RESUMO
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.
Assuntos
Espectrometria de Massa com Cromatografia Líquida , Proteômica , Animais , Proteômica/métodos , Cromatografia Líquida , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Schistosoma mansoni/química , Schistosoma mansoni/metabolismoRESUMO
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.
Assuntos
Esquistossomose , Vacinas , Animais , Proteômica/métodos , Proteínas de Helminto , Schistosoma , Esquistossomose/parasitologia , Esquistossomose/prevenção & controle , Proteoma/genéticaRESUMO
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.
Assuntos
5'-Nucleotidase/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Caveolina 1/metabolismo , Células Dendríticas/imunologia , Leishmaniose/imunologia , Microdomínios da Membrana/imunologia , Receptor A2B de Adenosina/metabolismo , Animais , Células Dendríticas/metabolismo , Células Dendríticas/parasitologia , Células Dendríticas/patologia , Imunidade , Imunomodulação , Leishmania/imunologia , Leishmaniose/metabolismo , Leishmaniose/parasitologia , Leishmaniose/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Macrófagos/patologia , Masculino , Microdomínios da Membrana/parasitologia , Microdomínios da Membrana/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Trato Gastrointestinal/metabolismo , Proteínas de Helminto/metabolismo , Proteômica/métodos , Schistosoma mansoni/metabolismo , Animais , Esôfago/metabolismo , Ontologia Genética , Proteínas de Helminto/análise , Proteínas de Helminto/genética , Masculino , Camundongos , Schistosoma mansoni/patogenicidade , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Anticorpos Anti-Helmínticos/análise , Antígenos de Helmintos/análise , Técnicas de Laboratório Clínico/métodos , Fezes/parasitologia , Schistosoma mansoni , Esquistossomose mansoni/diagnóstico , Animais , Brasil/epidemiologia , Doenças Endêmicas , Humanos , Técnicas Imunoenzimáticas , Contagem de Ovos de Parasitas , Schistosoma mansoni/imunologia , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/epidemiologia , Sensibilidade e Especificidade , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Proteomics is an important tool for the investigation of dynamic physiological responses of microbes under heavy metal stress. To gain insight into how bacteria respond to manganese (II) and identify the proteins involved in Mn (II) oxidation, the shotgun proteomics approach was applied to a potential Mn (II)-oxidizing Serratia marcescens strain cultivated in the absence and presence of Mn (II). RESULTS: The LG1 strain, which grew equally well in the two conditions, was found to express a set of proteins related to cellular processes vital for survival, as well as proteins involved in adaptation and tolerance to Mn (II). The multicopper oxidase CueO was identified, indicating its probable participation in the Mn (II) bio-oxidation; however, its expression was not modulated by the presence of Mn (II). A set of proteins related to cell and metabolic processes vital to the cells were downregulated in the presence of Mn (II), while cell membrane-related proteins involved in the maintenance of cell integrity and survival under stress were upregulated under this condition. CONCLUSIONS: These findings indicate that the LG1 strain may be applied successfully in the bioremediation of Mn (II), and the shotgun approach provides an efficient means for obtaining the total proteome of this species.
Assuntos
Proteínas de Bactérias/metabolismo , Manganês/metabolismo , Serratia marcescens/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteoma/química , Proteoma/genética , Proteoma/metabolismo , Proteômica , Serratia marcescens/química , Serratia marcescens/genética , Serratia marcescens/crescimento & desenvolvimentoRESUMO
The PR-11 peptide corresponds to the N-terminal and active region of the endogenously synthesized PR-39 molecule, of porcine origin. It is known to possess various biological effects including antimicrobial properties, angiogenic and anti-inflammatory activities. Apart from its reported activity as a proteasome inhibitor, a more comprehensive understanding of its function, at the molecular level, is still lacking. In this study, we used a label-free shotgun strategy to evaluate the proteomic alterations caused by exposure of cultured fibroblasts to the peptide PR-11. This approach revealed that more than half of the identified molecules were related to signalling, transcription and translation. Proteins directly associated to regulation of angiogenesis and interaction with the hypoxia-inducible factor 1-α (HIF-1α) were significantly altered. In addition, at least three differentially expressed molecules of the NF-κB pathway were detected, suggesting an anti-inflammatory property of PR-11. At last, we demonstrated novel potential ligands of PR-11, through its immobilization for affinity chromatography. Among the eluted molecules, gC1qR, a known complement receptor, appeared markedly enriched. This provided preliminary evidence of a PR-11 ligand possibly involved in the internalization of this peptide. Altogether, our findings contributed to a better understanding of the cellular pathways affected by PR-39 derived molecules.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Imobilizadas/metabolismo , Proteínas Imobilizadas/farmacologia , Ligantes , Espectrometria de Massas , Proteínas Mitocondriais/metabolismo , NF-kappa B/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Proteômica , Ratos , Ratos Wistar , SuínosRESUMO
VIP36 is a protein described as an L-type lectin in animals, responsible for the intracellular transport of glycoproteins within the secretory pathway, and also localized on the plasma membrane. Schistosoma mansoni has a complex system of vesicles and protein transport machinery to the cell surface. The excreted/secreted products of the larvae and eggs are known to be exposed to the host immune system. Hence, characterizing the role and action of SmVIP36 in the S. mansoni life cycle is important for a better understanding of the parasite-host relationship. To this purpose, we firstly performed in silico analysis. Analysis of SmVIP36 in silico revealed that it contains a lectin leg-like domain with a jellyroll fold as seen by its putative 3D tertiary structure. Additionally, it was also observed that its CRD contains calcium ion-binding amino acids, suggesting that the binding of SmVIP36 to glycoproteins is calcium-dependent. Finally, we observed that the SmVIP36 predicted amino acid sequence relative to its orthologs was conserved. However, phylogenetic analysis revealed that SmVIP36 follows species evolution, forming a further cluster with its definitive host Homo sapiens. Moreover, q-PCR analysis in the S. mansoni life cycle points to a significant increase in gene expression in the eggs, schistosomulae, and female adult stages. Similarly, protein expression increased in eggs, cercariae, schistosomulae, and adult worm stages. These results suggest that SmVIP36 might participate in the complex secretory activity within the egg envelope and tegument proteins, both important for the stages of the parasite that interact with the host.
Assuntos
Proteínas de Helminto/genética , Lectinas/genética , Proteínas de Membrana/genética , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/genética , Sequência de Aminoácidos , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Feminino , Expressão Gênica , Proteínas de Helminto/metabolismo , Humanos , Lectinas/metabolismo , Estágios do Ciclo de Vida , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Filogenia , Transporte Proteico , Schistosoma mansoni/classificação , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/parasitologiaRESUMO
Schistosomes are parasitic flatworms that infect >200 million people worldwide, causing the chronic, debilitating disease schistosomiasis. Unusual among parasitic helminths, the long-lived adult worms, continuously bathed in blood, take up nutrients directly across the body surface and also by ingestion of blood into the gut. Recent proteomic analyses of the body surface revealed the presence of hydrolytic enzymes, solute, and ion transporters, thus emphasising its metabolic credentials. Furthermore, definition of the molecular mechanisms for the uptake of selected metabolites (glucose, certain amino acids, and water) establishes it as a vital site of nutrient acquisition. Nevertheless, the amount of blood ingested into the gut per day is considerable: for males â¼100 nl; for the more actively feeding females â¼900 nl, >4 times body volume. Ingested erythrocytes are lysed as they pass through the specialized esophagus, while leucocytes become tethered and disabled there. Proteomics and transcriptomics have revealed, in addition to gut proteases, an amino acid transporter in gut tissue and other hydrolases, ion, and lipid transporters in the lumen, implicating the gut as the site for acquisition of essential lipids and inorganic ions. The surface is the principal entry route for glucose, whereas the gut dominates amino acid acquisition, especially in females. Heme, a potentially toxic hemoglobin degradation product, accumulates in the gut and, since schistosomes lack an anus, must be expelled by the poorly understood process of regurgitation. Here we place the new observations on the proteome of body surface and gut, and the entry of different nutrient classes into schistosomes, into the context of older studies on worm composition and metabolism. We suggest that the balance between surface and gut in nutrition is determined by the constraints of solute diffusion imposed by differences in male and female worm morphology. Our conclusions have major implications for worm survival under immunological or pharmacological pressure.
Assuntos
Interações Hospedeiro-Parasita/fisiologia , Schistosoma/fisiologia , Animais , Feminino , Humanos , Masculino , Esquistossomose/fisiopatologiaRESUMO
Several signaling molecules that govern development in higher animals have been identified in the parasite Schistosoma mansoni, including the transforming growth factor ß, protein tyrosine kinases, nuclear hormone receptors, among others. The Notch pathway is a highly conserved signaling mechanism which is involved in a wide variety of developmental processes including embryogenesis and oogenesis in worms and flies. Here we aimed to provide the molecular reconstitution of the Notch pathway in S. mansoni using the available transcriptome and genome databases. Our results also revealed the presence of the transcripts coded for SmNotch, SmSu(H), SmHes, and the gamma-secretase complex (SmNicastrin, SmAph-1, and SmPen-2), throughout all the life stages analyzed. Besides, it was observed that the viability and separation of adult worm pairs were not affected by treatment with N-[N(3,5)-difluorophenacetyl)-L-Alanyl]-S-phenylglycine t-butyl ester (DAPT), a Notch pathway inhibitor. Moreover, DAPT treatment decreased the production of phenotypically normal eggs and arrested their development in culture. Our results also showed a significant decrease in SmHes transcript levels in both adult worms and eggs treated with DAPT. These results provide, for the first time, functional validation of the Notch pathway in S. mansoni and suggest its involvement in parasite oogenesis and embryogenesis. Given the complexity of the Notch pathway, further experiments shall highlight the full repertoire of Notch-mediated cellular processes throughout the S. mansoni life cycle.
Assuntos
Genoma Helmíntico/genética , Receptores Notch/genética , Schistosoma mansoni/genética , Esquistossomose mansoni/parasitologia , Transdução de Sinais , Transcriptoma , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Animais , Biologia Computacional , Diaminas/farmacologia , Feminino , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Óvulo/efeitos dos fármacos , Receptores Notch/metabolismo , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/fisiologia , Caramujos , Tiazóis/farmacologiaRESUMO
Dibenzothiophene (DBT) and its oxidized derivative dibenzothiophene sulfone (DBTO2) are important representatives of polycyclic aromatic hydrocarbons (PAHs). Due to the importance of PAHs in oncogenesis and the lack of toxicological investigations related to DBT and DBTO2, this work proposes to assess their toxic and molecular effects caused by chronic treatment of Wistar rats. In parallel, their effects were compared to those caused by treatment with 1,2-dimethylhydrazine (DMH), a classic mutagenic agent. At the 14th day post-treatment, the animals were sacrificed and blood withdrawn for hematology and evaluation of liver and pancreatic functions. No significant alterations were observed. Nevertheless, histopathological analyses revealed dysplastic lesions in the intestines of animals treated with DBT and DBTO2. CD44 and carcinoembryonic antigen (CEA) staining demonstrated an approximately 3-fold increase in expression of both tissue markers for animals administered DBT, DBTO2, and DMH. A comparative two-dimensional gel analysis revealed additional 23 proteins exhibiting altered levels in the small intestines caused by exposure to DBT and DBTO2. At last, a protein-metabolite interaction map provided major insights into the metabolism of the dysplastic tissues. Our results provided strong evidence that DBT and its derivative could potentially act as cancer inducers, highlighting their toxicological and environmental relevance.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Tiofenos/toxicidade , Alanina Transaminase/sangue , Amilases/sangue , Animais , Proteínas de Arabidopsis , Aspartato Aminotransferases/sangue , Antígeno Carcinoembrionário/metabolismo , Ciclinas , Eletroforese em Gel Bidimensional , Processamento de Imagem Assistida por Computador , Intestino Delgado/patologia , Ratos , Ratos WistarRESUMO
Small ubiquitin-like modifier (SUMO) conjugation of proteins occurs through a concert action of enzymes using a similar ubiquitination mechanism. After a C-terminal peptide is cleaved from the SUMO precursor by a protease to reveal a di-glycine motif, SUMO is activated by an E1 enzyme (Aos1/Uba2) and conjugated to target proteins by the sole E2 enzyme (Ubc9) guided to the appropriate substrates by the SUMO E3 ligase. Previous reports from our group showed that Schistosoma mansoni has two paralogs of SUMO: one E2 conjugation Ubc9 and two SUMO-specific proteases (SENPs). The differential gene expression profile observed for SUMO pathway genes throughout the S. mansoni life cycle attests for the distinct patterns of SUMO conjugates observed during parasite development particularly during the cercariae to schistosomula transition. To continue this investigation, we here analysed the repertoire of SUMO E3 ligases and their expression profiles during cercariae/schistosomula transition. In silico analysis through S. mansoni databases showed two conserved SUMO E3 ligases: protein inhibitor of activated STAT (PIAS) and Ran-binding protein 2 (RanBP2). Furthermore, expression levels of the SUMO E3 ligases were measured by qRT-PCR using total RNA from cercariae, adult worms and mechanically transformed schistosomula. Our data showed an up-regulation of expression in lung schistosomula and adult worm stages. In conclusion, the differential expression of SmPIAS and SmRanBP2 during schistosomula development was similar to the expression levels of all genes related to SUMO conjugation, thereby suggesting that the control of protein activity, localisation or stability during cercariae to schistosomula transition is SUMO-dependent.
Assuntos
Pneumopatias Parasitárias/enzimologia , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/enzimologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Biologia Computacional , Regulação Enzimológica da Expressão Gênica/fisiologia , Pneumopatias Parasitárias/metabolismo , Pneumopatias Parasitárias/patologia , Camundongos , Schistosoma mansoni/genética , Esquistossomose mansoni/metabolismo , Esquistossomose mansoni/patologia , Ativação Transcricional , Transcriptoma , Enzimas de Conjugação de Ubiquitina/genética , Regulação para CimaRESUMO
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.
Assuntos
Celulose , Saccharum , Celulose/metabolismo , Proteômica , Saccharum/metabolismo , Espectrometria de Massas em Tandem , Proteínas Fúngicas/metabolismo , Fibras na Dieta/metabolismo , HidróliseRESUMO
The ubiquitin-proteasome system is responsible for degradation of the majority of intracellular proteins in eukaryotic cells. The 26S proteasome proteolytic complex is composed of a 20S core particle responsible for protein degradation and the 19S lid which plays a role in the recognition of polyubiquitinated substrates. The 19S regulatory particle (Rps) is composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. In this study, we analyzed the expression profile of 19S Rpt subunits in the larvae and adult stage of the Schistosoma mansoni life cycle. Conventional reverse transcriptase polymerase chain reaction (RT-PCR) revealed that the majority of the 19S Rpt subunits amplified at the expected molecular masses for various investigated stages. In addition, SmRpt1, SmRpt2, and SmRpt6 transcript levels were increased in 3 h-cultured schistosomula and reasonably maintained until 5 h in culture, as revealed by qRT-PCR. Phylogenetic analysis of 19S Rpt subunits showed high structural conservation in comparison to other Rpt orthologues. The mRNA expression profile of 19S Rpt subunits did not correlate with 26S proteasome proteolytic activity as judged by a (14)C-casein-degrading assay, in the early cultured schistosomula. Taken together, these results revealed a differential expression profile for 19S Rpt subunits whose transcript levels could not be directly associated to 26S proteasome activity.
Assuntos
Regulação da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/genética , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Adenosina Trifosfatases/genética , Animais , Sequência Conservada , Perfilação da Expressão Gênica , Humanos , Larva/enzimologia , Larva/genética , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Subunidades Proteicas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The flatworm Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease that occurs throughout the developing world. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control schistosomiasis is through immunization with an antischistosomiasis vaccine combined with drug treatment. In the search for potential vaccine candidates, numerous tegument antigens have been assessed. As the major interface between parasite and mammalian host, the tegument plays crucial roles in the establishment and further course of schistosomiasis. Herein, we evaluated the potential of a GPI fraction, containing representative molecules located on the outer surface of adult worms, as vaccine candidate. Immunization of mice with GPI-anchored proteins induced a mixed Th1/Th2 type of immune response with production of IFN-γ and TNF-α, and low levels of IL-5 into the supernatant of splenocyte cultures. The protection engendered by this vaccination protocol was confirmed by 42% reduction in worm burden, 45% reduction in eggs per gram of hepatic tissue, 29% reduction in the number of granulomas per area, and 53% reduction in the granuloma fibrosis. Taken together, the data herein support the potential of surface-exposed GPI-anchored antigens from the S. mansoni tegument as vaccine candidate.
Assuntos
Glicosilfosfatidilinositóis/imunologia , Proteínas de Helminto/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/prevenção & controle , Vacinas/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Feminino , Interferon gama/biossíntese , Interleucina-5/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Esquistossomose mansoni/imunologia , Células Th1/imunologia , Células Th2/imunologia , Fator de Necrose Tumoral alfa/biossíntese , VacinaçãoAssuntos
Apirase/imunologia , Epitopos/imunologia , Imunoglobulina E/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Sequência de Aminoácidos , Animais , Apirase/genética , Reações Cruzadas/imunologia , Interações Hospedeiro-Parasita/imunologia , Imunoglobulina E/sangue , Camundongos Endogâmicos BALB C , Peptídeos/genética , Peptídeos/imunologia , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Esquistossomose mansoni/sangue , Esquistossomose mansoni/parasitologia , Homologia de Sequência de AminoácidosRESUMO
SUMO-dependent post-translational modification is implicated in a variety of cellular functions including gene expression regulation, nuclear sub-localization, and signal transduction. Conjugation of SUMO to other proteins occurs in a similar process to ubiquitination, which involves three classes of enzymes: an E1 activating, an E2 conjugating, and an E3 target-specific ligase. Ubc9 is the unique SUMO E2 enzyme known to conjugate SUMO to target substrates. Here, we present the molecular characterization of this enzyme and demonstrate its expression profile during the S. mansoni life cycle. We have used bioinformatic approaches to identify the SUMO-conjugating enzyme, the SmUbc9-like protein, in the Schistosoma mansoni databases. Quantitative RT-PCR was employed to measure the transcript levels of SUMO E2 in cercariae, adult worms, and in vitro cultivated schistosomula. Furthermore, recombinant SmUbc9 was expressed using the Gateway system, and antibodies raised in rats were used to measure SmUbc9 protein levels in S. mansoni stages by Western blotting. Our data revealed upregulation of the SmUbc9 transcript in early schistosomula followed by a marked differential gene expression in the other analyzed stages. The protein levels were maintained fairly constant suggesting a post-transcriptional regulation of the SmUbc9 mRNA. Our results show for the first time that S. mansoni employs a functional SUMO E2 enzyme, for the conjugation of the SUMO proteins to its target substrates.
Assuntos
Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Schistosoma mansoni/metabolismo , Alinhamento de Sequência , Caramujos/parasitologia , Sumoilação , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
The study aimed to determine the potential of schistosomula crude antigen (SCA) as a diagnostic target for anti-S. mansoni antibody detection. Cercariae were transformed into schistosomula, homogenized through sonication, and then centrifuged to obtain the SCA. SCA was evaluated using ELISA and dot blots immunoassays on 30 S. mansoni infected sera samples obtained from chronic patients and 30 non-infected humans' sera samples. Either Kato-Katz or saline gradient method or both were employed as the diagnostic reference. Dot blots immunoassay was further performed on protein eluted from 10 to 12 kDa immunoreactive band identified by Western blot analysis. The area under the ROC curve was 0.95 (AUC 0.95, CI 0.88-1.01, p < 0.0001). The sensitivity and specificity of SCA-ELISA and dot blots assays were 96.67% and 86.67% respectively. The human IgG-specific response against SCA was significantly higher in S. mansoni infected individuals (OD = 0.678 ± 0.249) compared to the non-infected population (OD = 0.235 ± 0.136) (p < 0.0001). Our study showed that SCA and its 10-12 kDa component could be useful as diagnostic tools for chronic schistosomiasis.
Assuntos
Antígenos de Helmintos/sangue , Schistosoma mansoni/imunologia , Esquistossomose mansoni/diagnóstico , Adolescente , Adulto , Idoso , Animais , Western Blotting , Estudos de Casos e Controles , Criança , Pré-Escolar , Eletroforese em Gel de Poliacrilamida , Doenças Endêmicas , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Esquistossomose mansoni/imunologia , Sensibilidade e Especificidade , Adulto JovemRESUMO
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.
Assuntos
Ascomicetos , Proteômica , Biomassa , Cromatografia Líquida , Hidrólise , Espectrometria de Massas em TandemRESUMO
Eggs of Schistosoma mansoni trapped in human liver can lead to fibrosis. Since liver fibrosis requires activation of hepatic stellate cells (HSC) from a quiescent to a myofibroblastic phenotype, we investigated the effects of S. mansoni eggs on this process using in vitro co-cultures with human HSC and evaluated established biomarkers for activation and fibrosis. HSC demonstrate significantly reduced expression of alpha-smooth muscle actin (p<0.001), connective tissue growth factor (p<0.01) and type I collagen (p<0.001) but significantly increased expression of peroxisome proliferator-activated receptor-gamma (p<0.01). Morphologically, HSC exhibited elongated fine cellular processes and reduced size, increased accumulation of lipid droplets and reduced expression and organization of alpha-smooth muscle actin and F-actin stress fibres. Additionally, schistosome eggs prevented the HSC fibrogenic response to exogenous transforming growth factor-beta. In summary, schistosome eggs blocked fibrogenesis in HSC, a finding which may have implications for our understanding of the fibrotic pathology in S. mansoni infections.