Introducing fluorescence resonance energy transfer‐based biosensors for the analysis of cAMP‐PKA signalling in the fungal pathogen Candida glabrata

The cyclic adenosine monophosphate‐protein kinase A (cAMP‐PKA) pathway is central to signal transduction in many organisms. In pathogenic fungi such as Candida albicans, this signalling cascade has proven to be involved in several processes, such as virulence, indicating its potential importance in antifungal drug discovery. Candida glabrata is an upcoming pathogen of the same species, yet information regarding the role of cAMP‐PKA signalling in virulence is largely lacking. To enable efficient monitoring of cAMP‐PKA activity in this pathogen, we here present the usage of two FRET‐based biosensors. Both variations in the activity of PKA and the quantity of cAMP can be detected in a time‐resolved manner, as we exemplify by glucose‐induced activation of the pathway. We also present information on how to adequately process and analyse the data in a mathematically correct and physiologically relevant manner. These sensors will be of great benefit for scientists interested in linking the cAMP‐PKA signalling cascade to downstream processes, such as virulence, possibly in a host environment.     

Protein Kinase A Regulates Growth, Sporulation, and Pigment Formation in Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic human pathogenic fungus causing severe infections in immunocompromised patients. Cyclic AMP (cAMP) signal transduction plays an important role in virulence. A central component of this signaling cascade is protein kinase A (PKA), which regulates cellular processes by phosphorylation of specific target proteins. Here we describe the generation and analysis of A. fumigatus mutants expressing the gene encoding the catalytic subunit of PKA, pkaC1, under control of an inducible promoter. Strains overexpressing pkaC1 showed high PKA activity, reduced growth, sporulation deficiency, and formation of a dark pigment in the mycelium. These data indicate that cAMP-PKA signaling is involved in the regulation of important processes, such as growth, asexual reproduction, and biosynthesis of secondary metabolites. Furthermore, elevated PKA activity led to increased expression of the pksP gene. The polyketide synthase PksP is an essential enzyme for production of dihydroxynaphthalene-melanin in A. fumigatus and contributes to virulence. Our results suggest that increased pksP expression is responsible for pigment formation in the mycelium. Comparative proteome analysis of the pkaC1-overexpressing strain and the wild-type strain led to the identification of proteins regulated by the cAMP-PKA signal transduction pathway. We showed that elevated PKA activity resulted in activation of stress-associated proteins and of enzymes involved in protein biosynthesis and glucose catabolism. In contrast, proteins which were involved in nucleotide and amino acid biosynthesis were downregulated, as were enzymes involved in catabolism of carbon sources other than glucose.     

A Schizosaccharomyces pombe gene, ksg1, that shows structural homology to the human phosphoinositide-dependent protein kinase PDK1, is essential for growth, mating and sporulation

Fission yeast (Schizosaccharomyces pombe) requires inositol for growth, mating and sporulation. To define putative genes that are involved in the processing and transduction of the inositol signal, mutants that are temperature sensitive for growth and sporulation were selected on a medium containing non-limiting amounts of inositol. Two such mutants (ksg1-208 and ksg1-358) were analyzed, which are impaired in mating and sporulation at 30° C and undergo growth arrest in the G2 phase of the cell cycle at 35° C. The ksg1 gene was isolated by functional complementation. It maps on the left arm of chromosome II and encodes a putative 592-amino acid protein which exhibits good structural homology to a human 3-phosphoinositide-dependent protein kinase (PDK1) and its rat and Drosophila homologues. The two mutants have the same substitution at amino acid position 159: a glycine residue is replaced by glutamic acid. Deletion of the gene is lethal for haploid cells. We propose that ksg1 is involved in one or several phosphoinositide signalling processes that are responsible for control of the life cycle. Received: 24 September 1998 / Accepted: 8 November 1998