electrophoresis (2DE), tandem matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF) mass spectrometry, and data mining was carried out. The 2DE map revealed a set of 270 secreted protein spots from which 42 were confidently identified and classified into 11 categories according to Gene Ontology (GeneDB database) and KEEG Ontology annotation of biological processes. Parasite promastigotes were able to secrete/release proteins involved in immunomodulation, signal transduction, and intracellular survival, such as HSP70, acid phosphatase, activated protein kinase C receptor (LACK), elongation factor 1beta, and tryparedoxin peroxidase. Data mining showed that approximately 5% of identified proteins present a classical secretion signal whereas approximately 57% were secreted following non-classical secretion mechanisms, indicating that protein export in this primitive eukaryote might proceed mainly by unconventional pathways. This study reports a suitable approach to identify secreted proteins in the culture supernatant of L. braziliensis and provides new perspectives for the study of molecules potentially involved in the early stages of infection.

Cysteine peptidase expression in Trichomonas vaginalis isolates displaying high- and low-virulence phenotypes.

In the present study, we identified and characterized the cysteine peptidase (CP) profiles of Trichomonas vaginalis isolates exhibiting high- and low-virulence phenotypes using a combination of two-dimensional SDS-PAGE (2DE), tandem mass spectrometry (MS/MS), and data mining. Seven of the eight CPs identified belong to Clan CA, family C1, cathepsin L-like CP, and one belongs to Clan CD, family C13, asparaginyl endopeptidase-like CP. Quantitative and qualitative differences in CP expression were detected between the isolates. BLAST analysis followed by CLUSTAL alignment of amino acid sequences of differentially expressed CPs showed identity or high homology to previously described CP cDNA clones CP1, CP3, CP4, and to a secreted CP fraction of 30 kDa involved in apoptosis of vaginal epithelial cells. One- and two-dimensional-substrate gel analyses revealed the differential CP profiles between the isolates, indicating that the combination of zymography with 2DE and MS/MS might be a powerful experimental approach to map and identify active peptidases in T. vaginalis. Toxicity exerted upon HeLa cells by high- and low-virulence isolates was 98.3% and 31%, respectively. Pretreatment of parasites with specific Clan CA papain-like CP inhibitor l-3-carboxy-2,3-trans-epoxypropionyl-leucylamido(4-guanidino)butane (E-64) drastically reduced the cytotoxic effect to 21.7% and 0.8%, respectively, suggesting that T. vaginalis papain-like CPs are the main factors involved in the cellular damage.

A zymographic study of metalloprotease activities in extracts and extracellular secretions of Leishmania (Viannia) braziliensis strains

Proteolytic activities of 5 strains of Leishmania (Viannia) braziliensis isolated from Brazilian and Colombian patients, presenting distinct clinical manifestations, were characterized and compared using whole-promastigote extracts and extracellular secretions. Zymographic assays concerning whole-cell extracts and supernatants resulted in the detection of high molecular weight bands, ranging from 50 to 125 kDa. Proteolytic activities from both whole-cell extracts and supernatants were optimal in a pH range 5.5 to 9.0 for all analysed strains. Such protease activities were inhibited when 10 mM 1,10-phenanthroline was assayed, strongly suggesting that the enzymes responsible for hydrolysis of the substrate belong to the metalloproteases class. Distinct profiles of metalloproteases were observed among the studied L. (V.) braziliensis strains. Differences among the microorganisms might be related to the geographical origin of the strains and/or to the clinical presentation.

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae)

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate-SDS-PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6-8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl-methyl sulfonyl-fluoride and N-α-Tosyl-L-lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin-like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin-like serine proteases occur during the developmental cycle of A. aegypti.

T-Cell Populations and Cytokine Expression Are Impaired in Thymus and Spleen of Protein Malnourished BALB/c Mice Infected with Leishmania infantum

Visceral leishmaniasis (VL) is a parasitic infectious disease that causes significant morbidity and mortality in the tropical and subtropical regions of the world. Although infections with visceralizing Leishmania may be asymptomatic, factors such as undernutrition increase the likelihood of progressing to clinical disease. Protein malnutrition, the most deleterious cause of malnutrition in developing countries, has been considered as a primary risk factor for the development of clinical VL. However, data regarding the immunological basis of this association are scarce. With the aim to analyze the effects of protein malnutrition on Leishmania infantum infection, we used BALB/c mice subjected to control or low protein isocaloric diets. Each animal group was divided into two subgroups and one was infected with L. infantum resulting in four study groups: animals fed 14% protein diet (CP), animals fed 4% protein diet (LP), animals fed 14% protein diet and infected (CPi), and animals fed 4% protein diet and infected (LPi).The susceptibility to L. infantum infection and immune responses were assessed in terms of body and lymphoid organ weight, parasite load, lymphocyte subpopulations, and cytokine expression. LPi mice had a significant reduction of body and lymphoid organ weight and exhibited a severe decrease of lymphoid follicles in the spleen. Moreover, LPi animals showed a significant decrease in CD4+CD8+ T cells in the thymus, whereas there was an increase of CD4+ and CD8+ T cells percentages in the spleen. Notably, the cytokine mRNA levels in the thymus and spleen of protein malnourished-infected animals were altered compared to the CP mice. Protein malnutrition results in a drastic dysregulation of T cells and cytokine expression in the thymus and spleen of L. infantum-infected BALB/c mice, which may lead to defective regulation of the thymocyte population and an impaired splenic immune response, accelerating the events of a normal course of infection.

Proteolytic profiling and comparative analyses of active trypsin-like serine peptidases in preimaginal stages of Culex quinquefasciatus

BackgroundThe mosquito Culex quinquefasciatu s, a widespread insect in tropical and sub-tropical regions of the world, is a vector of multiple arboviruses and parasites, and is considered an important risk to human and veterinary health. Proteolytic enzymes play crucial roles in the insect physiology including the modulation of embryonic development and food digestion. Therefore, these enzymes represent important targets for the development of new control strategies. This study presents zymographic characterization and comparative analysis of the proteolytic activity found in eggs, larval instars and pupae of Culex quinquefasciatus.MethodsThe proteolytic profiles of eggs, larvae and pupa of Cx. quinquefasciatus were characterized by SDS-PAGE co-polymerized with 0.1% gelatin, according to the pH, temperature and peptidase inhibitor sensitivity. In addition, the proteolytic activities were characterized in solution using 100 μM of the fluorogenic substrate Z-Phe-Arg-AMC.ResultsComparison of the proteolytic profiles by substrate-SDS-PAGE from all preimaginal stages of the insect revealed qualitative and quantitative differences in the peptidase expression among eggs, larvae and pupae. Use of specific inhibitors revealed that the proteolytic activity from preimaginal stages is mostly due to trypsin-like serine peptidases that display optimal activity at alkaline pH. In-solution, proteolytic assays of the four larval instars using the fluorogenic substrate Z-Phe-Arg-AMC in the presence or absence of a trypsin-like serine peptidase inhibitor confirmed the results obtained by substrate-SDS-PAGE analysis. The trypsin-like serine peptidases of the four larval instars were functional over a wide range of temperatures, showing activities at 25°C and 65°C, with an optimal activity between 37°C and 50°C.ConclusionThe combined use of zymography and in-solution assays, as performed in this study, allowed for a more detailed analysis of the repertoire of proteolytic enzymes in preimaginal stages of the insect. Finally, differences in the trypsin-like serine peptidase profile of preimaginal stages were observed, suggesting that such enzymes exert specific functions during the different stages of the life cycle of the insect.

Protein expression in the midgut of sugar-fed Aedes albopictus females

BackgroundAedes albopictus is a vector for several fatal arboviruses in tropical and sub-tropical regions of the world. The midgut of the mosquito is the first barrier that pathogens must overcome to establish infection and represents one of the main immunologically active sites of the insect. Nevertheless, little is known about the proteins involved in the defense against pathogens, and even in the processing of food, and the detoxification of metabolites. The identification of proteins exclusively expressed in the midgut is the first step in understanding the complex physiology of this tissue and can provide insight into the mechanisms of pathogen-vector interaction. However, identification of the locally expressed proteins presents a challenge because the Ae. albopictus genome has not been sequenced.MethodsIn this study, two-dimensional electrophoresis (2DE) was combined with liquid chromatography in line with tandem mass spectrometry (LC-MS/MS) and data mining to identify the major proteins in the midgut of sugar-fed Ae. albopictus females.ResultsFifty-six proteins were identified by sequence similarity to entries from the Ae. aegypti genome. In addition, two hypothetical proteins were experimentally confirmed. According to the gene ontology analysis, the identified proteins were classified into 16 clusters of biological processes. Use of the STRING database to investigate protein functional associations revealed five functional networks among the identified proteins, including a network for carbohydrate and amino acid metabolism, a group associated with ATP production and a network of proteins that interact during detoxification of toxic free radicals, among others. This analysis allowed the assignment of a potential role for proteins with unknown function based on their functional association with other characterized proteins.ConclusionOur findings represent the first proteome map of the Ae. albopictus midgut and denotes the first steps towards the description of a comprehensive proteome map of this vector. In addition, the data contributes to the functional annotation of Aedes spp. genomes using mass spectrometry-based proteomics data combined with complementary gene prediction methods.

In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus

BackgroundCulex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. This species transmits a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. However, the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the Culicidae family and most likely because they are low abundance proteins.MethodsWe used zymography for the biochemical characterization of the peptidase profile of the midgut from C. quinquefasciatus females fed on sugar. Protein samples were also submitted to SDS-PAGE followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis for peptidase identification. The peptidases sequences were analyzed with bioinformatics tools to assess their distinct features.ResultsZymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar diet. After denaturation in SDS-PAGE, eight trypsin-like serine peptidases were identified by LC-MS/MS. These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far.ConclusionWe demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes.

Proteomics reveals major components of oogenesis in the reproductive tract of sugar-fed Anopheles aquasalis

Anopheles (Nyssorhynchus) aquasalis is a malaria vector mainly distributed along the coastal regions of South and Central America. In the absence of an effective vaccine against malaria, strategies for controlling the vector are the main tool for interrupting parasite transmission. Mechanisms of oogenesis and embryogenesis in anautogenous mosquitoes are mainly modulated by blood feeding. However, the expression, at the protein level, of genes involved in such mechanisms in sugar-fed females is unknown. In this work, total protein extracts of the reproductive tract of female An. aquasalis that were fed sugar were analyzed using liquid chromatography followed by mass spectrometry for protein identification and bioinformatic tools for data mining. We identified 922 proteins expressed in the organ, and using several databases, we attributed biological meaning for several of them. Remarkably, nine proteins involved in oogenesis were identified in females fed sugar. Putative vitellogenins, vitellogenin receptor, lipid storage droplet, transferrin, ferritin, and apolipoprotein, identified here, are proteins involved in egg development. Proteins involved in embryonic development, such as paxillin, exuperantia, several growth factors, and dorsal switch protein, were identified. Interestingly, in this study, we identified 15 peptidases of various classes such as aminopeptidases, carboxypeptidases, serine protease, cathepsin, and metalloprotease that could potentially interact with male seminal components. Here, we demonstrated that the reproductive tract of female An. aquasalis fed on sugar expresses proteins involved in oogenesis and embryonic development. These findings reveal unknown aspects of the physiology of this organ under the given nutritional conditions.

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis.