ABSTRACT
Paracoccidioides brasiliensis is a thermally dimorphic fungus agent of paracoccidioidomycosis, a deep-seated systemic infection of humans with high prevalence in Latin America. Until now no vaccine has been reported. Ionizing radiation can be used to attenuate pathogens for vaccine development and we have successfully attenuated yeast cells of P. brasiliensis by gamma irradiation. The aim of the present study was to examine at ultrastructural level the effects of gamma irradiation attenuation on the morphology of P. brasiliensis yeast cells. Paracoccidioides brasiliensis (strain Pb-18) cultures were irradiated with a dose of 6.5 kGy. The irradiated cells were examined by scanning and also transmission electron microscopy. When examined 2 h after the irradiation by scanning electron microscopy, the 6.5 kGy irradiated cells presented deep folds or were collapsed. These lesions were reversible since when examined 48 h after irradiation the yeast had recovered the usual morphology. The transmission electron microscopy showed that the irradiated cells plasma membrane and cell wall were intact and preserved. Remarkable changes were found in the nucleus that was frequently in a very electrondense form. An extensive DNA fragmentation was produced by the gamma irradiation treatment.
Subject(s)
Cell Membrane/radiation effects , Cell Wall/radiation effects , DNA, Fungal/radiation effects , Gamma Rays , Paracoccidioides/radiation effects , Paracoccidioides/ultrastructure , Cell Membrane/ultrastructure , Cell Wall/ultrastructure , DNA Fragmentation , Fungal Vaccines , Paracoccidioides/cytology , Paracoccidioidomycosis/microbiology , Vaccines, AttenuatedABSTRACT
Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America, and currently there is no effective vaccine. The aim of this study was to attenuate the yeast form of P. brasiliensis by gamma irradiation for further studies on vaccine research. Paracoccidioides brasiliensis (strain Pb 18) cultures were irradiated at doses between 0.5 and 8.0 kGy. After each dose the viability, reproductive ability and protein metabolism were evaluated. The comparison between the antigenic profile of irradiated and control yeast was made by Western blot and the virulence evaluated by the inoculation in C(57)Bl/J6 mice. At 6.5 kGy the yeast lost its reproductive capacity. The viability and the incorporation of [L-(35)S]-methionine were the same in control and up to 6.5 kGy irradiated cells, but 6.5 kGy-irradiated yeast secreted 40% less proteins. The Western blot profile was clearly similar in control and 6.5 kGy-irradiated yeast. No colony-forming unit (CFU) could be recovered from the tissues of the mice infected with the radioattenuated yeast. We concluded that for P. brasiliensis yeast it is possible to find a dose in which the pathogen loses its reproductive ability and virulence, while retaining its viability, metabolic activity and the antigenic profile.
