Abstract
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.
Highlights
Paracoccidioides brasiliensis is one of a group of six phylogenetically related ascomycete fungi that, from more than a hundred-thousand fungi in the environment, have adapted for survival in mammalian hosts [1,2,3]
We used a PbTPK2-GFP fusion construct to screen for S. cerevisiae transformant colonies that produced green fluorescence; these transformants demonstrate that the P. brasiliensis TPK2 gene complemented the SGY446 and XPY5a/α phenotypes, to grow at 37°C (Fig 1A), and to produce pseudohyphae (Fig 1B), respectively
In order to confirm that the ability of P. brasiliensis TPK2 to complemented the SGY446 and XPY5a/α phenotypes was attributable to its kinase activity, rather than indirectly by sequestering the ScBcy1 regulatory proteins to release ScTpk1 and 3 in the S. cerevisiae cells, a mutation (e.g. K301R) was introduced into PbTPK2 to render the protein derivative kinase inactive [44]
Summary
Paracoccidioides brasiliensis is one of a group of six phylogenetically related ascomycete fungi that, from more than a hundred-thousand fungi in the environment, have adapted for survival in mammalian hosts [1,2,3] These six fungi are dimorphic, undergoing extensive changes that allow them to switch from a nonpathogenic filamentous mycelium, usually found in soil, to pathogenic single-cellular yeast that every year causes infections in millions of people across the globe. Infection is the result of hypha-fragments or spores released from mycelium, which are inhaled by the host, exposing them to an increased temperature that triggers the morphological switch The pathogenicity of these fungi is intimately linked to the morphological change since strains that are unable to transform from mycelium to yeast are often avirulent [3,4]. One of the best-studied fungal c-AMP-signaling pathways that control morphological changes is that in Saccharomyces cerevisiae, which produces pseudohyphae in response to nitrogen limitation in the presence of a rapidly fermentable sugar [20,21]
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