RESUMEN

The phytopathogenic fungus Chrysoporthe cubensis is a relevant source of lignocellulolytic enzymes. This work aimed to compare the profile of lignocellulose-degrading proteins secreted by C. cubensis grown under semi-solid state fermentation using wheat bran (WB) and sugarcane bagasse (SB). The exoproteomes of the fungus grown in wheat bran (WBE) and sugarcane bagasse (SBE) were qualitative and quantitatively analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Data are available via ProteomeXchange with identifier PXD046075. Label-free proteomic analysis of WBE and SBE showed that the fungus produced a spectrum of carbohydrate-active enzymes (CAZymes) with exclusive characteristics from each extract. While SBE resulted in an enzymatic profile directed towards the depolymerization of cellulose, the enzymes in WBE were more adaptable to the degradation of biomass rich in hemicellulose and other non-lignocellulosic polymers. Saccharification of alkaline pre-treated sugarcane bagasse with SBE promoted glucose release higher than commercial cocktails (8.11 g L-1), while WBE promoted the higher release of xylose (5.71 g L-1). Our results allowed an in-depth knowledge of the complex set of enzymes secreted by C. cubensis responsible for its high lignocellulolytic activity and still provided the identification of promising target proteins for biotechnological applications in the context of biorefinery.

Asunto(s)

Celulosa , Saccharum , Celulosa/metabolismo , Proteómica , Saccharum/metabolismo , Espectrometría de Masas en Tándem , Proteínas Fúngicas/metabolismo , Fibras de la Dieta/metabolismo , Hidrólisis

RESUMEN

Schistosomes are blood flukes with specialised tissues and organs, each one playing a pivotal role in perpetuating the parasite life cycle. Herein, we describe a detailed methodology for preserving the proteome of adult Schistosoma mansoni worms during manual dissection for enrichment of tissues associated with the parasite's alimentary tract. We provide step-by-step directions for specimen storage and dissection while in preservative solution, tissue homogenisation, protein extraction and digestion using a methodology fully compatible with downstream quantitative liquid chromatography-mass spectrometry analysis. Our methodology uses label-free and QconCAT-based absolute quantification for detection of S. mansoni oesophageal gland products proposed as vaccine candidates. Through stabilisation of the proteome and minimising sample degradation during dissection our approach has allowed us to access the hidden proteome of target tissues not readily available from total lysates because of their small volume. This protocol can be replicated or adapted to other Schistosoma species lacking quantitative proteomics characterisation of specialised tissues for discovery of proteins with potential diagnostic and therapeutic utility.

Asunto(s)

Cromatografía Líquida con Espectrometría de Masas , Proteómica , Animales , Proteómica/métodos , Cromatografía Liquida , Proteoma/metabolismo , Espectrometría de Masas en Tándem , Schistosoma mansoni/química , Schistosoma mansoni/metabolismo

RESUMEN

INTRODUCTION: Schistosomes are long-lived blood dwelling helminth parasites using intricate mechanisms to invade, mature, and reproduce inside their vertebrate hosts, whilst simultaneously deploying immune evasion strategies. Their multi-tissue organization and solid body plan presents particular problems for the definition of sub-proteomes. AREAS COVERED: Here, we focus on the two host-parasite interfaces of the adult worm accessible to the immune system, namely the tegument and the alimentary tract, but also on the secretions of the infective cercaria, the migrating schistosomulum and the mature egg. In parallel, we introduce the concepts of "leakyome' and 'disintegrome' to emphasize the importance of interpreting data in the context of schistosome biology so that misleading conclusions about the distinct proteome compositions are avoided. Lastly, we highlight the possible clinical implications of the reviewed proteomic findings for pathogenesis, vaccine design and diagnostics. EXPERT OPINION: Proteomics has provided considerable insights into the biology of schistosomes, most importantly for rational selection of novel vaccine candidates that might confer protective immunity, but also into the pathogenesis of schistosomiasis. However, given the increasing sensitivity of mass spectrometric instrumentation, we stress the need for care in data interpretation since schistosomes do not deviate from the fundamental rules of eukaryotic cell biology.

Asunto(s)

Esquistosomiasis , Vacunas , Animales , Proteómica/métodos , Proteínas del Helminto , Schistosoma , Esquistosomiasis/parasitología , Esquistosomiasis/prevención & control , Proteoma/genética

RESUMEN

Extracellular adenosine plays important roles in modulating the immune responses. We have previously demonstrated that infection of dendritic cells (DC) by Leishmania amazonensis leads to increased expression of CD39 and CD73 and to the selective activation of the low affinity A2B receptors (A2B R), which contributes to DC inhibition, without involvement of the high affinity A2A R. To understand this apparent paradox, we now characterized the alterations of both adenosine receptors in infected cells. With this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Fluorescence microscopy revealed that L. amazonensis infection stimulates the recruitment of A2B R, but not of A2A R, to the surface of infected DC, without altering the amount of mRNA or the total A2B R density, an effect dependent on lipophosphoglycan (LPG). Log-phase promastigotes or axenic amastigotes of L. amazonensis do not stimulate A2B R recruitment. A2B R clusters are localized in caveolin-rich lipid rafts and the disruption of these membrane domains impairs A2B R recruitment and activation. More importantly, our results show that A2B R co-localize with CD39 and CD73 forming a "purinergic cluster" that allows for the production of extracellular adenosine in close proximity with these receptors. We conclude that A2B R activation by locally produced adenosine constitutes an elegant and powerful evasion mechanism used by L. amazonensis to down-modulate the DC activation.

Asunto(s)

5'-Nucleotidasa/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Caveolina 1/metabolismo , Células Dendríticas/inmunología , Leishmaniasis/inmunología , Microdominios de Membrana/inmunología , Receptor de Adenosina A2B/metabolismo , Animales , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Células Dendríticas/patología , Inmunidad , Inmunomodulación , Leishmania/inmunología , Leishmaniasis/metabolismo , Leishmaniasis/parasitología , Leishmaniasis/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/parasitología , Macrófagos/patología , Masculino , Microdominios de Membrana/parasitología , Microdominios de Membrana/patología , Ratones , Ratones Endogámicos C57BL

RESUMEN

The phytopathogenic fungus Chrysoporthe cubensis has a great capacity to produce highly efficient enzymes for the hydrolysis of lignocellulosic biomass. The bioinfosecretome of C. cubensis was identified by computational predictions of secreted proteins combined with protein analysis using 1D-LC-MS/MS. The in silico secretome predicted 562 putative genes capable of encoding secreted proteins, including 273 CAZymes. Proteomics analysis confirmed the existence of 313 proteins, including 137 CAZymes classified as Glycosyl Hydrolases (GH), Polysaccharide Lyases (PL), Carbohydrate Esterases (CE) and Auxiliary Activities enzymes (AA), which indicates the presence of classical and oxidative cellulolytic mechanisms. The enzymes diversity in the extract shows fungal versatility to act in complex biomasses. This study provides an insight into the lignocellulose-degradation mechanisms by C. cubensis and allows the identification of the enzymes that are potentially useful in improving industrial process of bioconversion of lignocellulose. SIGNIFICANCE: Chrysoporthe cubensis is an important deadly canker pathogen of commercially cultivated Eucalyptus species. The effective depolymerisation of the recalcitrant plant cell wall performed by this fungus is closely related to its high potential of lignocellulolytic enzymes secretion. Since the degradation of biomass occurs in nature almost exclusively by enzyme secretion systems, it is reasonable to suggest that the identification of C. cubensis lignocellulolytic enzymes is relevant in contributing to new sustainable alternatives for industrial solutions. As far as we know, this work is the first accurate proteomic evaluation of the enzymes secreted by this species of fungus. The integration of the gel-based proteomic approach, the bioinformatic prediction of the secretome and the analyses of enzymatic activity are powerful tools in the evaluation of biotechnological potential of C. cubensis in producing carbohydrate-active enzymes. In addition, analysis of the C. cubensis secretome grown in wheat bran draws attention to this plant pathogen and its extracellular enzymatic machinery, especially regarding the identification of promising new enzymes for industrial applications. The results from this work allowed for explanation and reinforce previous research that revealed C. cubensis as a strong candidate to produce enzymes to hydrolyse sugarcane bagasse and similar substrates.

Asunto(s)

Ascomicetos , Proteómica , Biomasa , Cromatografía Liquida , Hidrólisis , Espectrometría de Masas en Tándem

RESUMEN

The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity against Schistosoma mansoni. Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 55 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyzer software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritizing vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.

RESUMEN

Peptide-mediated targeting to colorectal cancer can increase selectivity and specificity of this cancer diagnosis acting as biomarkers. The present work aimed to select peptides using the phage display technique and associate the peptides with polymeric nanospheres in order to evaluate their cytotoxicity and selectivity during cell interaction with Caco-2 human colon tumor cell line. Two peptides identified by phage display (peptide-1 and peptide-2) were synthesized and exhibited purity higher than 84%. Poly(lactic acid)-block-polyethylene glycol nanospheres were prepared by nanoprecipitation and double emulsion methods in order to load the two peptides. Nanoparticles ranged in size from 114 to 150 nm and peptide encapsulation efficiency varied from 16 to 32%, depending on the methodology. No cytotoxic activity was observed towards Caco-2 tumor cell line, either free or loaded peptides in concentrations up to 3 µM at incubation times of 6 and 24 h, indicating safety as biomarkers. Fluorescein isothiocyanate-labeled peptides allowed evaluating selective interactions with Caco-2 cells, where peptide-1 entrapped in nanospheres showed greater intensity of co-localized cell fluorescence, in comparison to peptide-2. Peptide-1 loaded in nanospheres revealed promising to be investigated in further studies of selectivity with other human colon rectal cells as a potential biomarker.Graphical abstract.

Asunto(s)

Adenocarcinoma , Neoplasias Colorrectales , Nanosferas , Péptidos , Adenocarcinoma/diagnóstico , Bacteriófagos , Biomarcadores , Células CACO-2 , Técnicas de Visualización de Superficie Celular , Neoplasias Colorrectales/diagnóstico , Humanos , Tamaño de la Partícula , Poliésteres , Polietilenglicoles

RESUMEN

NTPDases (EC 3.6.1.5) are enzymes belonging to a protein family which have as a common feature the ability to hydrolyze di- and triphosphate nucleotides (ADP and ATP) to monophosphate nucleosides (AMP) in the presence of Ca+2 and Mg+. The potato apyrase has been the first protein of the NTPDase family to be purified. In mammals, these enzymes are involved in physiologic and sick processes as thromboregulation, inflammatory and immunologic responses. In this study, we investigated the in vitro potential of synthetic chalcones on the inhibition of potato apyrase purified from Solanum tuberosum. The protein was purified with high grade purity and its identity was confirmed by electrophoresis, western blot, and LC-MS/MS. Five out of the eight chemically synthetized chalcones analyzed in this study showed significant inhibition of the apyrase activity. The compound with the best rate of inhibition of ATP hydrolytic activity was able to promote 54% inhibition with a concentration of 3.125 µM. Ticlopidine, used as an inhibition drug control, was able to promote inhibitions around 50% of the activity (IC50 = 2.167 µM). Our results with the potato apyrase inhibition with the synthetic chalcones suggest that these compounds may use as potential lead candidates for the treatment of some diseases associated with nucleotides.

Asunto(s)

Adenosina Trifosfato/química , Apirasa/antagonistas & inhibidores , Chalconas/química , Adenosina Trifosfato/genética , Secuencia de Aminoácidos/genética , Antígenos CD/química , Antígenos CD/genética , Apirasa/química , Apirasa/genética , Biotecnología , Chalconas/farmacología , Cromatografía Liquida , Humanos , Hidrólisis/efectos de los fármacos , Ingeniería de Proteínas , Solanum tuberosum/enzimología , Espectrometría de Masas en Tándem

RESUMEN

In experimental infection with Strongyloides venezuelensis, the acute and recovery phases can be distinguished, unlike human infections caused by Strongyloides stercoralis. The objective of this study was to evaluate the production of anti-Strongyloides IgG antibodies and the recognition of immunogenic protein bands during the acute and the recovery phases in rats experimentally infected with S. venezuelensis. Rats were infected subcutaneously with 400 or 4,000 S. venezuelensis infective larvae. The acute phase was characterized by elimination of a large number of eggs in the faeces on days 6-14 post infection; the recovery phase was characterized by the resolution of the infection between days 30 and 35 post infection. Differences in IgG levels were observed in the acute and the recovery phases. Different antigenic fractions were recognized in both phases of infection. It is concluded that proteins within the 30-40 kDa range are immunoreactive markers for both the acute and the recovery phases in rats experimentally infected with S. venezuelensis, particularly using membrane antigen.

Asunto(s)

Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/inmunología , Proteínas del Helminto/inmunología , Inmunoglobulina G/inmunología , Estrongiloidiasis/inmunología , Enfermedad Aguda , Animales , Western Blotting , Reacciones Cruzadas , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Heces/parasitología , Masculino , Ratas Wistar , Factores de Tiempo

RESUMEN

The Asian invasive species Limnoperna fortunei (Dunker, 1857), known as the golden mussel, causes great economic and environmental damage due to its fixative capacity and accelerated proliferation. Molecular studies for the control of larval and adult forms are of great economic, scientific and technological interest. Here, we first report on the compositional analysis of the L. fortunei proteome obtained through shotgun analysis using LC-MS/MS. Among those 2790 proteins identified, many of them related to secretory processes and membrane receptors. Our second approach consisted in exposing the mollusc to the molluscicide niclosamide to evaluate the induced proteomic alterations. Exposure to niclosamide at 0.25 mg/L for 24 h resulted in a pronounced differential abundance of proteins when compared to those obtained when exposure was reduced to 4 h at 2.3 mg/L. In total, 342 proteins were found differentially expressed in the responsive individuals as revealed by label-free quantitative proteomics. Regarding the affected cell processes were: cell division and differentiation, cytoskeletal organization and compartment acidification (upregulated), and energy metabolism (downregulated). Our findings constitute the first inventory of the expressed proteome of the golden mussel and have the potential to contribute with a more rational proposition of molecular targets for control and monitoring of this species. SIGNIFICANCE: With the recent availability of transcriptomic and genomic data applied to L. fortunei the timing is right to interrogate its putative gene repertoire using proteomic techniques. These have the potential to validate the existence of the predicted genes, infer their relative abundance and quantify their levels as a response to environmental stressors or various agents. Here we provided an inventory of the golden mussel proteome and evaluated its response to the molluscicide niclosamide. The obtained results open new avenues for intervention aimed at its control or elimination, particularly by targeting the various cellular processes that were uncovered.

Asunto(s)

Niclosamida , Proteoma , Animales , Cromatografía Liquida , Proteómica , Espectrometría de Masas en Tándem

RESUMEN

Cardiovascular diseases (CVD) are the leading cause of death worldwide. Growth factor therapy has emerged as novel therapeutic strategy under investigation for CVD. In this sense, adrenomedullin-2 (ADM-2) has been recently identified as a new angiogenic factor able to regulate the regional blood flow and cardiovascular function. However, the therapeutic value of ADM-2 is limited by its short biological half-life and low plasma stability. Poly (lactic-co-glycolic acid) (PLGA) micro- and nanoparticles have been investigated as growth factor delivery systems for cardiac repair. In this study, we aimed to develop PLGA nanoparticles containing ADM-2 intended for therapeutic angiogenesis. PLGA nanoparticles containing ADM-2 were prepared by a double emulsion modified method, resulting in 300 nm-sized stable particles with zeta potential around - 30 mV. Electron microscopy analysis by SEM and TEM revealed spherical particles with a smooth surface. High encapsulation efficiency was reached (ca.70%), as quantified by ELISA. ADM-2 associated to polymer nanoparticles was also determined by EDS elemental composition analysis, SDS-PAGE and LC-MS/MS for peptide identification. In vitro release assays showed the sustained release of ADM-2 from polymer nanoparticles for 21 days. Cell viability experiments were performed in J774 macrophages and H9c2 cardiomyocyte cells, about which PLGA nanoparticles loaded with ADM-2 did not cause toxicity in the range 0.01-1 mg/ml. Of note, encapsulated ADM-2 significantly induced cell proliferation in EA.hy926 endothelial cells, indicating the ADM-2 bioactivity was preserved after the encapsulation process. Collectively, these results demonstrate the feasibility of using PLGA nanoparticles as delivery systems for the angiogenic peptide ADM-2, which could represent a novel approach for therapeutic angiogenesis in CVD using growth factor therapy.

Asunto(s)

Inductores de la Angiogénesis/administración & dosificación , Proliferación Celular/efectos de los fármacos , Portadores de Fármacos , Células Endoteliales/efectos de los fármacos , Hormonas Peptídicas/administración & dosificación , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Inductores de la Angiogénesis/química , Inductores de la Angiogénesis/toxicidad , Animales , Línea Celular , Preparaciones de Acción Retardada , Composición de Medicamentos , Liberación de Fármacos , Humanos , Cinética , Ratones , Nanopartículas , Hormonas Peptídicas/química , Hormonas Peptídicas/toxicidad , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/toxicidad , Ratas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Solubilidad

RESUMEN

Schistosomes are blood-dwelling helminth parasites that cause schistosomiasis, a debilitating disease resulting in inflammation and, in extreme cases, multiple organ damage. Major challenges to control the transmission persist, and the discovery of protective antigens remains of critical importance for vaccine development. Rhesus macaques can self-cure following schistosome infection, generating antibodies that target proteins from the tegument, gut, and esophagus, the last of which is the least investigated. We developed a dissection technique that permitted increased sensitivity in a comparative proteomics profiling of schistosome esophagus and gut. Proteome analysis of the male schistosome esophagus identified 13 proteins encoded by microexon genes (MEGs), 11 of which were uniquely located in the esophageal glands. Based on this and transcriptome information, a QconCAT was designed for the absolute quantification of selected targets. MEGs 12, 4.2, and 4.1 and venom allergen-like protein 7 were the most abundant, spanning over 245 million to 6 million copies per cell, while aspartyl protease, palmitoyl thioesterase, and galactosyl transferase were present at <1 million copies. Antigenic variation by alternative splicing of MEG proteins was confirmed together with a specialized machinery for protein glycosylation/secretion in the esophagus. Moreover, some gastrodermal secretions were highly enriched in the gut, while others were more uniformly distributed throughout the parasite, potentially indicating lysosomal activity. Collectively, our findings provide a more rational, better-oriented selection of schistosome vaccine candidates in the context of a proven model of protective immunity.

Asunto(s)

Tracto Gastrointestinal/metabolismo , Proteínas del Helminto/metabolismo , Proteómica/métodos , Schistosoma mansoni/metabolismo , Animales , Esófago/metabolismo , Ontología de Genes , Proteínas del Helminto/análisis , Proteínas del Helminto/genética , Masculino , Ratones , Schistosoma mansoni/patogenicidad , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem

RESUMEN

The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity against Schistosoma mansoni. Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 55 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyzer software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritizing vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.

Asunto(s)

Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/inmunología , Mapeo Epitopo , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Animales , Antígenos Helmínticos/genética , Ratones , Ratones Noqueados , Schistosoma mansoni/genética , Esquistosomiasis mansoni/genética , Esquistosomiasis mansoni/prevención & control , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología

RESUMEN

Strongyloides venezuelensis is an important alternative source of antigen for the serologic diagnosis of human strongyloidiasis. Proteomics techniques applied to the analysis of the protein content of infective third stage larvae (iL3) of S. venezuelensis provide a powerful tool for the discovery of new candidates for immunodiagnosis. This study presents an overview of the protein iL3 S. venezuelensis focusing on the diagnosis of strongyloidiasis. A total of 877 proteins were identified by shotgun proteomics. Many of these proteins are involved in different cellular processes, metabolic as well as structural maintenance. Our results point to a catalog of possible diagnostic targets for human strongyloidiasis and highlight the need for evaluation of uncharacterized proteins, especially the proteins within the CAP domain, transthyretin, and BTPI inhibitor domains, as a repertoire as yet unexplored in the context of strongyloidiasis diagnostic markers. We believe that the protein profile presented in this shotgun analysis extends our understanding of the protein composition within the Strongyloides genus, opening up new perspectives for research on biomarkers that may help with the diagnosis of human strongyloidiasis. Data are available via ProteomeXchange with identifier PXD013703.

Asunto(s)

Biomarcadores/metabolismo , Larva/metabolismo , Proteoma , Strongyloides/metabolismo , Estrongiloidiasis/diagnóstico , Animales , Catepsinas/metabolismo , Galectinas/metabolismo , Interacciones Huésped-Parásitos , Humanos , Pruebas Inmunológicas , Metaloproteasas/metabolismo , Patología Molecular , Proteómica

RESUMEN

The population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations.

Asunto(s)

Anticuerpos Antihelmínticos/análisis , Antígenos Helmínticos/análisis , Técnicas de Laboratorio Clínico/métodos , Heces/parasitología , Schistosoma mansoni , Esquistosomiasis mansoni/diagnóstico , Animales , Brasil/epidemiología , Enfermedades Endémicas , Humanos , Técnicas para Inmunoenzimas , Recuento de Huevos de Parásitos , Schistosoma mansoni/inmunología , Schistosoma mansoni/aislamiento & purificación , Esquistosomiasis mansoni/epidemiología , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad

RESUMEN

BACKGROUND: Despite decades of use of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there needs to be better diagnostic tools to detect low-intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schistosome-specific immune responses in hopes of developing sensitive and specific new methods for immunodiagnosis. METHODS AND FINDINGS: Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S. mansoni-infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1-10 eggs per gram of feces that were undiagnosed by KK parasitological technique. CONCLUSIONS: The serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed.

Asunto(s)

Antígenos Helmínticos/sangre , Proteínas del Helminto/sangre , Proteoma/metabolismo , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos Helmínticos/inmunología , Biomarcadores/sangre , Brasil , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Heces/parasitología , Femenino , Proteínas del Helminto/inmunología , Humanos , Inmunoglobulina G/sangre , Lactante , Masculino , Persona de Mediana Edad , Óvulo/inmunología , Recuento de Huevos de Parásitos , Proteoma/inmunología , Proteómica , Proteínas Recombinantes/inmunología , Esquistosomiasis mansoni/sangre , Sensibilidad y Especificidad , Pruebas Serológicas/métodos

RESUMEN

Organisms, in general, respond to environmental stress by altering their pattern of protein expression (proteome), as an alternative to growing in stressful conditions. A strain of Meyerozyma guilliermondii resistant to manganese was isolated from a sample of water collected from mine drainage in southeastern Minas Gerais (Brazil), and demonstrated manganese detoxification capacity. Protein extracts containing the soluble fractions were obtained after growth of the strain in the absence and presence of MnSO4. Tryptic peptides recovered from samples were analyzed by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Shotgun/bottom-up analyses of the soluble fractions revealed a total of 1257 identified molecules. Treatment with Mn did not affect the growth of yeast but induced changes in the protein profile, with 117 proteins expressed in the absence of Mn and 69 expressed only in its presence. Most of these are annotated as related to DNA repair, oxidoreductase activity, and remodeling of gene expression. This is the first proteomic report of M. guilliermondii, with promising characteristics for Mn bioremediation, and the first of the genus Meyerozyma. This proteomic characterization may help in the understanding of molecular regulatory mechanisms associated with tolerance to excess Mn, and the potential use of biomass in bioremediation processes. SIGNIFICANCE: Environmental pollution by heavy metals such as manganese (Mn2+) has increased as it is a by-product of the mining industry and a potential environmental contaminant. Many studies have explored the use of bacteria for manganese bioremediation, but yeasts have emerged as a promising alternative, displaying faster growth and greater removal efficiency. Previous works of our laboratory showed that Meyerozyma guilliermondii, a non-pathogenic haploid yeast (ascomycete), has excellent removal and accumulation capacity of Mn2+, potentially useful in bioremediation. Nowadays efforts have been devoted to understanding the physiology of metal hyperaccumulation to gain insights into the molecular basis of hyperaccumulation. To obtain a comprehensive understanding of the molecular mechanism of Mn2+ hyperaccumulation in M. guilliermondii, proteomic approaches were employed yielding the first compositional proteomic map of total soluble proteins and their differential expression in the presence of Mn2+. We believe our findings are of biotechnological interest concerning the utilization of M. guilliermondii for bioremediation purposes.

Asunto(s)

Ascomicetos/metabolismo , Proteínas Fúngicas/biosíntesis , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Compuestos de Manganeso/farmacología , Estrés Oxidativo/efectos de los fármacos , Sulfatos/farmacología , Ascomicetos/genética , Proteínas Fúngicas/genética , Manganeso/farmacología , Proteómica

RESUMEN

The population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations.

Asunto(s)

Humanos , Schistosoma mansoni , Inmunoensayo

RESUMEN

BACKGROUND: Trypanosoma cruzi, a hemoflagellate protozoan parasite and the etiological agent of Chagas disease (CD), exhibits great genetic and biological diversity. Infected individuals may present clinical manifestations with different levels of severity. Several hypotheses have been proposed to attempt to correlate the diversity of clinical signs and symptoms to the genetic variability of T. cruzi. This work aimed to investigate the differential expression of proteins from two distinct genetic groups of T. cruzi (discrete typing units TcI and TcII), isolated from chronically infected individuals displaying the cardiac form of CD. For this purpose, epimastigote forms of the two isolates were cultured in vitro and the cells recovered for protein extraction. Comparative two-dimensional (2D) gel electrophoreses were performed and differentially expressed spots selected for identification by mass spectrometry, followed by database searching and protein categorization. RESULTS: The 2D electrophoretic profiles revealed the complex composition of the T. cruzi extracted proteome. Protein spots were distributed along the entire pH and molecular mass ranges attesting for the integrity of the protein preparations. In total, 46 differentially expressed proteins were identified present in 40 distinct spots found in the comparative gel analyses. Of these, 16 displayed upregulation in the gel from TcI-typed parasites and 24 appeared overexpressed in the gel from TcII-typed parasites. Functional characterization of differentially expressed proteins revealed major alterations associated with stress response, lipid and amino acid metabolism in parasites of the TcII isolate, whilst those proteins upregulated in the TcI sample were primarily linked to central metabolic pathways. CONCLUSIONS: The comparative 2D-gel electrophoresis allowed detection of major differences in protein expression between two T. cruzi isolates, belonging to the TcI and TcII genotypes. Our findings suggest that patients displaying the cardiac form of the disease harbor parasites capable of exhibiting distinct proteomic profiles. This should be of relevance to disease prognosis and treatment.

Asunto(s)

Cardiomiopatía Chagásica/parasitología , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Electroforesis en Gel Bidimensional , Femenino , Variación Genética , Genotipo , Humanos , Proteómica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/química , Trypanosoma cruzi/aislamiento & purificación , Trypanosoma cruzi/metabolismo