The regulation of the CAMP signalling pathway in the human pathogenic fungus, Paracoccidioides brasiliensis

Paracoccioides brasiliensis (Pb) is the causative agent of the disease Paracoccioidomycosis (PCM), which is one of the most prevalent systemic mycoses in Latin Amercia (Borges-Walmsley et al., 2002). P. brasiliensis is a thermally dimorphic fungus which undergoes morphological changes from a mycelial form at 26°C (environment) to a pathogenic yeast form at 37°C (human body) after inhalation of spores/conidia into the lungs of a human host (Nemecek et al., 2006). The cAMP pathway controls this morphological transformation in several fungi (Borges-Walmsley and Walmsley, 2000; Kronstad et al., 1998).

G proteins are guanine-nucleotide (GDP or GTP) binding proteins that are generally associated with the cytoplasmic side of the plasma membrane. They receive signals from G protein-coupled receptors (GPCR). Adenylyl cyclase acts downstream of these G proteins. Get subunits are required to regulate the activity of adenylyl cyclase (AC), which controls the level of cellular cAMP (lvey and Hoffman, 2005). Protein Kinase A (PKA), which is activated by cAMP, is required for morphogenesis and virulence (Durrenberger et al., 1998; Sonnebom et al., 2000).

The cAMP pathway in P. hrasiliensis is poorly understood. However, recently the genes encoding a number of the components of the cAMP pathway have been cloned in our lab: these include the genes encoding three Ga proteins, Gpal-3, a Gf3 protein, Gpbl; a Gy protein, Gpgl; Ras; adenylyl cyclase, Cyrl; and the catalytic subunit of PKA, Tpk2. Two hybrid analyses confirmed that Gpal and Gpgl interact with Gpbl. These data indicate the formation of a Ga(3y trimer complex. A GST pull-down assay confirmed that Gpal and Gpbl interacted with the N-tenninus of adenylyl cyclase. Our hypothesis is that Gpal and Gpbl modulate the activity of the AC/Tpk2 signalling pathway. Consistent with this hypothesis, we found changes in intracellular cAMP levels during the mycelium to yeast transformation that con-elated with changing transcript levels of the signalling genes (Chen et al., 2007).

We have established that Tpk2 interacts with the N-terminus of adenylyl cyclase, the G protein (3 subunit Gpbl and with the co-repressor Tup I by both two-hybrid and GST pull-down analyses. This suggests that Tpk.2 activity is required for feedback regulation of adenylyl cyclase to reduce cAMP levels. P. brasiliensis Tpk2-C-terminal 226oS3-GFP and Tpk2 full length (FL) complemented the growth defect of a S. cerevisiae tpkl temperature sensitive mutant strain SGY446 and induced the formation of pseudohyphae in the 5. cerevisiae tpkl mutant diploid strain XPY5a/a. Tpk2 C-terminus has been over expressed in E. coli and in vitro PKA activity was measured. On the other hand we have also analysed the second catalytic subunit Tpkl. which failed to induce pseudohyphae in S. cerevisiae tpk2 mutant strain and is localised to the cytoplasm.

Interestingly, the Pb G(3 subunit Gpbl inhibited the development of pseudohyphae in TPK2 FL transformed yeast cells. Tpk2 C-terminus and Tpk2 FL co-transformed with Gpb-GFP were localized in the nucleus. Our hypothesis is that Gpbl down regulates the activity of Tpk2, because Gpbl binds to the catalytic C-terminal domain of Tpk2.