Abstract
Recent evidence suggests that Paracoccidioides species have the potential to undergo sexual reproduction, although no sexual cycle has been identified either in nature or under laboratory conditions. In the present work we detected low expression levels of the heterothallic MAT loci genes MAT1-1 and MAT1-2, the α-pheromone (PBα) gene, and the α- and a-pheromone receptor (PREB and PREA) genes in yeast and mycelia forms of several Paracoccidioides isolates. None of the genes were expressed in a mating type dependent manner. Stimulation of P. brasiliensis MAT1-2 strains with the synthetic α-pheromone peptide failed to elicit transcriptional activation of MAT1-2, PREB or STE12, suggesting that the strains tested are insensitive to α-pheromone. In order to further evaluate the biological functionality of the pair α-pheromone and its receptor, we took advantage of the heterologous expression of these Paracoccidioides genes in the corresponding S. cerevisiae null mutants. We show that S. cerevisiae strains heterologously expressing PREB respond to Pbα pheromone either isolated from Paracoccidioides culture supernatants or in its synthetic form, both by shmoo formation and by growth and cell cycle arrests. This allowed us to conclude that Paracoccidioides species secrete an active α-pheromone into the culture medium that is able to activate its cognate receptor. Moreover, expression of PREB or PBα in the corresponding null mutants of S. cerevisiae restored mating in these non-fertile strains. Taken together, our data demonstrate pheromone signaling activation by the Paracoccidioides α-pheromone through its receptor in this yeast model, which provides novel evidence for the existence of a functional mating signaling system in Paracoccidioides.
Highlights
Paracoccidioides species are thermodimorphic ascomycete fungi that occur in a non-pathogenic mycelial form at environmental temperatures, and switch to a pathogenic multiplebudding yeast-form at the mammalian host temperature (37uC)
To evaluate the biological significance of this in silico predicted pheromone gene we confirmed the sequence of cDNA generated from P. lutzii Pb01 and P. brasiliensis ATCC60855 mycelium cultures (Figure S1)
Ability of the Corresponding S. cerevisiae Null Mutants We have demonstrated that the PBa gene from Paracoccidioides encodes a functional a-pheromone that signals through its cognate receptor PreB, using S. cerevisiae as a host model
Summary
Paracoccidioides species are thermodimorphic ascomycete fungi that occur in a non-pathogenic mycelial form at environmental temperatures (below 25uC), and switch to a pathogenic multiplebudding yeast-form at the mammalian host temperature (37uC) This fungus is the etiological agent of paracoccidioidomycosis (PCM), a systemic mycosis that is prevalent in Latin America and has high incidence in Colombia, Venezuela and Brazil [1,2]. A study by Pyrzak et al [16] showed that low transcriptional activity of mating-type regulators might be the cause of the low fertility observed in A. fumigatus These studies suggest that the discovery of sexual reproduction in asexual fungi may require specific culturing conditions, and show as well that fungi may have regulatory constraints that hinder expression of the mating system
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