Paracoccioides brasiliensis (Pb) is the causative agent of the disease Paracoccioidomycosis (PCM), which is one of the most prevalent systemic mycoses in Latin Amercia (BorgesWalmsley 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 si~e of the plasma membrane. They receive signals from G protein-coupled receptors (GPCR). Adenylyl cyclase acts downstream of these G proteins. Ga subunits are required to regulate the activity of adenylyl cyclase (AC), which controls the level of cellular cAMP (Ivey and Hoffman, 2005). Protein Kinase A (PKA), which is activated by cAMP, is required for morphogenesis and virulence (Durrenberger et al., 1998; Sonneborn et al., 2000). The cAMP pathway in P. brasiliensis 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, Gpa1-3, a Gp protein, Gpb1; a Gy protein, Gpg1; Ras; adenylyl cyclase, Cyrl; and the catalytic subunit of PKA, Tpk2. Twohybrid analyses confirmed that Gpa1 and Gpg1 interact with Gpbl. These data indicate the formation of a Gapy trimer complex. A GST pull-down assay confirnled that Gpa1 and Gpb1 interacted with the N-telminus of adenylyl cyclase. Our hypothesis is that Gpal and Gpb1 modulate the activity of the AC/Tpk2 signalling pathway. Consistent with this hypothesis, we I found changes in intracellular cAMP levels during the mycelium to yeast transformation that correlated with changing transcript levels of the signalling genes (Chen et al., 2007). We have established that Tpk2 interacts with the N-terrninus of adenylyl cyclase, the G protein Psubunit Gpb1 and with the co-repressor Tup1 by both two-hybrid and GST pulldown analyses. This suggests that Tpk2 activity is required for feedback regulation of adenylyl cyclase to reduce cAMP levels. P. brasiliensis Tpk2-C-terrninal 226-583-GFP and Tpk2 full length (FL) complemented the growth defect of a S. cerevisiae tpk2 temperature sensitive mutant strain SGY446 and induced the formation of pseudohyphae in the S. cerevisiae tpk2 mutant diploid strain XPY5a/a. Tpk2 C-terrninus 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 Gp subunit Gpbl inhibited the development of pseudohyphae in TPK2 FL transformed yeast cells. Tpk2 C-temlinus and Tpk2 FL co-transformed with GpbGFP were localized in the nucleus. Our hypothesis is that Gpb1 down regulates the activity of Tpk2, because Gpbl binds to the catalytic C-terrninal domain of Tpk2.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:505150 |
| Date | January 2008 |
| Creators | Janganan, Thamarai kannan |
| Publisher | Durham University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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