Functionality of the <em>Paracoccidioides</em> Mating α-Pheromone-Receptor System

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.现有研究证据表明，副球孢子菌属（Paracoccidioides）物种具备有性生殖潜力，但目前尚未在自然环境或实验室条件下观测到其完整有性周期。本研究中，我们在多株副球孢子菌分离株的酵母相与菌丝相中，检测到异宗配合交配型位点（MAT loci）基因MAT1-1、MAT1-2，α-信息素（PBα）基因，以及α-和a-信息素受体（PREB与PREA）基因呈现低水平表达。所有上述基因的表达均未表现出交配型依赖性。用合成α-信息素肽刺激巴西副球孢子菌（P. brasiliensis）MAT1-2菌株时，未能诱导MAT1-2、PREB或STE12基因的转录激活，提示受试菌株对α-信息素不具有响应性。为进一步评估α-信息素与其受体配对的生物学功能，我们采用了将副球孢子菌相关基因在酿酒酵母（S. cerevisiae）对应缺失突变株中进行异源表达的实验体系。实验结果显示，异源表达PREB的酿酒酵母菌株，无论是用从副球孢子菌培养上清中分离的Pbα信息素，还是合成形式的Pbα信息素刺激，均可通过施莫突起（shmoo）形成、生长抑制及细胞周期阻滞产生响应。这一结果证实，副球孢子菌属物种会向培养基中分泌具有活性的α-信息素，该物质可激活其同源受体。此外，在酿酒酵母的对应缺失突变株中表达PREB或PBα，可恢复这些非可育菌株的交配能力。综合上述实验结果，我们的数据证实，副球孢子菌属的α-信息素可通过其受体在该酵母模型中激活信息素信号通路，这为副球孢子菌属中存在功能性交配信号系统提供了新的实验证据。