Global regulatory roles of the cAMP/PKA pathway revealed by phenotypic, transcriptomic and phosphoproteomic analyses in a null mutant of the PKA catalytic subunit in Candida albicans

Abstract

The conserved cAMP-dependent protein kinase (PKA) plays critical roles in the regulation of morphological transitions and virulence in the human fungal pathogen Candida albicans. It has long been thought that the PKA catalytic subunit is essential for cell viability in this fungus. Paradoxically, the single adenylyl cyclase-encoding gene, CYR1, which is required for the production of cAMP in C. albicans, is not essential for cell growth. Here, a double mutant of TPK1 and TPK2 (tpk2/tpk2 tpk1/tpk1, t2t1), which encode two isoforms of the PKA catalytic subunit was successfully generated, suggesting that this subunit is not essential for cell viability. Inactivation of the PKA catalytic subunit blocked filamentation and dramatically attenuated white-to-opaque switching, but promoted sexual mating. Comparative transcriptomic analyses demonstrated that the t2t1 and cyr1/cyr1 mutants exhibited similar global gene expression profiles. Compared with the WT strain, the general transcriptional activity and metabolism were significantly decreased in both the t2t1 and cyr1/cyr1 mutants. Using combined phosphoproteomic and bioinformatic analyses, we identified 181 potential PKA phosphorylation targets, which represent 148 unique proteins involved in a wide spectrum of biological processes. The study sheds new insights into the global regulatory features of the cAMP/PKA pathway in C. albicans.

Multiple roles and diverse regulation of the Ras/cAMP/protein kinase A pathway in Candida albicans

“…Global transcriptional profile analysis demonstrates that the tpk1/tpk1 tpk2/tpk2 and cyr1/cyr1 mutants share a high similarity in the regulation of hyphal development, metabolism and stress response‐related genes (Harcus et al ., ; Cao et al ., ). The discrepancy between the Bockmuhl study and ours could be due to the different parental strains used for the generation of the TPK mutants (Bockmuhl et al ., ; Cao et al ., ). However, it is unknown whether the URA3‐IRO1 locus is involved in this regulation.…”

“…Under several culture conditions including Lee’s glucose, Lee’s GlcNAc and Spider media, the deletion of either TPK1 or TPK2 does not affect the filamentous growth. However, no hyphal growth was observed in the tpk1/tpk1 tpk2/tpk2 double mutant under all inducing conditions tested, suggesting that the catalytic subunit is essential for hyphal growth (Cao et al ., ). Global transcriptional profile analysis demonstrates that the tpk1/tpk1 tpk2/tpk2 and cyr1/cyr1 mutants share a high similarity in the regulation of hyphal development, metabolism and stress response‐related genes (Harcus et al ., ; Cao et al ., ).…”

“…However, no hyphal growth was observed in the tpk1/tpk1 tpk2/tpk2 double mutant under all inducing conditions tested, suggesting that the catalytic subunit is essential for hyphal growth (Cao et al ., ). Global transcriptional profile analysis demonstrates that the tpk1/tpk1 tpk2/tpk2 and cyr1/cyr1 mutants share a high similarity in the regulation of hyphal development, metabolism and stress response‐related genes (Harcus et al ., ; Cao et al ., ). The discrepancy between the Bockmuhl study and ours could be due to the different parental strains used for the generation of the TPK mutants (Bockmuhl et al ., ; Cao et al ., ).…”

“…It had long been thought that the catalytic subunit of PKA was essential for cell viability in C. albicans (Bockmuhl et al ., ). Taking advantage of a newly developed gene deletion strategy and a multiple autotrophic strain (Noble and Johnson, ), we have successfully generated a tpk1/tpk1 tpk2/tpk2 double mutant in C. albicans (Cao et al ., ). Similar to the cyr1/cyr1 mutant (Rocha et al ., ), the tpk1/tpk1 tpk2/tpk2 double mutant exhibits an extremely slow growth rate (Cao et al ., ).…”

“…Activation of the Ras/cAMP/PKA pathway by overexpressing a constitutively active form of Ras (Ras1V13), the deletion of PDE2 or the deletion of BCY1 promotes filamentation in C. albicans (Feng et al ., ; Jung et al ., ; Ding et al ., ). Consistently, inactivation of this pathway by the deletion of CYR1 or both TPK1 and TPK2 completely blocks the filamentous growth in response to many strong inducers including serum, N‐acetylglucosamine (GlcNAc) and high levels of CO 2 (Klengel et al ., ; Cao et al ., ).…”