Cell signalling in Paracoccidioides brasiliensis
Paracocidioides brasiliensis (P. brasiliensis or Pb) is the etiological agent of paracocidioidomycosis which is the most prevalent systemic mycosis in South America. About 60% of the clinical cases are found concentrated in Brazil and the disease is a major threat for the public health there, p. brasiliensis is a dimorphic fungus because it undergoes a morphological switching from a mycelium to yeast form after shifting the temperature from 26 c to 37 c. Similar morphological changes have been implicated to be important in the pathogenicity of other dimorphic fungi and they appear to be under the control of the с AMP signalling transduction pathway. In order to establish the relationship of с AMP signalling pathway and the morphological change in p. brasiliensis, a project was initiated to clone the key components of the с AMP signalling pathway and analyze their functions. To this end, a genomic DNA library and two cDNA libraries oiPb were constructed. Degenerate primers were synthesized based on the sequence of genes of several other important fungi in GenBank. After amplification, specific PCR products were obtained and sequenced. The specific PCR products were used for labeling to screen libraries and their sequences were used for designing specific primers to do genomic walking and RACE-PCR. Both cDNA and genomic DNA sequences have been determined for the key components of the с AMP signalling pathway including adenylate cyclase (PbCYRl), three G protein α subunits (PbGPAl,PbGPA2 and PbGPA3), a G protein β subunit (PbGPBl), a G protein γ subunit (PbGPGl), a с AMP dependent protein kinase catalytic subunit (PbTPKJ), a с AMP depentdent protein kinase-like gene (PbTPKLl) and a TUP gene (PbTUPA).The interactions of the proteins encoded by the genes in the с AMP signaling pathway were studied with Clontech's Matchmaker yeast-two-hybrid System III. This approach demonstrated that the N-terminal third of adenylate cyclase PbCyrl 1 •678 can interact with PbGpal and PbGpbl. To further test if PbGpa2, PbGpa3, PbRasl, and PbActin can interact with adenylate cyclase, random mutagenesis libraries were made for these genes and screened with PbCyr1 1 •678, PbCyr1 600- 1316, PbCyri 1302 1876 and PbCyr1 1648-2100. It was demonstrated that PbCyrl 1-678, where the Ras association domain resides, can interact with truncated versions ofPbGpa2, PbGpa3, PbRasl, and PbActin, i.e., PbGpa21 -102, PbGpa21-183, PbGpa3 1-160, PbGpa3 1-213, PbRas1 1-83,PbRas 126-238, and PbActin 1-314. For the first time, the major components of Pb cAMP signaling pathway have been cloned and characterized; and we provide direct evidence that the G protein a subunits, G protein p subunit, RAS protein and actin interact directly with adenylate cyclase in fungal biology. This thesis funds the basis for the further study of the cAMP signalling pathway in P. brasiliensis. It may facilitate delineation of the mechanism for the dimorphic switching and the development of potentially novel antifungal drugs.