Resumo

The methylcitrate cycle (MCC) is responsible for the propionyl-CoA to pyruvate conversion in some microorganisms. At high intracellular concentrations, this secondary metabolite is toxic to the cell and can be generated by the odd-chain fatty acids degradation and from metabolism of some amino acids, such as isoleucine, valine and methionine - carbon sources widely available in host tissues. However, no studies of this pathway in the human pathogenic fungus Paracoccidioides brasiliensis have been done. Previous studies have shown that specific enzymes of MCC are upregulated in infection-mimicking conditions. Therefore, in order to investigate the role of MCC in P. brasiliensis the growth and viability was monitored in different concentrations and times of incubation in different carbon sources: glucose, oleic acid, acetate, propionate and amino acids. Thereafter, it was performed real-time PCR assays of the genes coding for the three MCC specific enzymes 2-methylcitrate synthase (MCS), methylcitrate dehydratase (MCD) and 2-methylisocitrate lyase (MCL). The genes coding for MCS and MCL were highly expressed in propionate, acetate and amino acids. On the other hand, the gene coding for MCD showed only high expression in the early hours of incubation in propionate and amino acids. During macrophage infection, the genes coding for MCS and MCD displayed high expression. The heterologous recombinant MCS enzyme was obtained in order to investigate its ability in recognize propionyl-CoA as a substrate. The recombinant MCS displayed both, citrate and methylcitrate synthase activity suggesting its role in the Krebs cycle and MCC. Moreover, the Km determination of the MCS confirmed its higher affinity to propionyl-CoA than to acetyl-CoA. The fungus proteome with propionate as the only carbon source are under progress. Given that the methylcitrate cycle is responsible propionyl-CoA detoxification in the cells of some microorganisms, understand the mechanism of this pathway in P. brasiliensis will provide data for understanding some metabolic mechanisms that this fungus takes hand during infection.