ABSTRACT
Although colony-forming unit (CFU) counting is widely used to quantify fungal load in tissue from animal experimentally infected with Paracoccidioides brasiliensis, several technical disadvantages have been described. Here we developed highly accurate quantitative PCR (qPCR) assays to determine the relative P brasiliensis load in lungs from infected mice. SYBR Green- and TaqMan-based assays using primers and probe for the 43-kDa glycoprotein (gp43) gene detected as little as 270 gene copies (about 2 fg of DNA) per reaction. Although qPCR assays cannot distinguish between living and dead yeasts, we found a highly positive linear correlation between CFU and qPCR.
Subject(s)
Lung Diseases, Fungal/microbiology , Lung/microbiology , Paracoccidioides , Paracoccidioidomycosis/microbiology , Animals , Colony Count, Microbial , Disease Models, Animal , Male , Mice , Mice, Inbred BALB C , Real-Time Polymerase Chain Reaction , Reproducibility of Results , Stem CellsABSTRACT
The genus Paracoccidioides comprises species of dimorphic fungi that cause paracoccidioidomycosis (PCM), a systemic disease prevalent in Latin America. Here, we investigated whether administration of native 60-kDa heat shock protein of P. brasiliensis (nPbHsp60) or its recombinant counterpart (rPbHsp60) affected the course of experimental PCM. Mice were subcutaneously injected with nPbHsp60 or rPbHsp60 emulsified in complete's Freund Adjuvant (CFA) at three weeks after intravenous injection of P. brasiliensis yeasts. Infected control mice were injected with CFA or isotonic saline solution alone. Thirty days after the nPbHsp60 or rPbHsp60 administration, mice showed remarkably increased fungal load, tissue inflammation, and granulomas in the lungs, liver, and spleen compared with control mice. Further, rPbHsp60 treatment (i) decreased the known protective effect of CFA against PCM and (ii) increased the concentrations of IL-17, TNF-α, IL-12, IFN-γ, IL-4, IL-10, and TGF-ß in the lungs. Together, our results indicated that PbHsp60 induced a harmful immune response, exacerbated inflammation, and promoted fungal dissemination. Therefore, we propose that PbHsp60 contributes to the fungal pathogenesis.
