RESUMO
Trichophyton rubrum is the primary causative agent of dermatophytosis worldwide. This fungus colonizes keratinized tissues and uses keratin as a nutritional source during infection. In T. rubrum-host interactions, sensing a hostile environment triggers the adaptation of its metabolic machinery to ensure its survival. The glyoxylate cycle has emerged as an alternative metabolic pathway when glucose availability is limited; this enables the conversion of simple carbon compounds into glucose via gluconeogenesis. In this study, we investigated the impact of stuA deletion on the response of glyoxylate cycle enzymes during fungal growth under varying culture conditions in conjunction with post-transcriptional regulation through alternative splicing of the genes encoding these enzymes. We revealed that the ΔstuA mutant downregulated the malate synthase and isocitrate lyase genes in a keratin-containing medium or when co-cultured with human keratinocytes. Alternative splicing of an isocitrate lyase gene yielded a new isoform. Enzymatic activity assays showed specific instances where isocitrate lyase and malate synthase activities were affected in the mutant strain compared to the wild type strain. Taken together, our results indicate a relevant balance in transcriptional regulation that has distinct effects on the enzymatic activities of malate synthase and isocitrate lyase.
Assuntos
Arthrodermataceae , Fatores de Transcrição , Humanos , Isocitrato Liase/genética , Malato Sintase/genética , Gluconeogênese/genética , Processamento Alternativo , Carbono , Glucose , Queratinas , GlioxilatosRESUMO
In the present study, we examined the characteristics of cDNA, the regulation of the gene expression of Paracoccidioides brasiliensis MLS (Pbmls), and the enzymatic activity of the protein P. brasiliensis MLS (PbMLS) from the P. brasiliensis Pb01 isolate. Pbmls cDNA contains 1617 bp, encoding a protein of 539 amino acids with a predicted molecular mass of 60 kDa. The protein presents the MLSs family signature, the catalytic residues essential for enzymatic activity and the peroxisomal/glyoxysomal targeting signal PTS1. The high level of Pbmls transcript observed in the presence of two-carbon (2C) sources suggests that in P. brasiliensis, the primary regulation of carbon flux into the glyoxylate cycle (GC) was at the level of the Pbmls transcript. The gene expression, protein level, and enzymatic activity of Pbmls were highly induced by oxalurate in the presence of glucose and by proline in the presence of acetate. In the presence of glucose, the gene expression, protein level, and enzymatic activity of Pbmls were mildly stimulated by proline. Our results suggested that PbMLS condenses acetyl-CoA from both 2C sources (GC) and nitrogen sources (from proline and purine metabolism) to produce malate. The regulation of Pbmls by carbon and nitrogen sources was reinforced by the presence of regulatory motifs CREA and UIS found in the promoter region of the gene.
Assuntos
Alantoína/metabolismo , Glioxilatos/metabolismo , Malato Sintase/fisiologia , Redes e Vias Metabólicas/fisiologia , Paracoccidioides/enzimologia , Sequência de Aminoácidos , Carbono/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Malato Sintase/genética , Modelos Biológicos , Dados de Sequência Molecular , Nitrogênio/metabolismo , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Alinhamento de SequênciaRESUMO
The humoral responses to recombinant MPT-51 and GlcB was determined by using an enzyme-linked immunosorbent assay. Levels of immunoglobulin M (IgM) against MPT-51 and IgG against GlcB were higher among tuberculosis (TB) patients than among control individuals. When the MPT-51 and GlcB assays were combined, 90.8% specificity and 75.5% sensitivity were observed. MPT-51 and GlcB were recognized in the humoral responses of Brazilian TB patients.