RESUMO
Sporothrix schenckii is the etiological agent of sporotrichosis, a mycosis of humans and other mammals. Little is known about the responses of this thermodimorphic pathogen to perturbations in the cell wall (CW) by different stress conditions. Here we describe the effect of Congo Red (CR) on the fungal growth, morphogenesis and activity of glucosamine-6-phosphate (GlcN-6-P) synthase. Under conditions of yeast development, 15 µM CR abolished conidia (CN) germination, but when yeast cells were first obtained in the absence of the dye and then post-incubated in its presence, yeasts rapidly differentiated into mycelial cells. On the other hand, under conditions of mycelium development, 150 µM CR did not affect CN germination, but filamentous cells underwent structural changes characterized by a distorted CW contour, the loss of polarity and the formation of red-pigmented, hyphal globose structures. Under these conditions, CR also induced a significant and transient increase in the activity of GlcN-6-P synthase, an essential enzyme in CW biogenesis.
Assuntos
Vermelho Congo/farmacologia , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Sporothrix/crescimento & desenvolvimento , Sporothrix/metabolismo , Animais , Parede Celular/química , Humanos , Hifas/crescimento & desenvolvimento , Micélio/crescimento & desenvolvimento , Sporothrix/enzimologia , Esporotricose/microbiologiaRESUMO
Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Glucosamina/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/isolamento & purificação , Plasmídeos/metabolismo , Saccharomyces cerevisiae/genética , Sporothrix/química , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Fumaratos/química , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expressão Gênica , Teste de Complementação Genética , Biblioteca Genômica , Glucosamina/análogos & derivados , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/antagonistas & inibidores , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Cinética , Fases de Leitura Aberta , Plasmídeos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Sporothrix/enzimologia , beta-Alanina/análogos & derivados , beta-Alanina/químicaRESUMO
Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis in eukaryotes, was stabilized and purified from trophozoites of the parasite protozoan E. histolytica. Analytical electrophoresis revealed the presence in the purified preparations of a major polypeptide of 45 kDa and barely detectable amounts of two other proteins of 70 and 120 kDa. Both the 45 and 70 kDa polypeptides were recognized by a mouse anti-ODC monoclonal antibody. The major polypeptide exhibited amino terminal sequence homology in the range of 40-73% with ODCs from other organisms. The immunoreactive polypeptide of 70 kDa was not identified. The molecular masses of 216 and 45 kDa determined for the native enzyme by gel filtration and for the major polypeptide by SDS-PAGE, respectively, suggest that the amoeba ODC is a homopentamer. Dialysis against hydroxylamine rendered the enzyme activity fully dependent on pyridoxal 5'-phosphate (PLP). As expected for an oligomeric enzyme, ODC activity exhibited sigmoidal kinetics when it was measured as a function of increasing concentrations of L-ornithine and PLP yielding S(0.5) values of 0.45 and 0.18 mM, respectively. Purified ODC was inhibited by 1,3-diaminopropane and 2,4-diamino-2-butanone but was largely insensitive to inhibition by alpha-difluoromethylornithine (DFMO), indicating that the enzyme may not be a suitable target for this anti-parasitic drug. Other features of the amoeba ODC were common with the enzyme from prokaryotes and eucaryotes.