RESUMO
BACKGROUND: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. RESULTS: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style. CONCLUSIONS: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.
Assuntos
Doenças do Gato/microbiologia , Proteínas Fúngicas/genética , Sporothrix/genética , Esporotricose/transmissão , Fatores de Virulência/genética , Adaptação Biológica , Animais , Doenças do Gato/transmissão , Gatos , Evolução Molecular , Especiação Genética , Genoma Mitocondrial , Humanos , Filogenia , Sporothrix/classificação , Sporothrix/patogenicidade , Esporotricose/microbiologia , Esporotricose/veterináriaRESUMO
In the present work we examine the contribution of 5-lipoxygenase- (5-LO-) derived lipid mediators to immune responses during the acute phase of Trypanosoma cruzi infection in 5-LO gene knockout (5-LO(-/-)) mice and wild-type (WT) mice. Compared with WT mice, the 5-LO(-/-) mice developed less parasitemia/tissue parasitism, less inflammatory cell infiltrates, and a lower mortality. This resistance of 5-LO(-/-) mice correlated with several differences in the immune response to infection, including reduced PGE2 synthesis; sustained capacity of splenocytes to produce high levels of interleukin (IL)-12 early in the infection; enhanced splenocyte production of IL-1ß, IL-6, and IFN-γ; rapid T-cell polarization to secrete high quantities of IFN-γ and low quantities of IL-10; and greater numbers of CD8(+)CD44(high)CD62L(low) memory effector T cells at the end of the acute phase of infection. The high mortality in WT mice was associated with increased production of LTB4/LTC4, T cell bias to produce IFN-γ, high levels of serum nitrite, and marked protein extravasation into the peritoneal cavity, although survival was improved by treatment with a cys-LT receptor 1 antagonist. These data also provide evidence that 5-LO-derived mediators negatively affect host survival during the acute phase of T. cruzi infection.
Assuntos
Doença de Chagas/enzimologia , Doença de Chagas/patologia , Trypanosoma cruzi/patogenicidade , Animais , Araquidonato 5-Lipoxigenase , Doença de Chagas/genética , Doença de Chagas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Knockout , Nitritos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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 , VacinaçãoRESUMO
Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis.
Assuntos
Micélio/fisiologia , Oxirredutases/biossíntese , Paracoccidioides/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Antifúngicos/farmacologia , Antimicina A/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Humanos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Oxirredução , Estresse Oxidativo , Paracoccidioides/citologia , Paracoccidioides/crescimento & desenvolvimento , Proteínas de Plantas , Cianeto de Potássio/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regulação para CimaRESUMO
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. Several papers on Schistosoma mansoni vaccine and drug development have been published in the past few years, representing an important field of study. The advent of technologies that allow large-scale studies of genes and proteins had a remarkable impact on the screening of new and potential vaccine candidates in schistosomiasis. In this postgenomic scenario, bioinformatic technologies have emerged as important tools to mine transcriptomic, genomic, and proteomic databases. These new perspectives are leading to a new round of rational vaccine development. Herein, we discuss different strategies to identify potential S. mansoni vaccine candidates using computational vaccinology.
Assuntos
Antiparasitários/imunologia , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Proteínas de Helminto/imunologia , Schistosoma mansoni/imunologia , Esquistossomose/prevenção & controle , Vacinas/imunologia , Animais , Proteínas de Helminto/química , Proteínas de Helminto/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteômica/métodos , Análise de Sequência de DNA/métodos , Análise de Sequência de Proteína/métodos , Vacinação , Vacinas/química , Vacinas/genéticaRESUMO
Current schistosomiasis control strategies are mainly based on chemotherapy, but the development of a vaccine against this parasitic disease would contribute to a long-lasting decrease in disease spectrum and transmission. When it comes to vaccine candidates, several genes encoding Schistosoma mansoni proteins expressed at the mammalian host-parasite interface have been tested. Among the most promising molecules are the proteins present on the tegument and digestive tract of the parasite. In this study, we evaluate the potential of SmKI-1, the first Kunitz-type protease inhibitor functionally characterized in S. mansoni, as a vaccine candidate. Bioinformatic analysis points to the C-terminal fragment as the main region of the molecule responsible for the development of a potential protective immune response induced by SmKI-1. Therefore, for the vaccine formulations, we produced the recombinant (r) SmKI-1 and two different fragments, its Kunitz (KI) domain and its C-terminal tail. First, we demonstrate that mice immunized with recombinant SmKI-1 (rSmKI-1) or its fragments, formulated with Freund's adjuvant, induced the production of IgG-specific antibodies. Further, all vaccine formulations tested here also induced a Th1-type of immune response, as suggested by the production of IFN-γ and TNF-α by protein-stimulated cultured splenocytes. However, the protective effect conferred by vaccination was only observed in groups which received rSmKI-1 or C-terminal domain vaccines. Mice administered with rSmKI-1 demonstrated reduction of 47% in worm burden, 36% in egg number in mouse livers, and 33% in area of liver granulomas. Additionally, mice injected with C-terminal domain showed reduction of 28% in worm burden, 38% in egg number in liver, and 25% in area of liver granulomas. In contrast, KI domain immunization was unable to reduce worm burden and ameliorate liver pathology after challenge infection. Taken together, our data demonstrated that SmKI-1 is a potential candidate for use in a vaccine to control schistosomiasis, and its C-terminal tail seems to be the main region of the molecule responsible for protection conferred by this antigen.
Assuntos
Resistência à Doença/imunologia , Proteínas de Helminto/imunologia , Interações Hospedeiro-Parasita/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/parasitologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/química , Antígenos de Helmintos/imunologia , Citocinas/metabolismo , Mapeamento de Epitopos , Epitopos/imunologia , Feminino , Proteínas de Helminto/química , Imunização , Imunoglobulina G/imunologia , Camundongos , Carga Parasitária , Inibidores de Proteases , Domínios e Motivos de Interação entre Proteínas/imunologia , Proteínas Recombinantes/imunologia , Esquistossomose mansoni/metabolismo , Esquistossomose mansoni/prevenção & controle , Vacinas/imunologiaRESUMO
BACKGROUND: The parasitic flatworm Schistosoma mansoni is a blood fluke that causes schistosomiasis. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Numerous antigens that are expressed at the interface between the parasite and the mammalian host have been assessed. Among the most promising molecules are the proteins present in the tegument and digestive tract of the parasite. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we evaluated the potential of Sm10.3, a member of the micro-exon gene 4 (MEG-4) family, for use as part of a recombinant vaccine. We confirmed by real-time PCR that Sm10.3 was expressed at all stages of the parasite life cycle. The localization of Sm10.3 on the surface and lumen of the esophageal and intestinal tract in adult worms and lung-stage schistosomula was confirmed by confocal microscopy. We also show preliminary evidence that rSm10.3 induces erythrocyte agglutination in vitro. Immunization of mice with rSm10.3 induced a mixed Th1/Th2-type response, as IFN-γ, TNF-α, and low levels of IL-5 were detected in the supernatant of cultured splenocytes. The protective effect conferred by vaccination with rSm10.3 was demonstrated by 25.5-32% reduction in the worm burden, 32.9-43.6% reduction in the number of eggs per gram of hepatic tissue, a 23.8% reduction in the number of granulomas, an 11.8% reduction in the area of the granulomas and a 39.8% reduction in granuloma fibrosis. CONCLUSIONS/SIGNIFICANCE: Our data suggest that Sm10.3 is a potential candidate for use in developing a multi-antigen vaccine to control schistosomiasis and provide the first evidence for a possible role for Sm10.3 in the blood feeding process.
Assuntos
Aglutinação , Antígenos de Helmintos/imunologia , Eritrócitos/parasitologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/prevenção & controle , Vacinação/métodos , Vacinas de Subunidades Antigênicas/imunologia , Estruturas Animais/química , Animais , Antígenos de Helmintos/análise , Antígenos de Helmintos/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Carga Parasitária , Esquistossomose mansoni/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: Schistosomiasis is a neglected tropical disease caused by several species of trematode of the genus Schistosoma. The disease affects more than 200 million people in the world and causes up to 280,000 deaths per year, besides having high morbidity due to chronic illness that damages internal organs. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Among the most promising molecules as vaccine candidates are the proteins present in the tegument and digestive tract of the parasite. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we describe for the first time Schistosoma mansoni syntenin (SmSynt) and we evaluate its potential as a recombinant vaccine. We demonstrate by real-time PCR that syntenin is mainly expressed in intravascular life stages (schistosomula and adult worms) of the parasite life cycle and, by confocal microscopy, we localize it in digestive epithelia in adult worms and schistosomula. Administration of siRNAs targeting SmSynt leads to the knock-down of syntenin gene and protein levels, but this has no demonstrable impact on parasite morphology or viability, suggesting that high SmSynt gene expression is not essential for the parasites in vitro. Mice immunization with rSmSynt, formulated with Freund's adjuvant, induces a Th1-type response, as suggested by the production of IFN-γ and TNF-α by rSmSynt-stimulated cultured splenocytes. The protective effect conferred by vaccination with rSmSynt was demonstrated by 30-37% reduction of worm burden, 38-43% reduction in the number, and 35-37% reduction in the area, of liver granulomas. CONCLUSIONS/SIGNIFICANCE: Our report is the first characterization of syntenin in Schistosoma mansoni and our data suggest that this protein is a potential candidate for the development of a multi-antigen vaccine to control schistosomiasis.
Assuntos
Antígenos de Helmintos , Schistosoma mansoni/imunologia , Esquistossomose mansoni , Sinteninas , Vacinas/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/classificação , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Schistosoma mansoni/genética , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/prevenção & controle , Alinhamento de Sequência , Sinteninas/química , Sinteninas/classificação , Sinteninas/genética , Sinteninas/imunologiaRESUMO
Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.
Assuntos
Proteínas Contráteis/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Vacúolos/metabolismo , Western Blotting , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Anotação de Sequência Molecular , Transporte Proteico , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Trypanosoma cruzi/citologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/ultraestrutura , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/ultraestruturaRESUMO
Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I-V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH-ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD(+)-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast's exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.