RESUMO
Paracoccidioides brasiliensis is the major etiologic agent of Paracoccidioidomycosis (PCM), the most frequent human deep mycosis in Latin America. It is proposed that masking of ß-glucan in P. brasiliensis cell wall is a critical virulence factor that contributes to the development of a chronic disease characterized by a long period of treatment, which is usually toxic. In this context, the search for immunomodulatory agents for therapeutic purposes is highly desirable. One strategy is to use pattern recognition receptors (PRRs) ligands to stimulate the immune response mediated by phagocytes. Here, we sought to evaluate if Zymosan, a ß-glucan-containing ligand of the PRRs Dectin-1/TLR-2, would enhance phagocyte function and the immune response of mice challenged with P. brasiliensis. Dendritic cells (DCs) infected with P. brasiliensis and treated with Zymosan showed improved secretion of several proinflammatory cytokines and expression of maturation markers. In addition, when cocultured with splenic lymphocytes, these cells induced the production of a potential protective type 1 and 17 cytokine patterns. In macrophages, Zymosan ensued a significant fungicidal activity associated with nitric oxide production and phagolysosome acidification. Importantly, we observed a protective effect of Zymosan-primed DCs delivered intranasally in experimental pulmonary PCM. Overall, our findings support the potential use of ß-glucan-containing compounds such as Zymosan as an alternative or complementary antifungal therapy. LAY SUMMARY: We report for the first time that Paracoccidioides brasiliensis-infected phagocytes treated with Zymosan (cell wall extract from bakers' yeast) show enhanced cytokine production, maturation, and fungal killing. Also, Zymosan-primed phagocytes induce a protective immune response in infected mice.
Assuntos
Paracoccidioides/imunologia , Paracoccidioidomicose/tratamento farmacológico , Fagócitos/efeitos dos fármacos , Zimosan/farmacologia , Animais , Camundongos , Paracoccidioides/patogenicidade , Paracoccidioidomicose/imunologia , Fagócitos/imunologia , Virulência , Zimosan/uso terapêuticoRESUMO
Anastomotic dehiscence is the most severe complication of colorectal surgery. Metalloproteinases (MMPs) and interleukins (ILs) can be used to analyze the healing process of anastomosis. To evaluate the effects of bromopride on MMP and cytokine gene expression in left colonic anastomoses in rats with or without induced abdominal sepsis, 80 rats were divided into two groups for euthanasia on the third or seventh postoperative day (POD). They were then divided into subgroups of 20 rats for sepsis induction or not, and then into subgroups of 10 rats for administration of bromopride or saline. Left colonic anastomosis was performed and abdominal sepsis was induced by cecal ligation and puncture. A colonic segment containing the anastomosis was removed for analysis of gene expression of MMP-1α, MMP-8, MMP-13, IL-β, IL-6, IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). On the third POD, bromopride was associated with increased MMP-1α, MMP-13, IL-6, IFN-γ, and IL-10 gene expression. On the seventh POD, all MMP transcripts became negatively modulated and all IL transcripts became positively modulated. In the presence of sepsis, bromopride administration increased MMP-8 and IFN-γ gene expression and decreased MMP-1, TNF-α, IL-6, and IL-10 gene expression on the third POD. On the seventh POD, we observed increased expression of MMP-13 and all cytokines, except for TNF-α. In conclusion, bromopride interferes with MMP and IL gene expression during anastomotic healing. Further studies are needed to correlate these changes with the healing process.
Assuntos
Animais , Masculino , Antieméticos/farmacologia , Colo Descendente/cirurgia , Expressão Gênica/efeitos dos fármacos , Interleucinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Metoclopramida/análogos & derivados , Anastomose Cirúrgica , Ceco/cirurgia , Interferon gama/análise , Interleucina-1beta/análise , /análise , /análise , Interleucinas/genética , Ligadura , Metaloproteinase 1 da Matriz/análise , /análise , /análise , Metaloproteinases da Matriz/genética , Metoclopramida/farmacologia , Punções , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/etiologia , Fator de Necrose Tumoral alfa/análise , Cicatrização/efeitos dos fármacosRESUMO
Anastomotic dehiscence is the most severe complication of colorectal surgery. Metalloproteinases (MMPs) and interleukins (ILs) can be used to analyze the healing process of anastomosis. To evaluate the effects of bromopride on MMP and cytokine gene expression in left colonic anastomoses in rats with or without induced abdominal sepsis, 80 rats were divided into two groups for euthanasia on the third or seventh postoperative day (POD). They were then divided into subgroups of 20 rats for sepsis induction or not, and then into subgroups of 10 rats for administration of bromopride or saline. Left colonic anastomosis was performed and abdominal sepsis was induced by cecal ligation and puncture. A colonic segment containing the anastomosis was removed for analysis of gene expression of MMP-1α, MMP-8, MMP-13, IL-ß, IL-6, IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). On the third POD, bromopride was associated with increased MMP-1α, MMP-13, IL-6, IFN-γ, and IL-10 gene expression. On the seventh POD, all MMP transcripts became negatively modulated and all IL transcripts became positively modulated. In the presence of sepsis, bromopride administration increased MMP-8 and IFN-γ gene expression and decreased MMP-1, TNF-α, IL-6, and IL-10 gene expression on the third POD. On the seventh POD, we observed increased expression of MMP-13 and all cytokines, except for TNF-α. In conclusion, bromopride interferes with MMP and IL gene expression during anastomotic healing. Further studies are needed to correlate these changes with the healing process.
Assuntos
Antieméticos/farmacologia , Colo Descendente/cirurgia , Expressão Gênica/efeitos dos fármacos , Interleucinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Metoclopramida/análogos & derivados , Anastomose Cirúrgica , Animais , Ceco/cirurgia , Interferon gama/análise , Interleucina-10/análise , Interleucina-1beta/análise , Interleucina-6/análise , Interleucinas/genética , Ligadura , Masculino , Metaloproteinase 1 da Matriz/análise , Metaloproteinase 13 da Matriz/análise , Metaloproteinase 8 da Matriz/análise , Metaloproteinases da Matriz/genética , Metoclopramida/farmacologia , Punções , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/etiologia , Fator de Necrose Tumoral alfa/análise , Cicatrização/efeitos dos fármacosRESUMO
Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl ß-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.
Assuntos
Celulose 1,4-beta-Celobiosidase/metabolismo , Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Fungos Mitospóricos/metabolismo , Proteínas Recombinantes/metabolismo , Clonagem Molecular/métodos , Meios de Cultura/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Fungos Mitospóricos/enzimologia , TemperaturaRESUMO
The expression enhancement by cytomegalovirus promoter and different intron A (IA) variants were evaluated in CHO-K1, HepG2, HEK-293 and COS-7 cells by assessing the levels of luciferase activity. This data along with mRNA levels measurement indicated that the construct harboring an IA variant with a 200-nucleotide deletion (Δ200) had the greatest impact on increasing luciferase expression among all constructs evaluated. Based on these results, we redesigned pCMV-IA variants and cloned them into plasmids expressing a humanized antibody. These plasmids were then used to transfect CHO-K1 cells. Production of the antibody was not augmented with the Δ200 promoter variant. The 600-nucleotide deletion (Δ600) and whole IA promoter variants expressed similar levels of the recombinant protein. These data indicate that the IA-based enhanced expression of transgenes depends on a small region within the intron.
Assuntos
Citomegalovirus/genética , Íntrons , Proteínas Recombinantes/biossíntese , Transgenes , Animais , Biotecnologia , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Expressão Gênica , Células HEK293 , Humanos , Luciferases/análise , Luciferases/genética , Luciferases/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
AIMS: Penicillium echinulatum is effective for bioconversion processes. However, nothing is known about the molecular biology of its cellulolytic system. We describe for the first time the isolation, cloning and expression of a P. echinulatum cellulase cDNA (Pe-egl1) encoding a putative endoglucanase. METHODS AND RESULTS: Pe-egl1 cDNA was identified from random sequencing of a P. echinulatum cDNA library. The deduced EGL1 protein possibly belongs to the glycosyl hydrolase family 5A, with 387 amino acid residues and strong similarity with other fungal endoglucanases. The cDNA was heterologously expressed in Pichia pastoris. The recombinant EGL1 secreted by a Pic. pastoris recombinant strain revealed the characteristics of particular interest: an optimal activity over a broad pH range (5.0-9.0), and an optimal temperature of 60 degrees C. The recombinant EGL1 also showed high thermostability (84% of residual activity after 1 h of pre-incubation at 70 degrees C). Calcium exerted a strong stimulatory effect over EGL1 activity. CONCLUSIONS: Altogether, these results point to the potential application of this P. echinulatum endoglucanase in cellulose processing industries, particularly the textile one because of its biochemical properties. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization and heterologous expression of the first P. echinulatun cDNA inaugurates the exploitation of this potential industrial micro-organism.
Assuntos
Celulase/genética , Celulase/metabolismo , Regulação Fúngica da Expressão Gênica , Penicillium/enzimologia , Penicillium/genética , Sequência de Aminoácidos , Sequência de Bases , Cátions/farmacologia , Celulase/química , Celulose/metabolismo , Clonagem Molecular , DNA Complementar/genética , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genes Fúngicos/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Pichia/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , TemperaturaRESUMO
Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a facultative intracellular human pathogen that can persist within macrophage phagolysosomes, indicating that the fungus has evolved defense mechanisms in order to survive under nutritionally poor environments. The analysis of P. brasiliensis transcriptome revealed several virulence factor orthologs of other microorganisms, including the glyoxylate cycle genes. This cycle allows the utilization of two-carbon (C2) compounds as carbon source in gluconeogenesis. Semiquantitative RT-PCR analyses revealed that these genes were upregulated when P. brasiliensis was recovered from murine macrophages, without any additional in vitro growth. The induction of this cycle, in response to macrophage microenvironments, was shown to be coordinated with the upregulation of the gluconeogenic phosphoenolpyruvate carboxykinase gene. In addition, assays employing RNA extracted from P. brasiliensis grown in a medium with acetate instead of glucose also showed increased levels of glyoxylate cycle transcripts. Our main results suggest that P. brasiliensis uses the glyoxylate cycle as an important adaptive metabolic pathway.
Assuntos
Glioxilatos/metabolismo , Macrófagos/microbiologia , Paracoccidioides/fisiologia , Paracoccidioidomicose/metabolismo , Animais , DNA Fúngico/análise , Regulação Fúngica da Expressão Gênica , Macrófagos/fisiologia , Camundongos , Paracoccidioides/genética , Paracoccidioidomicose/imunologia , RNA Fúngico/genética , RNA Fúngico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus, which is found as mycelia at 22-26 degrees C and as yeasts at 37 degrees C. A remarkable feature common to several pathogenic fungi is their ability to differentiate from mycelium to yeast morphologies, or vice-versa. Although P. brasiliensis is a recognized pathogen for humans, little is known about its virulence genes. In this sense, we performed a search for putative virulence genes in the P. brasiliensis transcriptome. BLAST comparative analyses were done among P. brasilienses assembled expressed sequence tags (PbAESTs) and the sequences deposited in GenBank. As a result, the putative virulence PbAESTs were grouped into five classes, metabolism-, cell wall-, detoxification-related, secreted factors, and other determinants. Among these, we have identified orthologs of the glyoxylate cycle enzymes, a metabolic pathway involved in the virulence of bacteria and fungi. Besides the previously described alpha- and beta-glucan synthases, orthologs to chitin synthase and mannosyl transferases, also important in cell wall synthesis and stabilization, were identified. With respect to the enzymes involved in the intracellular survival of P. brasiliensis, orthologs to superoxide dismutase, thiol peroxidase and an alternative oxidase were also found. Among the secreted factors, we were able to find phospholipase and urease orthologs in P. brasiliensis transcriptome. Collectively, our results suggest that this organism may possess a vast arsenal of putative virulence genes, allowing the survival in the different host environments.
Assuntos
Humanos , Animais , Etiquetas de Sequências Expressas/metabolismo , Paracoccidioides/patogenicidade , Transcrição Gênica/genética , DNA Complementar , DNA Fúngico , Dados de Sequência Molecular , Paracoccidioides/enzimologia , Paracoccidioides/genética , Paracoccidioidomicose/virologia , Regulação Fúngica da Expressão Gênica , Sequência de Bases , Transcrição Gênica/fisiologia , Virulência/genéticaRESUMO
Paracoccidioides brasiliensis is a thermally dimorphic and a human pathogenic fungus. Our group has partially sequenced its transcriptome and generated a database of mycelial and yeast PbAESTs (P. brasiliensis assembled expressed sequence tags). In the present review we describe the identification of PbAESTs encoding molecular chaperones. These proteins, involved in protein folding and renaturation, are also implicated in several other biological processes, where the dimorphic transition is of particular interest. Another important issue concerning these proteins refers to their participation in the immunopathogenicity of infectious diseases. We have found 438 ESTs (184 in mycelium and 253 in yeast) encoding P. brasiliensis molecular chaperones and their co-chaperones, which were clustered in 48 genes. These genes were classified in families, corresponding to three small chaperones, nine HSP40s, 10 HSP60s, seven HSP70s, five HSP90s, four HSP100s, and 10 other chaperones. These results greatly increase the knowledge on P. brasiliensis molecular chaperones, since only eight of such proteins had been previously characterized.
Assuntos
Humanos , Chaperonas Moleculares/genética , Etiquetas de Sequências Expressas/química , Paracoccidioides/genética , Transcrição Gênica/genética , DNA Complementar , DNA Fúngico , Genes Fúngicos , Resposta ao Choque Térmico/genéticaRESUMO
The RNA biogenesis machinery of Paracoccidioides brasiliensis was assessed by comparative analyses of PbAESTs (P. brasiliensis assembled expressed sequence tags (ESTs)) with sequences from Saccharomyces cerevisiae MIPS database. PbAESTs related to almost all categories of S. cerevisiae RNA biogenesis were found. Two of the 12 S. cerevisiae RNA Pol II core subunits, Rpb3 and Rpb7, were found, probably reflecting the growth phase from which the cDNA libraries used in ESTs generation were constructed, as well as the low abundance of some of these transcripts. We have also found orthologs to TATA-box-binding protein (TBP), and at least one subunit of each TBP-associated factors (TFII) in P. brasiliensis transcriptome, except TFIIB. Genes associated to the chromatin remodeling complex, as well as transcription factors probably involved in the control of genes associated to a sexual cycle and virulence, were also identified. With respect to the pre-mRNA processing, 65 PbAEST orthologs to S. cerevisiae basal splicing machinery and 21 orthologs of 5'- and 3'-end formation processes were found. Components involved in RNA interference were detected, suggesting that this gene expression regulation mechanism is probably used by P. brasiliensis. Twelve PbAESTs related to Pol I and Pol III machineries were assigned as S. cerevisiae orthologs. Finally, 25 and 10 PbAESTs associated to rRNA and tRNA processing, respectively, were detected. Taken together, our results enable us to depict, for the first time, a global view of transcription and RNA processing in P. brasiliensis.