RESUMO
Candida parapsilosis is an important, emerging opportunistic fungal pathogen. Highly mannosylated fungal cell wall proteins are initial contact points with host immune systems. In Candida albicans, Och1 is a Golgi α1,6-mannosyltransferase that plays a key role in the elaboration of the N-linked mannan outer chain. Here, we disrupted C. parapsilosis OCH1 to gain insights into the contribution of N-linked mannosylation to cell fitness and to interactions with immune cells. Loss of Och1 in C. parapsilosis resulted in cellular aggregation, failure of morphogenesis, enhanced susceptibility to cell wall perturbing agents and defects in wall composition. We removed the cell wall O-linked mannans by ß-elimination, and assessed the relevance of mannans during interaction with human monocytes. Results indicated that O-linked mannans are important for IL-1ß stimulation in a dectin-1 and TLR4-dependent pathway; whereas both, N- and O-linked mannans are equally important ligands for TNFα and IL-6 stimulation, but neither is involved in IL-10 production. Furthermore, mice infected with C. parapsilosis och1Δ null mutant cells had significantly lower fungal burdens compared to wild-type (WT)-challenged counterparts. Therefore, our data are the first to demonstrate that C. parapsilosis N- and O-linked mannans have different roles in host interactions than those reported for C. albicans.
RESUMO
Protein glycosylation pathways are conserved metabolic processes in eukaryotic organisms and are required for cell fitness. In fungal pathogens, the N-linked glycosylation pathway is indispensable for proper cell wall composition and virulence. In Sporothrix schenckii sensu stricto, the causative agent of sporotrichosis, little is known about this glycosylation pathway. Here, using a genome-wide screening for putative members of the glycosyl hydrolase (CAZy - GH) families 47 and 63, which group enzymes involved in the processing step during N-linked glycan maturation, we found seven homologue genes belonging to family 47 and one to family 63. The eight genes were individually expressed in C. albicans null mutants lacking either MNS1 (for members of family 47) or CWH41 (for the member of family 63). Our results indicate that SsCWH41 is the functional ortholog of CaCWH41, whereas SsMNS1 is the functional ortholog of CaMNS1. The remaining genes of family 47 encode Golgi mannosidases and endoplasmic reticulum degradation-enhancing alpha-mannosidase-like proteins (EDEMs). Since these GH families gather proteins used as target for drugs to control cell growth, identification of these genes could help in the design of antifungals that could be used to treat sporotrichosis and other fungal diseases. In addition, to our knowledge, we are the first to report that Golgi mannosidases and EDEMs are expressed and characterized in yeast cells.
Assuntos
Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Sporothrix/enzimologia , Candida albicans/enzimologia , Candida albicans/genética , Candida albicans/metabolismo , Clonagem Molecular , Biologia Computacional , Expressão Gênica , Glicosídeo Hidrolases/genética , Glicosilação , Sporothrix/genéticaRESUMO
Candida albicans is the main causative agent of systemic candidiasis, a condition with high mortality rates. The study of the interaction between C. albicans and immune system components has been thoroughly studied and nowadays there is a model for the anti-C. albicans immune response; however, little is known about the sensing of other pathogenic species of the Candida genus. Sporothrix schenckii is the causative agent of sporotrichosis, a subcutaneous mycosis, and thus far there is limited information about its interaction with the immune system. In this paper, we review the most recent information about the immune sensing of species from genus Candida and S. schenckii. Thoroughly searches in scientific journal databases were performed, looking for papers addressing either Candida- or Sporothrix-immune system interactions. There is a significant advance in the knowledge of non-C. albicans species of Candida and Sporothrix immune sensing; however, there are still relevant points to address, such as the specific contribution of pathogen-associated molecular patterns (PAMPs) for sensing by different immune cells and the immune receptors involved in such interactions. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).
Assuntos
Candida/imunologia , Candidíase/imunologia , Interações Hospedeiro-Patógeno/imunologia , Sporothrix/imunologia , Esporotricose/imunologia , Imunidade Adaptativa , Animais , Antígenos de Fungos/imunologia , Parede Celular/imunologia , Humanos , Imunidade Celular , Hospedeiro Imunocomprometido , Subpopulações de Linfócitos/imunologia , Macrófagos/imunologia , Camundongos , Neutrófilos/imunologia , Esporotricose/microbiologiaRESUMO
Sporothrix schenckii is a fungal pathogen of humans and the etiological agent of sporotrichosis. In fungi, proper protein glycosylation is usually required for normal composition of cell wall and virulence. Upon addition of precursor oligosaccharides to nascent proteins in the endoplasmic reticulum, glycans are further modified by Golgi-glycosyl transferases. In order to add sugar residues to precursor glycans, nucleotide diphosphate sugars are imported from the cytosol to the Golgi lumen, the sugar is transferred to glycans, and the resulting nucleoside diphosphate is dephosphorylated by the nucleoside diphosphatase Gda1 before returning to cytosol. Here, we isolated the open reading frame SsGDA1 from a S. schenckii genomic DNA library. In order to confirm the function of SsGda1, we performed complementation assays in a Saccharomyces cerevisiae gda1∆ null mutant. Our results indicated that SsGDA1 restored the nucleotide diphosphatase activity to wild-type levels and therefore is a functional ortholog of S. cerevisiae GDA1.
Assuntos
Genes Fúngicos , Pirofosfatases/genética , Pirofosfatases/metabolismo , Sporothrix/enzimologia , Sporothrix/genética , Sequência de Aminoácidos , Parede Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Glicosilação , Complexo de Golgi/metabolismo , Dados de Sequência Molecular , Pirofosfatases/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
Sporothrix (Sp.) schenckii is a pathogenic fungus that infects humans and animals, and is responsible for the disease named sporotrichosis. The cell wall of this fungus has glycoproteins with a high content of mannose and rhamnose units, which are synthesized by endoplasmic reticulum- and Golgi-localized glycosyltransferases. Little is known about the enzymic machinery involved in the synthesis of these oligosaccharides in Sp. schenckii, or the genes encoding these activities. This is in part because of the lack of an available genome sequence for this organism. Using a partial genomic DNA library we identified SsMNT1, whose predicted product has significant similarity to proteins encoded by members of the Saccharomyces (Sa.) cerevisiae KRE2/MNT1 gene family. In order to biochemically characterize the putative enzyme, SsMNT1 was heterologously expressed in the methylotrophic yeast Pichia pastoris. Recombinant SsMnt1 showed Mn(2+)-dependent mannosyltransferase activity and the ability to recognize as acceptors α-methyl mannoside, mannose, Man(5)GlcNAc(2) oligosaccharide and a variety of mannobiosides. The characterization of the enzymic products generated by SsMnt1 revealed that the enzyme is an α1,2-mannosyltransferase that adds up to two mannose residues to the acceptor molecule. Functional complementation studies were performed in Sa. cerevisiae and Candida albicans mutants lacking members of the KRE2/MNT1 gene family, demonstrating that SsMnt1 is involved in both the N- and O-linked glycosylation pathways, but not in phosphomannan elaboration.
Assuntos
Manosiltransferases/genética , Manosiltransferases/metabolismo , Sporothrix/enzimologia , Candida albicans/enzimologia , Candida albicans/genética , Cátions Bivalentes/metabolismo , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Ativadores de Enzimas/metabolismo , Deleção de Genes , Expressão Gênica , Teste de Complementação Genética , Magnésio/metabolismo , Dados de Sequência Molecular , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sporothrix/genética , Sporothrix/metabolismo , Especificidade por SubstratoRESUMO
Yarrowia lipolytica lipase (YLL) was used as catalyst in the enzymatic ring-opening polymerization (ROP) of ε-caprolactone. This low-cost solid-state lipase produces low-molecular-weight polyesters with unique multiphase morphology as determined by carbon-13 NMR. YLL attaches sugar head groups to polycaprolactone in a one-pot biocatalytic pathway. Synthesis of α-ω-telechelic (polymer with two reactive hydroxyl end groups) PCL diols is achieved by enzymatic ROP with YLL immobilized on the macroporous resin Lewatit VPOC 1026, and in the presence of diethylene glycol or poly(ethylene glycol). Biodegradable linear polyester urethanes are prepared by polycondensation between synthesized PCL diols and hexamethylene-diisocyanate.
Assuntos
Materiais Biocompatíveis/síntese química , Proteínas Fúngicas/química , Lipase/química , Poliésteres/síntese química , Yarrowia/química , Biocatálise , Biodegradação Ambiental , Caproatos/química , Cianatos/química , Etilenoglicóis/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Imobilizadas/química , Proteínas Imobilizadas/isolamento & purificação , Isocianatos , Lactonas/química , Lipase/isolamento & purificação , Espectroscopia de Ressonância Magnética , Peso Molecular , Poliésteres/química , Polietilenoglicóis/químicaRESUMO
The cell surface of Candida albicans is enriched with highly glycosylated mannoproteins that are involved in the interaction with host tissues. N- and O-glycosylation are post-translational modifications that initiate in the endoplasmic reticulum, and finalize in the Golgi. The KRE2/MNT1 family encode a set of multifunctional mannosyltransferases that participate in O-, N- and phosphomannosylation. In order to gain insights into the substrate specificities of these enzymes, recombinant forms of Mnt1, Mnt2, and Mnt5 were expressed in Pichia pastoris and the enzyme activities characterized. Mnt1 and Mnt2 showed a high specificity for α-methylmannoside and α1,2-mannobiose as acceptor substrates. Notably, they also used Saccharomyces cerevisiaeO-mannans as acceptors and generated products with more than three mannose residues, suggesting than Mnt1 and Mnt2 could be the mannosyltransferases adding the fourth and fifth mannose residue to the O-mannans in C. albicans. Mnt5 only recognized α-methylmannoside as acceptor, suggesting that participates in the addition of the second mannose residues to the N-glycan outer chain.
Assuntos
Candida albicans/enzimologia , Proteínas Fúngicas/química , Mananas/biossíntese , Manosiltransferases/química , Proteínas Fúngicas/genética , Manosiltransferases/genética , Modelos Químicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Protein glycosylation is one of the most common post-translational modifications present in the eukaryotic cell. The N-linked glycosylation is a biosynthetic pathway where an oligosaccharide is added to asparagine residues within the endoplasmic reticulum. Upon addition of the N-linked glycan to nascent proteins, alpha-glucosidase I removes the outermost alpha1,2-glucose unit from the N-linked core Glc(3)Man(9)GlcNAc(2). We have previously demonstrated that the endoplasmic reticulum α-glucosidase I is required for normal cell wall composition, and virulence of the human pathogen Candida albicans. In spite of the importance of this enzyme for normal cell biology, little is known about its structure and the amino acids participating in enzyme catalysis. Here, a DNA fragment corresponding to the 3'-end fragment of C. albicans CWH41, the encoding gene for α-glucosidase I, was expressed in a bacterial system and the recombinant peptide showed alpha-glucosidase activity, despite lacking 419 amino acids from the N-terminal end. The biochemical characterisation of the recombinant enzyme showed that presence of hydroxyl groups at carbons 3 and 6, and orientation of hydroxyl moiety at C-2 are important for glucose recognition. Additionally, results suggest that cysteine rather than histidine residues are involved in the catalysis by the recombinant enzyme.
Assuntos
Candida albicans/enzimologia , Escherichia coli/genética , alfa-Glucosidases/metabolismo , Sequência de Aminoácidos , Candida albicans/classificação , Clonagem Molecular , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Expressão Gênica , Genes Fúngicos , Glicosilação , Glicoproteínas de Membrana , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/genética , alfa-Glucosidases/isolamento & purificaçãoRESUMO
Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, alpha1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one alpha1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound alpha-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-alpha1,2-mannosidase antibodies. The enzyme hydrolysed Man(9)GlcNAc(2) into Man(8)GlcNAc(2) isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This alpha1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised alpha1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi alpha1,2-mannosidases and therefore, the processing of N-glycans by alpha1,2-mannosidases is similar to that present in lower eukaryotes.
Assuntos
Retículo Endoplasmático/enzimologia , Manosidases/isolamento & purificação , Sporothrix/enzimologia , Manosidases/química , Sporothrix/classificação , Sporothrix/citologiaRESUMO
Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, á1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one á1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound á-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-á1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This á1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised á1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi á1,2-mannosidases and therefore, the processing of N-glycans by á1,2-mannosidases is similar to that present in lower eukaryotes.