A Gβ protein and the TupA Co-Regulator Bind to Protein Kinase A Tpk2 to Act as Antagonistic Molecular Switches of Fungal Morphological Changes

The human pathogenic fungus Paracoccidioides brasiliensis (Pb) undergoes a morphological transition from a saprobic mycelium to pathogenic yeast that is controlled by the cAMP-signaling pathway. There is a change in the expression of the Gβ-protein PbGpb1, which interacts with adenylate cyclase, during this morphological transition. We exploited the fact that the cAMP-signaling pathway of Saccharomyces cerevisiae does not include a Gβ-protein to probe the functional role of PbGpb1. We present data that indicates that PbGpb1 and the transcriptional regulator PbTupA both bind to the PKA protein PbTpk2. PbTPK2 was able to complement a TPK2Δ strain of S. cerevisiae, XPY5a/α, which was defective in pseudohyphal growth. Whilst PbGPB1 had no effect on the parent S. cerevisiae strain, MLY61a/α, it repressed the filamentous growth of XPY5a/α transformed with PbTPK2, behaviour that correlated with a reduced expression of the floculin FLO11. In vitro, PbGpb1 reduced the kinase activity of PbTpk2, suggesting that inhibition of PbTpk2 by PbGpb1 reduces the level of expression of Flo11, antagonizing the filamentous growth of the cells. In contrast, expressing the co-regulator PbTUPA in XPY5a/α cells transformed with PbTPK2, but not untransformed cells, induced hyperfilamentous growth, which could be antagonized by co-transforming the cells with PbGPB1. PbTUPA was unable to induce the hyperfilamentous growth of a FLO8Δ strain, suggesting that PbTupA functions in conjunction with the transcription factor Flo8 to control Flo11 expression. Our data indicates that P. brasiliensis PbGpb1 and PbTupA, both of which have WD/β-propeller structures, bind to PbTpk2 to act as antagonistic molecular switches of cell morphology, with PbTupA and PbGpb1 inducing and repressing filamentous growth, respectively. Our findings define a potential mechanism for controlling the morphological switch that underpins the virulence of dimorphic fungi.

人类致病真菌巴西副球孢子菌（Paracoccidioides brasiliensis，Pb）会经历从腐生菌丝体到致病酵母的形态转换，该过程受环腺苷酸（cAMP）信号通路调控。在这一形态转换进程中，与腺苷酸环化酶相互作用的Gβ蛋白（Gβ-protein）PbGpb1的表达水平会发生显著改变。本研究利用酿酒酵母（Saccharomyces cerevisiae）的cAMP信号通路不含Gβ蛋白这一特性，对PbGpb1的功能角色展开探究。实验数据表明，PbGpb1与转录调节因子PbTupA均可结合至蛋白激酶A（PKA）蛋白PbTpk2。PbTPK2能够互补酿酒酵母TPK2缺失菌株XPY5a/α的表型，该菌株存在假菌丝生长缺陷。尽管PbGPB1对亲本酿酒酵母菌株MLY61a/α无显著影响，但它可抑制转入PbTPK2的XPY5a/α菌株的丝状生长，该现象与絮凝蛋白FLO11（floculin FLO11）的表达水平降低密切相关。体外实验结果显示，PbGpb1能够降低PbTpk2的激酶活性，这提示PbGpb1对PbTpk2的抑制作用可降低FLO11的表达水平，进而拮抗细胞的丝状生长。与之相反，在转入PbTPK2的XPY5a/α细胞中表达共调节因子PbTUPA（未转化细胞则无此效应）可诱导超丝状生长，而通过共转入PbGPB1能够拮抗这一表型。PbTUPA无法诱导FLO8缺失菌株产生超丝状生长，这表明PbTupA需与转录因子Flo8（Flo8）协同发挥作用，以调控FLO11的表达。本研究数据证实，巴西副球孢子菌的PbGpb1与PbTupA均带有WD/β-螺旋桨结构（WD/β-propeller structures），二者可通过结合至PbTpk2，作为调控细胞形态的拮抗分子开关：PbTupA可诱导丝状生长，而PbGpb1则对该过程产生抑制。本研究结果阐明了一种潜在的调控机制，可用于控制支撑二形性真菌毒力的形态转换进程。