The influence of a copper efflux pump in Histoplasma capsulatum virulence.

During the infectious process, pathogenic microorganisms must obtain nutrients from the host in order to survive and proliferate. These nutritional sources include the metallic nutrient copper. Despite its essentiality, copper in large amounts is toxic. Host defense mechanisms use high copper poisoning as a fungicidal strategy to control infection. Transcriptional analyses showed that yeast cultured in the presence of copper or inside macrophages (24 h) had elevated expression of CRP1, a copper efflux pump, suggesting that Histoplasma capsulatum could be exposed to a high copper environment in macrophages during the innate immune stage of infection. Accordingly, macrophages cultured in high copper are more efficient in controlling H. capsulatum growth. Also, silencing of ATP7a, a copper pump that promotes the copper influx in phagosomes, increases fungal survival in macrophages. The rich copper environment faced by the fungus is not dependent on IFN-γ, since fungal CRP1 expression is induced in untreated macrophages. Appropriately, CRP1 knockdown fungal strains are more susceptible to macrophage control than wild-type yeasts. Additionally, CRP1 silencing decreases fungal burden in mice during the phase of innate immune response (4-day postinfection) and CRP1 is required for full virulence in a macrophage cell lines (J774 A.1 and RAW 264.7), as well as primary cells (BMDM). Thus, induction of fungal copper detoxifying genes during innate immunity and the attenuated virulence of CRP1-knockdown yeasts suggest that H. capsulatum is exposed to a copper-rich environment at early infection, but circumvents this condition to establish infection.

Propionate metabolism in a human pathogenic fungus: proteomic and biochemical analyses.

Fungi of the complex Paracoccidioides spp. are thermodimorphic organisms that cause Paracoccidioidomycosis, one of the most prevalent mycoses in Latin America. These fungi present metabolic mechanisms that contribute to the fungal survival in host tissues. Paracoccidioides lutzii activates glycolysis and fermentation while inactivates aerobic metabolism in iron deprivation, a condition found during infection. In lungs Paracoccidioides brasiliensis face a glucose poor environment and relies on the beta-oxidation to support energy requirement. During mycelium to yeast transition P. lutzii cells up-regulate transcripts related to lipid metabolism and cell wall remodeling in order to cope with the host body temperature. Paracoccidioides spp. cells also induce transcripts/enzymes of the methylcitrate cycle (MCC), a pathway responsible for propionyl-CoA metabolism. Propionyl-CoA is a toxic compound formed during the degradation of odd-chain fatty acids, branched chain amino acids and cholesterol. Therefore, fungi require a functional MCC for full virulence and the ability to metabolize propionyl-CoA is related to the virulence traits in Paracoccidioides spp. On this way we sought to characterize the propionate metabolism in Paracoccidioides spp. The data collected showed that P. lutzii grows in propionate and activates the MCC by accumulating transcripts and proteins of methylcitrate synthase (MCS), methylcitrate dehydratase (MCD) and methylisocitrate lyase (MCL). Biochemical characterization of MCS showed that the enzyme is regulated by phosphorylation, an event not yet described. Proteomic analyses further indicate that P. lutzii yeast cells degrades lipids and amino acids to support the carbon requirement for propionate metabolism. The induction of a putative propionate kinase suggests that fungal cells use propionyl-phosphate as an intermediate in the production of toxic propionyl-CoA. Concluding, the metabolism of propionate in P. lutzii is under regulation at transcriptional and phosphorylation levels and that survival on this carbon source requires additional mechanisms other than activation of MCC.

Metabolic Adaptation of Paracoccidioides brasiliensis in Response to in vitro Copper Deprivation.

Copper is an essential micronutrient for the performance of important biochemical processes such as respiration detoxification, and uptake of metals like iron. Studies have shown that copper deprivation is a strategy used by the host against pathogenic fungi such as Cryptoccocus neoformans and Candida albicans during growth and development of infections in the lungs and kidneys. Although there are some studies, little is known about the impact of copper deprivation in members of the Paracoccidioides genus. Therefore, using isobaric tag labeling (iTRAQ)-Based proteomic approach and LC-MS/MS, we analyzed the impact of in vitro copper deprivation in the metabolism of Paracoccidioides brasiliensis. One hundred and sixty-four (164) differentially abundant proteins were identified when yeast cells were deprived of copper, which affected cellular respiration and detoxification processes. Changes in cellular metabolism such as increased beta oxidation and cell wall remodeling were described.