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Owing to the importance and clinical diversity of Leishmania infantum, studying its virulence factors is promising for understanding the relationship between parasites and hosts. In the present study, differentially abundant proteins from strains with different degrees of virulence in promastigote and amastigote forms were compared using two quantitative proteomics techniques, differential gel electrophoresis and isobaric mass tag labeling, followed by identification by mass spectrometry. A total of 142 proteins were identified: 96 upregulated and 46 downregulated proteins in the most virulent strain compared to less virulent. The interaction between the proteins identified in each evolutionary form was predicted. The results showed that in the amastigote form of the most virulent strain, there was a large group of proteins related to glycolysis, heat shock, and ribosomal proteins, whereas in the promastigote form, the group consisted of stress response, heat shock, and ribosomal proteins. In addition, biological processes related to metabolic pathways, ribosomes, and oxidative phosphorylation were enriched in the most virulent strain (BH400). Finally, we noted several proteins previously found to play important roles in L. infantum infection, which showed increased abundance in the virulent strain, such as ribosomal proteins, HSP70, enolase, fructose 1,6-biphosphate aldolase, peroxidoxin, and tryparedoxin peroxidase, many of which interact with each other.

In Brazil, Leishmania amazonensis is one of the etiological agents of tegumentary leishmaniasis and can cause a wide spectrum of diseases in humans, resulting in cutaneous, mucosal, diffuse, and even visceral leishmaniasis. Besides, this species has also been reported to affect dogs, causing typical symptoms of visceral disease. Unfortunately, the diagnostic of the Leishmania species is not routinely performed due to the difficulties of the available methods. In view of this, different molecular methods have been used in an attempt to solve the problem of diagnosis. Loop-mediated isothermal amplification (LAMP) is a relatively new nucleic acid amplification method, which has been successfully applied in the diagnosis of Leishmania spp. infections. However, this is the first work that standardizes a specific LAMP reaction for L. amazonensis. The set of primers selected were designed from the kDNA minicircle sequence of the L. amazonensis (GenBank: U19810.1). The LAMP assay developed in the present study showed 100% specificity and 89% sensitivity when compared with conventional PCR and was more sensitive than qPCR. In addition, the LAMP reaction developed here was able to amplify a qPCR sample with a parasite load of only 28 parasites in 50 ng of DNA. Consequently, considering the LAMP reaction specific to L. amazonensis and several advantages of the method (such as high efficiency, sensitivity and specificity), we believe that this reaction can be used as a promising diagnostic tool in clinical practice, field studies, and research.

Leishmaniasis has been considered as emerging and re-emerging disease, and its increasing global incidence has raised concerns. The great clinical diversity of the disease is mainly determined by the species. In several American countries, tegumentary leishmaniasis (TL) is associated with both Leishmania amazonensis and L. braziliensis, while visceral leishmaniasis (VL) is associated with L. (L.) infantum. The major molecules that determine the most diverse biological variations are proteins. In the present study, through a DIGE approach, we identified differentially abundant proteins among the species mentioned above. We observed a variety of proteins with differential abundance among the studied species; and the biological networks predicted for each species showed that many of these proteins interacted with each other. The prominent proteins included the heat shock proteins (HSPs) and the protein network involved in oxide reduction process in L. amazonensis, the protein network of ribosomes in L. braziliensis, and the proteins involved in energy metabolism in L. infantum. The important proteins, as revealed by the PPI network results, enrichment categories, and exclusive proteins analysis, were arginase, HSPs, and trypanothione reductase in L. amazonensis; enolase, peroxidoxin, and tryparedoxin1 in L. braziliensis; and succinyl-CoA ligase [GDP -forming] beta-chain and transaldolase in L. infantum.

Visceral leishmaniasis (VL) is a neglected tropical disease with dogs serving as reservoirs for one of its etiological agents, Leishmania infantum. In Brazil, VL control involves culling of seropositive dogs, among other actions. However, the most employed serological tests lack accuracy, and are not able to detect canine visceral leishmaniasis (CVL) during the early stages of infection. Early detection of CVL is highly desirable in order to shorten the contact time between the infected reservoirs and the vectors. In this study, we investigated the ability of two multiepitope proteins, PQ10 and PQ20, to detect CVL at earlier stages than currently employed methods, including ITS-1 conventional PCR. Using serum samples from naturally infected dogs, we observed that ELISA-PQ10 and ELISA-PQ20 were able to detect Leishmania infection at earlier time points as compared with kDNA PCR-RFLP in anti-IgG and anti-IgM assays. Using sera from experimentally infected dogs, we monitored seroconversion using multiepitope proteins, ELISA-crude antigen, as well as ITS-1 conventional and real-time PCR. While seroconversion was detected by ELISA-crude antigen in 16.6% of the dogs, multiepitope proteins were able to detect seroconversion in more than 80% of them. Moreover, the ability of ELISA-PQ10 and ELISA-PQ20 to detect Leishmania infection at earlier time points as compared with conventional PCR was also confirmed in experimental infection dogs' sera. Immunofluorescence to Babesia canis and Ehrlichia canis did not show cross-reactions with ELISA-PQ10/PQ20 positive samples. Results of real-time PCR and ELISA with multiepitope proteins were very similar, with concordances between 80 and 100%. Furthermore, our findings indicated that PQ10 and PQ20 immunoassays can be related to parasite load. ELISA-PQ10 and ELISA-PQ20 are more sensitive diagnostic tools for early CVL detection as compared with other methods They could potentially be used in screening tests due to easy execution and low costs facilities.

Diagnostic tools are important for clinical management and epidemiological evaluation of Tegumentary (TL) and Visceral (VL) Leishmaniasis. Serology is not frequently used for the diagnosis of the TL form because low antibody titers and cross-reaction with VL. Therefore, it is crucial to identify specific and immunogenic antigens from species associated with the TL form. Here we employed a proteomic approach coupled to an in silico analysis and identified the most abundant and immunogenic proteins from Leishmania amazonensis, Leishmania braziliensis and Leishmania infantum. Of 16 species specific proteins, nine were from the species causative of the TL form (L. amazonensis and L. braziliensis). In silico analysis revealed 18 B-cell epitopes with 0% similarity to Trypanosoma cruzi orthologs and, therefore, less likely to crossreact with sera of patients with Chagas disease. Two proteins reacted exclusively with serum from TL patients and presented several B-cell epitopes without similarity to T. cruzi orthologs: the hypothetical protein GI 134063939 and the metallo-peptidase Clan MA(E)-Family M3. The immunoassay revealed nine peptides with strong reactivity to sera from TL patients. These proteins and peptides may be good candidates to improve the specificity and sensibility of serological tests aiming to diagnose the TL of this neglected human disease.As no gold-standard test for tegumentary leishmaniasis (TL) exists, a combination of different diagnostic techniques is often necessary to obtain precise results. Thus, the identification of species-specific, highly immunogenic and abundant proteins that stimulate the humoral immune response in the host should help in the development of serological tests for human TL. Herein we searched for these potential antigens in Leishmania species related to American Leishmaniasis (L. amazonensis, L. braziliensis and L. infantum). To this end, we employed an immunoproteomic approach using proteins from these Leishmania species and sera from TL and Visceral Leishmaniasis (VL) patients. Our study unveils specific proteins and peptides that may represent antigens that will help the efforts to improve the accuracy of serological tests to diagnose the TL form.

Leishmania spp have a wide range of hosts, and each host can harbor several Leishmania species. Dogs, for example, are frequently infected by Leishmania infantum, where they constitute its main reservoir, but they also serve as hosts for L. braziliensis and L. amazonensis. Serological tests for antibody detection are valuable tools for diagnosis of L. infantum infection due to the high levels of antibodies induced, unlike what is observed in L. amazonensis and L. braziliensis infections. Likewise, serology-based antigen-detection can be useful as an approach to diagnose any Leishmania species infection using different corporal fluid samples. Immunogenic and secreted proteins constitute powerful targets for diagnostic methods in antigen detection. As such, we performed immunoproteomic (2-DE, western blot and mass spectrometry) and bioinformatic screening to search for reactive and secreted proteins from L. amazonensis, L. braziliensis, and L. infantum. Twenty-eight non-redundant proteins were identified, among which, six were reactive only in L. amazonensis extracts, 10 in L. braziliensis extracts, and seven in L. infantum extracts. After bioinformatic analysis, seven proteins were predicted to be secreted, two of which were reactive only in L. amazonensis extracts (52kDa PDI and the glucose-regulated protein 78), one in L. braziliensis extracts (pyruvate dehydrogenase E1 beta subunit) and three in L. infantum extracts (two conserved hypothetical proteins and elongation factor 1-beta). We propose that proteins can be suitable targets for diagnostic methods based on antigen detection.

Brucellosis serodiagnosis is still a challenge and vaccination is the main measure used to control bovine brucellosis, being S19 and RB51 the most currently used vaccines. So, in order to contribute to brucellosis control, a bidimensional (2D) immunoblot-based approach was used to find immunogenic proteins to be used in serodiagnosis, particularly with ability to be employed in DIVA (Differentiating Infected from Vaccinated Animals) strategy. Immunoproteomic profile of Brucella abortus 2308 was analyzed in 2D western blotting using pooled sera from S19 vaccinated animals, RB51 vaccinated animals, B. abortus naturally infected animals and non-vaccinated seronegative animals. Evaluation of the antigens differentially immunoreactive against the groups of sera showed three proteins of particular importance: MDH (malate dehydrogenase) immunoreactive for S19-vaccinated animals, SOD (superoxide dismutase) reactive for infected animals and ABC transporter (multispecies sugar ABC transporter) reactive against sera from vaccinated animals (S19 and RB51). These three proteins were produced in E. coli and tested in an indirect ELISA (I-ELISA). For MDH, comparison between the vaccinated animals (independent of the vaccine used) and the seropositive and seronegative animals in I-ELISA showed significant differences. Data on the I-ELISA using SOD showed that sera from non-vaccinated naturally infected animals exhibited significant difference in comparison with all other groups. Otherwise, sera from vaccinated animals (S19 and RB51) and from non-vaccinated naturally infected animals did not show significant difference in OD values, but they were all significant different from non-vaccinated seronegative animals using ABC transporter as antigen in I-ELISA. In conclusion, together the 2D western blot analysis and the preliminary I-ELISA results suggest that the combined use of MDH and SOD could be successful employed in a LPS-free protein based serodiagnosis approach to detect bovine brucellosis and to discriminate vaccinated from naturally infected animals, in early post-vaccination stages.

Background Zika virus (ZIKV) and dengue virus (DENV) are mosquito-borne flaviviruses prevalent throughout tropical regions. Currently, management of ZIKV and DENV centers on control of the primary vector Aedes aegypti. This vector is highly anthropophilic and is therefore prevalent throughout densely urbanised landscapes. A new passive trap for gravid Ae. aegypti (Gravid Aedes Trap - GAT) was developed for mosquito surveillance. Here the different killing agents and the level of transmission of arboviruses that may occur in mosquitoes sampled by GATs are assessed for the first time. Methods Gravid Aedes traps (GATs) were deployed at the Federal University of Minas Gerais campus, in Belo Horizonte, Brazil to sample Ae. aegypti. Three different killing agents were evaluated within the GATs: sticky cards, long-lasting insecticide-impregnated nets (LLINs) and canola oil. Traps were monitored weekly for 14 weeks then mosquito specimens were identified to the species level and Ae. aegypti catches were pooled and submitted to qRT-PCR assays for to DENV and ZIKV virus detection, followed by Bayesian phylogenetic analysis of the ZIKV. Additionally, comparisons of means were performed on transformed weekly catch data (P = 0.05, t-tests) with the stats package of the R statistical software. Results In total, 1506 female Ae. aegypti were captured using GATs, with traps using sticky cards catching more mosquito than those using either LLINs or canola oil. Both ZIKV and DENV were detected in Ae. aegypti females captured over several weeks suggesting that this highly populated university campus may have served as a significant transmission hub. The infection rate for ZIKV was present in seven (8.5%) pools from four weeks while DENV was detected in four (4.9%) pools from four weeks. Phylogenetic analysis of ZIKV classified the strain as Asian genotype. Conclusions The Federal University of Minas Gerais and similar organizations must strongly consider monitoring Ae. aegypti populations and reinforcing personal protection of staff and students during seasons of high mosquito activity.

In the present study, we successfully demonstrated for the first time the existence of cardiac proteomic differences between non-selectively bred rats with distinct intrinsic exercise capacities. A proteomic approach based on two-dimensional gel electrophoresis coupled to mass spectrometry was used to study the left ventricle (LV) tissue proteome of rats with distinct intrinsic exercise capacity. Low running performance (LRP) and high running performance (HRP) rats were categorized by a treadmill exercise test, according to distance run to exhaustion. The running capacity of HRPs was 3.5-fold greater than LRPs. Protein profiling revealed 29 differences between HRP and LRP rats (15 proteins were identified). We detected alterations in components involved in metabolism, antioxidant and stress response, microfibrillar and cytoskeletal proteins. Contractile proteins were upregulated in the LVs of HRP rats (α-myosin heavy chain-6, myosin light chain-1 and creatine kinase), whereas the LVs of LRP rats exhibited upregulation in proteins associated with stress response (aldehyde dehydrogenase 2, α-crystallin B chain and HSPβ-2). In addition, the cytoskeletal proteins desmin and α-actin were upregulated in LRPs. Taken together, our results suggest that the increased contractile protein levels in HRP rats partly accounted for their improved exercise capacity, and that proteins considered risk factors to the development of cardiovascular disease were expressed in higher amounts in LRP animals.

Systemic lupus erythematosus (SLE) is an autoimmune disease of the connective tissue with a large spectrum of clinical manifestations. Immune deregulation leads to autoantibody and immune complexes overproduction, complement activation, and persistent tissue inflammation. Considering that the current diagnosis depends on the interpretation of the complex criteria established by the American College of Rheumatology and that the disease course is characterized by unpredictable activations and remissions, each patient develops different manifestations, and therefore, the discovery of specific biomarkers is urgently required. Therefore, this study aimed to identify putative biomarkers for active and inactive SLE potentially capable in distinguishing laboratorial SLE from other autoimmune diseases. The 2D-DIGE proteomics technique was used to evaluate the differential abundance of proteins between patients with active SLE, inactive SLE, patients with other autoimmune disease, and healthy individuals. Six proteins showed increased abundance in active SLE (A) and inactive SLE (I) compared to the C and O groups, but not between groups A and I. There were two transthyretin (TTR) fragments or proteins with a structure similar to TTR (accession numbers: PDB: 1GKO_A and 2PAB_A), retinol-binding protein 4 (RBP4) isoform X1 (no information in databases such as UNIPROT), and antibody fragments. Two proteins, APO-AIV and SP-40,40, were upregulated in group A than in O and C and in group I versus C, but not in group I versus O. Therefore, we suggest these proteins to be considered as candidates for the diagnosis of SLE.

Identification of novel antigens is essential for developing new diagnostic tests and vaccines. We used DIGE to compare protein expression in amastigote and promastigote forms of Leishmania chagasi. Nine hundred amastigote and promastigote spots were visualized. Five amastigote-specific, 25 promastigote-specific, and 10 proteins shared by the two parasite stages were identified. Furthermore, 41 proteins were identified in the Western blot employing 2-DE and sera from infected dogs. From these proteins, 3 and 38 were reactive with IgM and total IgG, respectively. The proteins recognized by total IgG presented different patterns in terms of their recognition by IgG1 and/or IgG2 isotypes. All the proteins selected by Western blot were mapped for B-cell epitopes. One hundred and eighty peptides were submitted to SPOT synthesis and immunoassay. A total of 25 peptides were shown of interest for serodiagnosis to visceral leishmaniasis. In addition, all proteins identified in this study were mapped for T cell epitopes by using the NetCTL software, and candidates for vaccine development were selected. Therefore, a large-scale screening of L. chagasi proteome was performed to identify new B and T cell epitopes with potential use for developing diagnostic tests and vaccines.

Previous studies showed that P1G10, a proteolytic fraction from Vasconcellea cundinamarcensis latex, reduced the tumor mass in animals bearing melanoma, increased in vitro DNA fragmentation and decreased cell adhesion. Here, we present some molecular and cellular events related to the antimetastatic effect induced by the CMS-2 fraction derived from P1G10 in metastatic melanoma B16-F10 and melanocyte Melan-a. Using difference gel electrophoresis and mass spectrometry, we identified four proteins overexpressed in tumor cells, all of them related to proliferation, survival, migration and cell invasion, that had their expression normalized upon treatment with CMS-2: nucleophosmin 1, heat shock protein 65, calcyclin binding protein and eukaryotic translation initiation factor 4H. In addition, some antioxidant and glycolytic enzymes show increased expression after exposure to CMS-2, along with an induction of melanogenesis (differentiation marker). The down regulation of cofilin 1, a protein involved in cell motility, may explain the inhibition of cell migration and dendritic-like outgrowth in B16-F10 and Melan-a, observed after CMS-2 treatment. Taken together, it is argued that CMS-2 modulates the expression of proteins related to metastatic development, driving the cell to a more differentiated-like state. These effects support the CMS-2 antimetastatic activity and place this fraction in the category of anticancer agent.

Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the beta-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease.

To improve transfection efficiency in Trypanosoma cruzi, we developed a new electroporation protocol and expression vectors which use luciferase and green and red fluorescent proteins as reporter genes. In transient transfections, the electroporation conditions reported here resulted in luciferase expression 100 times higher than the levels obtained with previously described protocols. To verify whether sequences containing different trans-splicing signals influence reporter gene expression, we compared DNA fragments corresponding to 5′ untranslated plus intergenic (5′ UTR plus Ig) regions from GAPDH, TcP2β, α- and β-tubulin and amastin genes. Vectors containing sequences derived from the first four genes presented similar efficiencies and resulted in luciferase expression in transiently transfected epimastigotes that was up to 10 times higher than that for a control vector. In contrast, the amastin 5′ UTR plus Ig resulted in lower levels of reporter gene expression. We also constructed a vector containing an expression cassette designed to be targeted to the tubulin locus of the parasite. Fil: DaRocha, Wanderson D.. Universidade Federal de Minas Gerais; Brasil Fil: Silva, Rosiane A.. Universidade Federal de Minas Gerais; Brasil Fil: Bartholomeu, Daniella C.. Universidade Federal de Minas Gerais; Brasil Fil: Pires, Simone F.. Universidade Federal de Minas Gerais; Brasil Fil: Freitas, Jorge M.. Universidade Federal de Minas Gerais; Brasil Fil: Macedo, Andrea M.. Universidade Federal de Minas Gerais; Brasil Fil: Vazquez, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; Argentina Fil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; Argentina Fil: Teixeira, Santuza M. R.. Universidade Federal de Minas Gerais; Brasil

Angiotensin-(1–7) has been described as a new potential therapeutic tool for the treatment and prevention of metabolic disorders by regulating several pathways in visceral white adipose tissue (vWAT). The aim of this study was to access the proteins differentially regulated by Ang-(1–7) using proteomic analysis of visceral adipose tissue. Male mice were divided into three groups and fed for 60 days, with each group receiving one of the following diets: standard diet + HPβCD (ST), high fat diet + HPβCD (HFD) and high fat diet + Ang-(1–7)/HPβCD (HFD + Ang-(1–7)). Body weight, fat weight and food intake were measured. At the end of treatment, Ang-(1–7) induced a decrease in body and fat weight. Differential proteomic analysis using two-dimensional electrophoresis (2-DE) combined with mass spectrometry were performed. Results of protein mapping of mesenteric adipose tissue using 2-DE revealed the presence of about 450 spots in each gel ( n = 3/treatment) with great reproducibility (>70%). Image analysis and further statistical analysis allowed the detection and identification of eight proteins whose expression was modulated in response to HFD when compared to ST. Among these, two proteins showed a sensitive response to Ang-(1–7) treatment (eno1 and aldehyde dehydrogenase). In addition, three proteins were expressed statistically different between HFD + Ang-(1–7) and HFD groups, and four proteins were modulated compared to standard diet. In conclusion, comparative proteomic analysis of a mice model of diet-induced obesity allowed us to outline possible pathways involved in the response to Ang-(1–7), suggesting that Ang-(1–7) may be a useful tool for the treatment of metabolic disorders.

Knowledge of Leishmania virulence is essential for understanding how the contact between the pathogen and host cells can lead to pathogenesis. Virulence in two L. infantum strains was characterized using macrophages and hamsters. Next, we used difference gel electrophoresis (DIGE) and mass spectrometry to identify the differentially expressed proteins. A total of 63 spots were identified corresponding to 36 proteins; 20 were up-regulated, in which 16 had been previously associated with Leishmania virulence. Considering our results and what has been reported before, we suggest the hypothesis that L. infatum virulence could be a result of the increased expression of KMP-11 and metallopeptidase, associated with an improved parasite-host interacting efficiency and degradation of the protective host proteins and peptides, respectively. Other factors are tryparedoxin peroxidase and peroxidoxin, which protect the parasite against the stress response, and 14-3-3 protein-like, which can prolong infected host cell lifetime. Proteins as chaperones and endoribonuclease L-PSP can increase parasite survival. Enolase is able to perform versatile functions in the cell, acting as a chaperone or in the transcription process, or as a plasminogen receptor or in cell migration events. As expected in more invasive cells with high replication rates, energy consumption and protein synthesis are higher, with up-regulation of Rieske iron-sulfur protein precursor, EF-2, S-adenosylhomocysteine, and phosphomannomutase.

The mechanism of miltefosine-resistance in Leishmania spp. has been partially determined in experimental resistant lines; however, studies using clinical isolates with different miltefosine susceptibilities are still needed. In our study, we used a proteomic 2D-DIGE/MS approach to study different protein abundances in miltefosine-sensitive and -resistant Leishmania infantum chagasi isolates from visceral leishmaniasis patients with different miltefosine treatment outcomes. The high-resolution proteome obtained from these isolates showed 823 matched spots and 46 spots exhibited different abundances between the isolates. Out of these differentially expressed spots, 26 (56.5%) showed greater and 20 (43.5%) showed lower expression of the resistant isolate compared to the sensitive isolate. MALDI/TOF–TOF mass spectrometry allowed the identification of 32 spots with unique protein identification correspondent to 22 non-redundant proteins. Most of the proteins up-regulated in the proteome miltefosine-resistant isolates were associated with redox homeostasis, stress response, protection to apoptosis, and drug translocation. These differentially expressed proteins are likely involved in miltefosine natural resistance and suggest that the miltefosine-resistance mechanism in Leishmania is multifactorial. Biological significance Visceral leishmaniasis (VL) is a serious disease with a challenging treatment plan requiring the prolonged and painful applications of poorly tolerated toxic drugs. Therefore, the identification of miltefosine, an effective and safe oral drug, was considered a significant advancement in leishmaniasis therapy. However, different sensitivities to miltefosine in Leishmania have been observed in clinically relevant species, and the biological mechanism by which clinical isolates of Leishmania acquire drug resistance is poorly understood. Our work aims to elucidate the mechanism of natural resistance to miltefosine in Leishmania by studying the isolates from VL patients who displayed different miltefosine treatment outcomes.

© 2014 Silva et al. Brucella ovis is a major cause of reproductive failure in rams and it is one of the few well-described Brucella species that is not zoonotic. Previous work showed that a B. ovis mutant lacking a species-specific ABC transporter (ΔabcBA) was attenuated in mice and was unable to survive in macrophages. The aim of this study was to evaluate the role of this ABC transporter during intracellular survival of B. ovis. In HeLa cells, B. ovis WT was able to survive and replicate at later time point (48 hpi), whereas an ΔabcBA mutant was attenuated at 24 hpi. The reduced survival of the ΔabcBA mutant was associated with a decreased ability to exclude the lysosomal marker LAMP1 from its vacuolar membrane, suggesting a failure to establish a replicative niche. The ΔabcBA mutant showed a reduced abundance of the Type IV secretion system (T4SS) proteins VirB8 and VirB11 in both rich and acid media, when compared to WT B. ovis. However, mRNA levels of virB1, virB8, hutC, and vjbR were similar in both strains. These results support the notion that the ABC transporter encoded by abcEDCBA or its transported substrate acts at a post-transcriptional level to promote the optimal expression of the B. ovis T4SS within infected host cells.

Aim: To identify immunoreactive proteins of Cryptococcus gattii genotype VGII and their B-cell epitopes. Materials & methods: We combined 2D gel electrophoresis, immunoblotting and mass spectrometry to identify immunoreactive proteins from four strains of C. gattii genotype VGII (CG01, CG02, CG03 and R265). Next, we screened the identified proteins to map B-cell epitopes. Results: Sixty-eight immunoreactive proteins were identified. The strains and the number of proteins we found were: CG01 (12), CG02 (12), CG03 (18) and R265 (26). In addition, we mapped 374 peptides potentially targeted by B cells. Conclusion: Both immunoreactive proteins and B-cell epitopes of C. gattii genotype VGII that were potentially targeted by a host humoral response were identified. Considering the evolutionary relevance of the identified proteins, we may speculate that they could be used as the initial targets for recombinant protein and peptide synthesis aimed at the development of immunodiagnostic tools for cryptococcosis.

Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation.

Bacteroides fragilis is the anaerobe most frequently isolated from clinical specimens and piperacillin/tazobactam is among the drugs that can be used to treat polymicrobial infections in which this bacteria is often involved. During antibiotic therapy, inhibitory concentrations of antibiotics are always followed by subinhibitory concentrations which can generate phenotypic changes in bacteria. So, in this study we aimed to evaluate changes in the proteomic profile of B. fragilis grown in a sub-MIC of PTZ, using 2-D electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight/time of-flight. Analysis of the 2-DE gels showed 18 spots with significantly different volume percentages between experimental conditions and 12 were successfully identified by MS/MS. Two proteins with decreased abundance in sub-MIC condition were involved in the glycolysis (glyceraldehyde-3-phosphate dehydrogenase and triose phosphate isomerase), others two involved in amino acid metabolism (Oxoacyl-(acyl-carrier protein) synthase II and dihydrodipicolinate reductase), and finally, one protein involved in fatty acid metabolism (UDP-N-acetylglucosamine acyltransferase). Among the proteins with increased abundance, we founded three ATP synthase (alpha, beta, and alpha type V), which could be involved in antibiotic bacterial resistance by efflux pump, one protein involved in glycolysis (enolase), and one involved in protein degradation (aminoacyl-histidine dipeptidase). In conclusion, our data show overall changes in the proteome of B. fragilis conducted by sub-MIC of PTZ, whose consequences on bacterial physiology deserve further investigation.

The worldwide emergence of antibiotic-resistant bacteria poses a serious threat to human health. In addition to the difficulties in controlling infectious diseases, the phenotype of resistance can generate metabolic changes which, in turn, can interfere with host-pathogen interactions. The aim of the present study was to identify changes in the subproteome of a laboratory-derived piperacillin/tazobactam-resistant strain of Escherichia coli (minimal inhibitory concentration [MIC] = 128 mg/L) as compared with its susceptible wild-type strain E. coli ATCC 25922 (MIC = 2 mg/L) using 2-D fluorescence difference gel electrophoresis (2D-DIGE) followed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF MS). In the resistant strain, a total of 12 protein species were increased in abundance relative to the wild-type strain, including those related to bacterial virulence, antibiotic resistance and DNA protection during stress. Fourteen proteins were increased in abundance in the wild-type strain compared to the resistant strain, including those involved in glycolysis, protein biosynthesis, pentose-phosphate shunt, amino acid transport, cell division and oxidative stress response. In conclusion, our data show overall changes in the subproteome of the piperacillin/tazobactam-resistant strain, reporting for the first time the potential role of a multidrug efflux pump system in E. coli resistance to piperacillin/tazobactam.

Sistemas de transformação estável vêm sendo descritos para várias espécies de tripanosomatídeos como Leishmania spp, Trypanosoma brucei e Trypanosoma cruzi . A transfecção pode ser mediada pela integração de genes exógenos no genoma da célula hospedeira, que ocorre por recombinação homóloga ou através da manutenção dosvetores transformados na forma episomal. Com o objetivo de gerar parasitos que expressassem proteínas fluorescentes de forma estável, formas epimstigotas do T. cruzi foram transfectadas com o vetor de expressão denominado pROCKGFP/RFPNeo, que leva a integração das proteínas verde (GFP) e vermelha (RFP) fluorescentes no loco de-tubulin via recombinação homóloga com esse loco. Clones dos parasitos transfectados foram obtidos por diluição seriada em placas de 96 poços e/ou placas de ágar-sangue. Foi avaliada a estabilidade na expressão de GFP e RFP entre as populações (parasitos não clonados) e os clones. Os parasitos foram cultivados por 1 e 2 meses na presença de G418 (200 e 400 g/mL) e na ausência da droga e em seguida analisados pela técnica de citometria de fluxo. Foi observado que os clones expressando GFP e RFP foram capazes de manter a porcentagem de parasitos fluorescentes na ausência do antibiótico,enquanto que nas populações, houve redução da porcentagem de células verdes e vermelhas fluorescentes. As análises de chromoblot mostraram a integração de GFP e RFP no loco de -tubulina. Os parasitos transfectados mostraram infectividade semelhante ao não transfectado em culturas de células Vero e a expressão das proteínasfluorescentes pôde ser facilmente visualizada nas três formas do ciclo de vida do parasito por microscopia confocal. Parasitos expressando GFP foram capazes de infectar camundongos GKO (interferon-gama knockout) e apresentaram alta parasitemia 12 dias após a infecção. Foram observados parasitos fluorescentes no coração e diafragma dos animais. Cultura de células co-infectadas com parasitos da cepa Tulahuén expressando as duas diferentes fluorescências, mostraram que dois parasitos diferentes podem infectar a mesma célula. Também foram observadas células co-infectadas com parasitos transfectados, derivados de cepas pertencentes às distintas linhagens de T. cruzi, Col1.7G2GFP (T. cruzi I) e TulahuénRFP (T. cruzi II). Os parasitos gerados expressando GFP e RFP constituem, portanto uma nova ferramenta para o estudo de vários aspectos da biologia do T. cruzi. Com eles podemos estudar mecanismos de invasão celular, troca genética, susceptibilidade às drogas e inúmeros aspectos dainteração parasito-hospedeiro invertebrado e vertebrado, neste último, enfatizando o modelo histotrópico-clonal da doença de Chagas em modelos animais. Stable transfection protocols have been described for a number of protozoan parasites such as Leishmania spp, Trypanosoma brucei and Trypanosoma cruzi. The transfection can be achieved by the integration of the foreign gene into the genome through homologous recombination, or by episomal maintenance of the transfected plasmid. In order to produce parasites expressing fluorescent proteins, epimastigotes forms of T.cruzi were transfected with the plasmid pROCKGFP/RFPNeo. This construction allowed the integration of green (GFP) and red (RFP) fluorescent protein genes by homologous recombination into -tubulin locus of several strains of the parasite. Parasites transfected clones were isolated by serial dilution and/or agar-blood plates. To evaluate the stability of the GFP and RFP markers in the transfected populations andclones, parasites were cultivated for 1 and 2 months in the absence or presence of G418 (200 g mL and 400 g mL) and were analyzed by FACS. The selected clones were able to maintain high expression levels of the fluorescent protein markers even in absence of G418, whereas in transfected population, expression of GFP and RFP markers wassignificantly reduced when cultured in the absence of the drug. The integration of the GFP and RFP markers in the tubulin locus was confirmed by chromoblot analyses. GFP and RFP expressing parasites were inoculated into Vero cells culture to evaluate the infectivity of the transfected population. When compared with the wild type population, no significant differences in the infectivity of Vero cells were found. Wedetected the fluorescent proteins in the tree forms of parasite life cicle by confocal and fluorescence microscopy. Furthermore, GFP expressing parasites were able produce significant levels of parasitemia 12 days after being inoculated into interferon-gamma knockout (GKO) mice. We observed fluorescent parasites in heart and diaphragm of infected animals. Cell cultures infected simultaneously with two cloned cell lines, each one expressing a distinct fluorescent marker, showed that two different parasites were able to infect the same cell. Double infected cells were also detected when GFP and RFP expressing parasites are derived from strains belonging to two distinct T. cruzi lineages Col1.7G2GFP (T. cruzi I) and TulahuénRFP (T. cruzi II). The parasites expressing GFP and RFP constitute a new tool for the study of various aspects of the T.cruzi biology. We can study the cellular invasion mechanism, genetic exchange, drug susceptibility and several aspects of interaction with invertebrate and vertebrate hosts, emphasizing the histotropic-clonal model of Chagas disease.

Trypanosoma cruzi, the causative agent of Chagas disease, is extremely resistant to ionizing radiation, enduring up to 1.5 kGy of gamma rays. Ionizing radiation can damage the DNA molecule both directly, resulting in double-strand breaks, and indirectly, as a consequence of reactive oxygen species production. After a dose of 500 Gy of gamma rays, the parasite genome is fragmented, but the chromosomal bands are restored within 48 hours. Under such conditions, cell growth arrests for up to 120 hours and the parasites resume normal growth after this period. To better understand the parasite response to ionizing radiation, we analyzed the proteome of irradiated (4, 24, and 96 hours after irradiation) and non-irradiated T. cruzi using two-dimensional differential gel electrophoresis followed by mass spectrometry for protein identification. A total of 543 spots were found to be differentially expressed, from which 215 were identified. These identified protein spots represent different isoforms of only 53 proteins. We observed a tendency for overexpression of proteins with molecular weights below predicted, indicating that these may be processed, yielding shorter polypeptides. The presence of shorter protein isoforms after irradiation suggests the occurrence of post-translational modifications and/or processing in response to gamma radiation stress. Our results also indicate that active translation is essential for the recovery of parasites from ionizing radiation damage. This study therefore reveals the peculiar response of T. cruzi to ionizing radiation, raising questions about how this organism can change its protein expression to survive such a harmful stress.

We have previously demonstrated that both parasite genetic variability and host genetic background were important in determining the differential tissue distribution of the Col1.7G2 and JG T. cruzi monoclonal strains after artificial infections in mice. We observed that the JG strain was most prevalent in hearts of mouse lineages with the MHC haplotype H-2(d) (BALB/c and DBA2), while Col1.7G2 was predominant in hearts from C57BL/6 mice, which have the H-2(b) haplotype. To assess whether the MHC gene region indeed influenced tissue tropism of T. cruzi, we used the same two parasite strains to infect C57BL/6 (H-2(b)) and C57BLKS/J (H-2(d)) mice; the latter strain results from the introgression of DBA2 MHC region into the C57BL/6 background. We also performed ex vivo infections of cardiac explants from four congenic mice lineages with the H-2(b) and H-2(d) haplotypes arranged in two different genetic backgrounds: C57BLKS/J (H-2(d)) versus C57BL/6 (H-2(b)) and BALB/c (H-2(d)) versus BALB/B10-H2(b) (H-2(b)). In agreement with our former observations, Col1.7G2 was predominant in hearts from C57BL/6 mice (H-2(b)), but we observed a clear predominance of the JG strain in hearts from C57BLKS/J animals (H-2(d)). In the ex vivo experiments Col1.7G2 also prevailed in explants from H-2(b) animals while no predominance of any of the strains was observed in H-2(d) mice explants, regardless of the genetic background. These observations clearly demonstrate that the MHC region influences the differential tissue distribution pattern of infecting T. cruzi strains, which by its turn may be in a human infection the determinant for the clinical forms of the Chagas disease.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A ampla distribuição e sobreposição de espécies de Leishmania e de diferentes formas clínicas das leishmanioses no país podem afetar diretamente o curso clínico da doença, diagnóstico, controle e tratamento. Com isso, o desenvolvimento de métodos de diagnóstico espécie-específicos e que possam ser utilizados na rotina são necessários e de grande importância. No Brasil, a espécie Leishmania amazonensis é agente etiológico principalmente da forma cutânea da doença, mas já foi também encontrada promovendo doença visceral em humanos e cães. Além disso, sua coexistência com outras espécies do parasito, como L. infantum, agente etiológico da leishmaniose visceral, torna de suma importância a identificação da espécie durante o diagnóstico da infecção por Leishmania spp. Para isso, no presente trabalho, como teste molecular, padronizamos a técnica de LAMP (Amplificação Isotérmica Mediada por Loop), a partir do desenho e utilização de iniciadores específicos para L. amazonensis com visualização do resultado utilizando gel de poliacrilamida e detecção colorimétrica (SYBR® Safe). A reação apresentou 100% de especificidade quando comparado as espécies L. infantum, L. braziliensis, L. major, L. mexicana, L. donovani, L. guyanensis, Trypanosoma cruzi, Babesia canis e Toxoplasma gondii e quando testado diferentes cepas de L. amazonensis. O limite de detecção da reação foi de 10pg de DNA de L. amazonensis. Apresentou 89% de sensibilidade quando comparada a PCR convencional utilizando amostras de DNA de pele de hamsters experimentalmente infectados com L. amazonensis (n=9), L. infantum (n=9) e coinfecção (n=9). Para o desenvolvimento de um teste sorológico, foi realizada uma abordagem imunoproteômica para seleção de proteínas antigênicas espécie-específicas, para isso, utilizamos extrato proteico de L. amazonensis fracionados em eletroforese bi-dimensional (2-DE) associados a western blot (WB) com pool de soros de hamsters experimentalmente infectados com L. amazonensis e L. infantum. A sobreposição entre as imagens de gel 2-DE e membranas de WB permitiu a seleção de 8 spots reativos somente a soros de animais infectados com L. amazonensis e não reativos a soros de infectados com L. infantum ou de não infectados. Foram identificadas duas proteínas em 6/8 spots. Uma delas, a HSP70, produzida de forma recombinante foi utilizada como antígeno em um teste de ELISA com pool de soros de hamsters infectados com L. amazonensis, L. infantum, coinfectados e não infectados. Houve diferença significativa (P

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior No Brasil a leishmaniose tegumentar (LT) e a leishmaniose visceral (LV) são endêmicas e amplamente distribuídas, sendo a LT frequentemente associada com as espécies Leishmania (Leishmania) amazonensis e L. (Viannia) braziliensis, enquanto a forma visceral é causada por L. (L.) infantum. As formas clínicas são determinadas por fatores do parasito e do hospedeiro, dentre estes se destaca a resposta imune. A arginase e a óxido nítrico sintase induzível (iNOS) são enzimas que compartilham do mesmo substrato, a L-arginina, para produzir dentre outros compostos L-ornitina e óxido nítrico (NO), respectivamente. Além da sua função na formação de substratos importantes para a proliferação de células parasitárias e hospedeiras, a arginase se destaca por desempenhar um papel regulatório na disponibilidade de L-arginina nas células que a expressam, podendo contribuir para o estabelecimento da infecção por Leishmania spp. Nesse sentido, foi nosso objetivo avaliar a abundância e os níveis de atividade da arginase em L. amazonensis, L. braziliensis, L. infantum, e também em macrófagos infectados por estes parasitos, além de associar com a carga parasitária. Inicialmente, a abundância e a atividade da arginase foi determinada em cada espécie nas formas promastigotas. Foi observado que L. amazonensis apresentou 2,6 vezes maior abundância de arginase quando comparada às espécies L. braziliensis e L. infantum. Além disso, os níveis de atividade da arginase também foram maiores em promastigotas procíclicas e metacíclicas de L. amazonensis quando comparadas com L. braziliensis e L. infantum. Em seguida, mensuramos a atividade de arginase, produção de NO e carga parasitária em macrófagos infectados pelas espécies estudadas. Não foi observada diferença significativa entre os níveis de atividade enzimática da arginase, de produção de NO ou no índice de infecção entre macrófagos infectados pelas três espécies estudadas. Nossos dados demonstram que maior abundância e atividade de arginase em promastigotas de L. amazonensis não influencia na infecção de macrófagos “in vitro”. In Brazil, tegumentary leishmaniasis (LT) and visceral leishmaniasis (LV) are endemic and widely distributed, and LT is frequently associated with the species Leishmania (Leishmania) amazonensis and L. (Viannia) braziliensis, whereas the visceral form is caused by L. (L.) Infantum. Clinical forms are determined by parasite and host factors, among which the immune response is highlighted. Arginase and inducible nitric oxide synthase (iNOS) are enzymes that share the same substrate, L-arginine, to produce among other compounds L-ornithine and nitric oxide (NO), respectively. In addition to its role in the formation of important substrates for the proliferation of parasite and host cells, arginase plays a key role in the availability of L-arginine in the cells expressing it, and may contribute to the establishment of Leishmania spp. In this sense, it was our objective to evaluate the abundance and levels of arginase activity in L. amazonensis, L. braziliensis, L. infantum, and also in macrophages infected by these parasites, in addition to correlating with the parasite load. Initially, abundance and arginase activity was determined in each species in promastigote forms. It was observed that L. amazonensis presented 2.6 times greater abundance of arginase when compared to L. braziliensis and L. infantum species. Furthermore, arginase activity levels were also higher in procyclic and metacyclic promastigotes of L. amazonensis when compared to L. braziliensis and L. infantum. Next, we measured arginase activity, NO production and parasite load between macrophages infected by the species studied. No significant difference was observed between levels of enzymatic activity of arginase, NO production or infection rate among macrophages infected by the three species studied. Our data demonstrate that increased abundance and arginase activity in L. amazonensis promastigotes does not influence in vitro macrophage infection.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior As Leishmanioses são doenças causadas por protozoários tripanosomatídeos do gênero Leishmania e, no Brasil, as espécies Leishmania amazonensis e Leishmania braziliensis são predominantemente associadas à Leishmaniose Tegumentar Americana (LTA) enquanto Leishmania infantum, é agente da Leishmaniose Visceral (LV). Estas espécies foram utilizadas neste estudo por sua importância clínica e epidemiológica no nosso país. O sequenciamento do genoma de algumas espécies de Leishmania mostrou que cada genoma pode conter cerca de oito mil genes que codificam proteínas, dos quais mais da metade são genes hipotéticos. Apesar desse grande número, existem poucos dados acerca das proteínas hipotéticas e suas funções biológicas, sendo indispensáveis estudos para caracterização. Em um trabalho anterior do nosso grupo, foi realizada uma análise proteômica comparativa entre cepas de L. infantum com diferença de virulência. Dentre as proteínas com diferença de abundância, foram selecionadas as proteínas de função desconhecida LmxM.36.6760, aumentada na cepa mais virulenta, e LinJ.30.3360, aumentada na cepa menos virulenta. Assim, no presente estudo, foi nosso objetivo caracterizar essas duas proteínas de função desconhecida. Inicialmente, utilizamos análises de bioinformática através dos servidores NCBI, ProtParam, UniProtKB, Expasy, que indicaram presença de sequências similares as das proteínas LmxM.36.6760 e LinJ.30.3360 nas espécies L. braziliensis, L. donovani, L. major, L. mexicana e L. infantum, e estas não apresentam peptídeo sinal, domínio transmembrana ou âncora GPI. A estrutura secundária predita para LmxM.36.6760 é de alfa-hélice enquanto para a proteína LinJ.30.3360 é de folha-beta. Observamos similaridade dos resultados obtidos entre os três preditores de localização celular: TargetP, WOLF PSORT e DeepLoc-1.0, que indicaram localização na mitocôndria para proteína LmxM.36.6760 e localização no citoplasma do parasito para proteína LinJ.30.3360. Os resultados de microscopia confocal mostraram-se inconclusivos quanto a localização celular das proteínas LmxM.36.6760 e LinJ.30.3360. Foi possível observar que ambas as proteínas apresentam abundancia similar entre as espécies L. amazonensis, L. braziliensis e L. infantum. Leishmaniasis is a disease caused by trypanosomatid protozoa of the genus Leishmania, and in Brazil the species Leishmania amazonensis and Leishmania braziliensis are predominantly associated with American Cutaneous Leishmaniasis (ACL) while Leishmania infantum is an agent of Visceral Leishmaniasis (VL). These species were used in this study because of their clinical and epidemiological importance in our country. Sequencing the genome of some Leishmania species has shown that each genome can contain about 8,000 genes encoding proteins, of which more than half are hypothetical genes. Despite this large number, there are few data on hypothetical proteins and their biological functions, and studies for characterization are indispensable. In a previous study of our group, a comparative proteomic analysis was performed between L. infantum strains with virulence difference. Among proteins with difference in abundance, proteins of unknown function LmxM.36.6760, which increased in the most virulent strain, and LinJ.30.3360, which increased in the less virulent strain, were selected. Thus, in the present study, it was our aim to characterize these two proteins of unknown function. Initially, we used bioinformatics analysis through the serves NCBI, ProtParam, UniProtKB, Expasy which indicated the presence of sequences similar to those of LmxM.36.6760 and LinJ.30.3360 in L. braziliensis, L. donovani, L. major, L. mexicana and L. infantum, and these do not present signal peptide, transmembrane domain or GPI anchor. The secondary structure predicted for LmxM.36.6760 is alpha-helix whereas for LinJ.30.3360 protein is beta-pleated. We observed similarity in the results obtained among the three cell localization predictors: TargetP, WOLF PSORT and DeepLoc-1.0, which indicated localization in the mitochondria for LmxM.36.6760 protein and localization in the cytoplasm of the parasite for protein LinJ.30.3360. Confocal microscopy results were inconclusive as to the cellular localization of LmxM.36.6760 and LinJ.30.3360 proteins. It was possible to observe that both proteins present similar abundance among the species L. amazonensis, L. braziliensis and L. infantum.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior As leishmanioses são doenças com ampla diversidade de formas clínicas, devido as diferentes espécies. A leishmaniose tegumentar (LT) está frequentemente associada à infecção por Leishmania (Leishmania) amazonensis e/ou L. (Viannia) braziliensis, enquanto a forma visceral (LV) é causada por L. (L.) infantum. As proteínas por sua vez, estão entre as principais moléculas que podem determinar as mais diversas variações biológicas. Outro aspecto relevante é que um diagnóstico eficiente contribui para a avaliação não só clínica, mas também da epidemiologia e controle da doença. No presente trabalho, por meio de abordagem proteômica de expressão diferencial em gel (DIGE), idenficamos proteínas diferencialmente abundantes entre as espécies de Leishmania mais frequentes no Brasil, L. (L.) amazonensis, L. (Viannia) braziliensis e L. (L.) infantum. Através da utilização de ferramentas de bioinformática, foram mostradas as interações dessas proteínas em redes biológicas, bem como os processos biológicos nos quais essas moléculas atuam. No nosso trabalho destacamos 6 dessas categorias funcionais: metabolismo do nitrogênio, resposta ao stress, organização do citoesqueleto, transporte, desenvolvimento de estruturas e vias associadas à regulação do sistema imune; sendo que, nessa última, o enriquecimento estava presente somente em L. braziliensis e L. infatum. Os dados revelaram que L.infatum, espécie com maior potencial de visceralização, apresentou maior quantidade de proteínas detectadas para quase todos os processos. Selecionamos dentre essas proteínas quais eram também antigênicas de acordo com a forma clínica, LT ou LV, dos soros utilizados no western blot. As proteínas antigênicas foram submetidas a uma análise in silico para a predição de epitopos para células B que não possuíam similaridades com ortólogos em Trypanosoma cruzi. Das 18 proteínas identificadas com essas características (mais abundantes, específicas e antigênicas em uma espécie em relação às outras) oito são das espécies causadoras da forma tegumentar da doença (L. amazonensis e L. braziliensis) e 10 na espécie que causa a forma visceral (L. infantum). Os 68 epitopos preditos simultaneamente pelos softwares ABCPred e BCPreds nessas proteínas, foram sintetizados em membrana de celulose e avaliados em relação a reatividade. Considerando os dados analisados, podemos destacar duas proteínas de L. amazonensis, que apresentam potencial para diagnóstico específico de LT: metalo-peptidase, Clan MA (E)-Familia M3 e uma proteína hipotética. Ambas são mais abundantes nessa espécie, apresentam reatividade exclusiva ao pool de soros de LT e a maioria dos epitopos não apresenta similaridade com os ortólogos em T. cruzi. Além disso, nove epitopos, provenientes das proteínas selecionadas por reatividade exclusiva de soros de pacientes com LT ao extrato de L. amazonensis, foram considerados como potenciais antígenos para o diagnóstico sorológico da LT em humanos. Leishmaniasis are disease with a wide diversity of clinical forms, due to the different species. The cutaneous leishmaniasis (LT) is frequently associated with Leishmania (Leishmania) amazonensis and / or L. (Viannia) braziliensis infection, while the visceral form (LV) is caused by L. (L.) infantum. Proteins are one of the main molecules that determinete the largest number of biological variations. Another relevant aspect is that an efficient diagnosis contributes not only to clinical evaluation, but also to disease epidemiology and control. In the present study, by means of a proteomic approach (DIGE), we identified differentially abundant proteins among the most frequent Leishmania species in our country, L (L.) amazonensis, L. (Viannia) braziliensis and L. (L.) infantum. Through the use of bioinformatics tools, the interactions of these proteins in biological networks, as well as the biological processes in which these molecules act, were shown. In our study we highlight 6 of these functional categories: nitrogen metabolism, stress response, cytoskeletal organization, transport, development of structures and pathways associated with regulation of the immune system; being in the latter the enrichment was present in L. braziliensis and L. infatum, only. Data revealed that L.infatum, species with greater visceral potential, showed higher amount of proteins detected for almost all the processes. In addition, we selected from these proteins which were also antigenic according to the clinical form, TL or VL, of the sera used in the western blot. Antigenic proteins were subjected to in silico analysis for the prediction of B cell epitopes without similarities with orthologs in Trypanosoma cruzi. Among 18 proteins identified with these characteristics (more abundant, specific and antigenic in one species relative the others) eight belongs to species that cause the cutaneous form of the disease (L. amazonensis and L. braziliensis) and 10 belongs to the species that cause the visceral form (L. infantum). The 68 epitopes predicted simultaneously by the ABCPred and BCPreds software in these proteins were synthesized in cellulose membrane and evaluated for reactivity. Considering the analyzed data, we highlight two proteins of L. amazonensis, which have potential for specific diagnosis of TL: metallopeptidase, Clan MA (E) -Familia M3 and a hypothetical protein. Both are more abundant in this species, they have exclusive reactivity to the sera from TL pool and most of the epitopes no show similarity to orthologs in T. cruzi. In addition, nine epitopes from proteins selected by exclusive reactivity of sera from TL patients to L. amazonensis extract were considered as potential antigens for the serological diagnosis of TL in humans.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Leishmania infantum é o protozoário responsável pela Leishmaniose Visceral nas Américas e na Europa, uma doença grave, podendo ser fatal se não tratada. Os casos humanos variam de infecção subclínica, com um número indeterminado de assintomáticos em áreas endêmicas, a um quadro crescente e de alta letalidade em crianças. Em cães infectados, menos de 50% apresentam sinais clínicos da doença, no entanto, alguns sucumbem em poucos meses após a infecção. Essa ampla diversidade clínica em humanos e em caninos sugere que fatores tanto do hospedeiro quanto do parasito podem estar envolvidos na variabilidade da doença. Assim, devido a sua importância e diversidade clínico-epidemiológica em nosso país, o estudo de fatores de virulência em L. infantum se mostra bastante promissor para o entendimento da relação parasito-hospedeiro. Em um estudo anterior foi caracterizada a virulência de duas cepas (BH46 e BH400) de L. infantum, e foi observado uma maior virulência e infectividade da cepa BH400. Além disso, através da análise proteômica da forma promastigota, um total de 63 spots foram identificados correspondendo a 36 proteínas das quais, 16 já haviam sido previamente associados à virulência de Leishmania spp. Dando continuidade a essa linha de pesquisa, no presente estudo incluímos as formas amastigotas axênicas e esplênicas com o objetivo comparar a abundância diferencial de proteínas entre as cepas BH46 e BH400. Para isso, foram utilizadas duas técnicas de proteômica quantitativa: marcação por fluoróforo (DIGE) e marcação por isóbaros (TMT), seguidas de identificação por Espectrometria de Massas. Foram identificadas um total de 414 proteínas únicas que apresentaram diferença de abundância, sendo 68,4% encontradas na técnica TMT, 19% no DIGE e 12,6% em ambas as técnicas. Na forma Promastigota, foi observado um total de 270 proteínas com diferença de abundância entre as cepas. Também foram encontradas 302 e 293 proteínas diferencialmente abundantes entre as cepas nas formas Amastigota Axênica e em Amastigota Esplênica, respectivamente. Na cepa BH400, foi observado 117 proteínas com aumento de abundância em Amastigota Esplênica, enquanto 79 proteínas apresentavam diminuição. Por outro lado, em Amastigota Axênica foi observado uma maior quantidade de proteínas com abundância diminuída (178) enquanto 66 proteínas apresentaram aumento de abundância. Em Promastigota de BH400 a quantidade de proteínas com abundância aumentada (114) e diminuída (115) foi similar. A predição de interação entre as proteínas identificadas em cada forma apontou as proteínas relacionadas com tradução e proteínas de resposta ao estresse oxidativo como importantes redes. Dentre todas as proteínas com diferença de abundância, foi observado que na forma Amastigotas Axênicas, nenhum processo biológico estava enriquecido, mas simultaneamente nas formas Promastigota e Amastigota Esplênica da cepa 400 (mais virulenta) estavam enriquecidos os processos metabólicos: Celular, de Ácido Carboxílico, de Substância Orgânica, e Processo de Resposta ao Estresse. Além desses, nas formas promastigotas de 400, estavam também enriquecidos os Processos Metabólico Primário e de Biossíntese Celular. Por outro lado, simultaneamente nas formas promastigotas e amastigotas esplênicas da cepa BH46 foi observado enriquecimento do processo de biossíntese de compostos nitrogenados, enquanto nas formas amastigotas esplênicas dessa cepa estavam enriquecidos também os processos de biossíntese celular, expressão gênica e tradução. Foi observado que 14 proteínas estavam simultaneamente aumentadas em pelo menos duas formas evolutivas da cepa 400, sendo 8 delas em promastigota e amastigota esplênica, 5 em promastigota e amastigota axênica e 1 nas formas amastigotas (axênica e esplênica). Da mesma forma, 20 proteínas estavam com abundância diminuída simultaneamente em duas formas da cepa 400, sendo, 6 em promastigota e amastigota esplênica, 2 em promastigota e amastigota axênica e 12 nas formas amastigotas (axênica e esplênica). Por fim, foi observado uma maior semelhança entre a abundância das proteínas em Promastigota e Amastigota Axênica do que em Amastigota Esplênica, independente da cepa. Os resultados alcançados contribuem para o avanço no conhecimento da biologia de L. infantum, e muitas das proteínas identificadas são promissoras para o melhor entendimento da relação parasito-hospedeiro nessa espécie, principalmente no estudo de fatores de virulência. Leishmania infantum is the protozoan responsible for Visceral Leishmaniasis in Americas and Europe, a serious disease that can be fatal if untreated. Human cases range from subclinical infection, with an undetermined number of asymptomatic in endemic areas, to a growing and highly lethal in children. In infected dogs, less than 50% have clinical signs of the disease, however, some succumb within a few months after infection. This clinical signs diversity in humans and canines suggests that both host and parasite factors may be involved in disease variability. Thus, due to its importance and clinical-epidemiological diversity in our country, the study of virulence factors in L. infantum is very promising for the understanding of the parasite-host relationship. In a previous study the virulence of two strains (BH46 and BH400) of L. infantum was characterized, and a higher virulence and infectivity of strain BH400 was observed. In addition, through proteomic analysis of the promastigote form, a total of 63 spots were identified corresponding to 36 proteins of which 16 had previously been associated with the virulence of Leishmania spp. Continuing this line of research, in the present study we included the axenic and splenic amastigote forms of both strains with the objective of comparing the differential abundance of proteins between strains BH46 and BH400. Two quantitative proteomics techniques were used: fluorescent labeling (DIGE) and isobaric labeling (TMT) followed by identification by mass spectrometer. A total of 414 unique proteins that showed differences on abundance were identified, being 68.4% found in the TMT technique, 19.1% in DIGE and 12.6% in both techniques. In Promastigote form, a total of 270 proteins with a difference of abundance between the strains were observed. We also found 302 and 293 differential abundant proteins among the strains in the Axenic Amastigote and in the Splenic Amastigote, respectively. In the BH400 strain, 117 proteins with increased abundance were observed in Splenic Amastigote, while 79 proteins showed decrease. On the other hand, in Axenic Amastigote was observed a greater amount of proteins with abundance diminished (178) while 66 proteins presented increase of abundance. In Promastigote of BH400 the amount of proteins with increased (114) and decreased (115) abundance was similar. The interaction prediction of the proteins identified in each form, demonstrate translation-related proteins and response to oxidative stress-related proteins, as important networks. Among all proteins with a difference of abundance, it was observed that in Axenic Amastigotes form, none biological process was enriched, but simultaneously in the Promastigota and Splenic Amastigote forms of BH400 strain (more virulent) were enriched the metabolic processes: Cellular, Carboxylic Acid, of Organic Substance, and Response to Stress. Furthemore, in the promastigote form of BH400, the Primary Metabolic and Cell Biosynthesis Processes were also enriched. On the other hand, in promastigotes and splenic amastigotes forms of the BH46 strain, the enrichment of the biosynthesis process of nitrogenous compounds was observed, whereas in amastigote forms, the cellular biosynthesis process, gene expression and translation were enriched. It was observed that 14 proteins were simultaneously increased in at least two evolutionary forms of BH400 strain, 8 in promastigote and splenic amastigote, 5 in promastigotes and axenic amastigotes and 1 in amastigotes (axenic and splenic) forms. Also, 20 proteins were simultaneously decreased in two evolutive forms of BH400, 6 of them in promastigote and splenic amastigote, 2 in axenic amastigote and promastigote and 12 in both amastigote forms (axenic and splenic). Finally, a greater similarity between the abundance of the proteins in Promastigote and Axenic Amastigote was observed than in Splenic Amastigote, independently of the strain. The results obtained contribute to the advances in the knowledge of the biology of L. infantum, and many of the identified proteins are promising for a better understanding of the parasite-host relationship in this species, especially in virulence factors study.

Leishmania infantum, sinonímia com L. chagasi, é o protozoário responsável pela Leishmaniose Visceral nas Américas e na Europa, uma doença grave, podendo ser fatal se não tratada. Os casos humanos variam desde infecção sub-clínica, com um número indeterminado de assintomáticos em áreas endêmicas, até um quadro crescente de co-infecção com HIV e alta letalidade em crianças. Em cães, a doença é severa, caracterizada por crônica e progressiva caquexia, hepatoesplenomegalia, linfadenopatia, onicogrifose e pancitopenia. Porém, menos de 60% dos cães infectados apresentam sinais clínicos da doença, sugerindo que tanto fatores do hospedeiro quanto do parasito podem estar envolvidos na variabilidade clínica da doença. Assim, devido a sua importância e diversidade clínico-epidemiológica em nosso país, o estudo de fatores de virulência em L. infantum mostra-se bastante promissor para um melhor entendimento da relação parasito-hospedeiro. Neste estudo, inicialmente foi caracterizada a virulência entre duas cepas de L. infantum (BH46 e BH400) e em seguida foi comparada a expressão diferencial de proteínas entre as cepas. Dessa forma, através de eletroforese bidimensional e Differential Gel Electrophoresis (DIGE), foram avaliados os perfis proteômicos das formas promastigota entre as diferentes cepas. Posteriormente, as proteínas foram identificadas por Espectrometria de Massas. Essas análises nos permitiram selecionar e identificar proteínas possivelmente associadas à diferença de virulência entre as cepas BH46 e BH400, entre elas estão a Triparredoxina, S-Adenosil Homocisteina Hidrolase, HSP- 70, Metalo-Peptidase, Clan MA(E), Família M3, Enolase, Fosfomanomutase, Peroxirredoxina, proteína 14-3-3 like, proteína Rab 1 ligante à GTP e KMP-11. Através da predição da rede de interações entre as proteínas, foi observado que muitas vezes as proteínas descritas como envolvidas na virulência interagem entre si, tornando importante o estudo de todos os complexos proteicos globais do organismo, e não apenas uma proteína individualmente. Assim, a caracterização de fatores envolvidos com a diferença de virulência entre BH46 e BH400 permitirá um melhor entendimento sobre a relação parasito-hospedeiro. Leishmania infantum, synonymy with L. chagasi, is the protozoan responsible for Visceral Leishmaniasis in the Americas and Europe, a serious disease that can be fatal if untreated. Human cases range from subclinical infection, with an undetermined number of asymptomatic patients in endemic areas, a growing cadre of co-infection with HIV and high mortality in children. In dogs, the disease is severe, characterized by chronic and progressive cachexia, hepatosplenomegaly, lymphadenopathy, and pancytopenia onychogryphosis. However, less than 60% of the infected dogs present clinical signs of the disease, suggesting that host and parasitefactores are involved in clinical variability of disease. Thus, due to its importance and clinical and epidemiological diversity in our country, the study of virulence factors in L. (L.) infantum appears to be quite promising for understanding the host-parasite relationship. This study compared the differential expression of proteins between two strains of L. (L.) infantum (BH46 and BH400) with difference in virulence. Therefore, using two-dimensional electrophoresis and Differential Gel Electrophoresis (DIGE) will be assessed proteomic profiles of promastigote forms between different strains. These analyzes allowed us to select and identify proteins possibly related to the difference in virulence between strains BH46 and BH400, among them are the Tryparedoxin, S-Adenosyl homocysteine Hydrolase, HSP-70, Metallo-Peptidase, Clan MA(E) Family M3, Enolase, Phosphomannomutase, Peroxiredoxin, 14-3-3 protein like protein, GTP binding protein Rab 1 and KMP-11. Through the prediction of the network of interactions between the proteins was observed that often described as proteins involved in virulence interact with each other making important to study all overall organism protein complexes, and not only a protein individually. Thus, the characterization of factors involved in the virulence difference between BH46 and BH400 will enable a better understanding of the host-parasite relationship.