Candida albicans Lacking the Gene Encoding the Regulatory Subunit of Protein Kinase A Displays a Defect in Hyphal Formation and an Altered Localization of the Catalytic Subunit

Abstract

The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.Candida albicans is an opportunistic human fungal pathogen of great medical significance in immunocompromised patients (25). This fungus has the capability of switching its mode of growth between budding yeast and hypha or pseudohypha in response to environmental signals. Genetic evidence indicates that the morphogenetic switch to the hyphal mode of growth, though associated with pathogenicity and virulence (20), is necessary but not sufficient to trigger disease (5). The relationship between morphology and pathogenicity has been the focus of intensive research devoted to the study of the developmental programs involved in the dimorphic transition.The remarkable conservation of signal transduction pathways in fungi allowed the identification of components of these pathways in several fungal species based on the insight gained from studying pseudohyphal differentiation in Saccharomyces cerevisiae. In C. albicans, two major pathways implicated in dimorphism could be established: the mitogen-activated protein kinase and the cyclic AMP (cAMP)/protein kinase A transduction pathways (for a review, see reference 19).Initial biochemical studies indicate that high cAMP levels promote the yeast-to-hypha transition in C. albicans (23,31). In addition, we have shown that in vivo inhibition of protein kinase A blocks hyphal growth induced by N-acetylglucosamine (GlcNAc) (6). Recent genetic studies allowed the identification of the genes involved in the cAMP/protein kinase A pathway. A transduction cascade similar to that of S. cerevisiae, with regard to location and function of the homologous components, has been established. Thus, CaRa...

Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

“…Thus, zygomycetes seem to be the only class of fungi that have more than one isoform for the R subunit (1,2,8,9,11,14,26,46,50,51,52,54). The corresponding mutants with mutations in the genes encoding the PKA regulatory subunit have been obtained from several of these fungi, and there does not seem to be a unique way in how fungi respond to the deletion of the R subunit, since some of them, such as A. niger (46), have the same level of PKA activity as in the wild-type cell, whereas in others, such as C. neoformans or A. fumigatus (11,54), the PKA activity is increased.…”

“…Sporulation or conidiation defects also appear to be common to these mutants in filamentous fungi, such as A. niger, A. fumigatus, or C. lagenarium (46,51,54). Some R subunit mutants also show a loss of growth polarity, as in A. niger, C. albicans, and N. crassa (2,8,46). In plant-and human-pathogenic fungi such as U. maydis, C. albicans, and C. neoformans, R subunits are also required for filamentous growth (8,11,12,14).…”

Fungal Morphogenesis

“…The cAMP-dependent protein kinase A (PKA) is a major target of Cyr1, consisting of one regulatory subunit (Bcy1) and two catalytic subunits (Tpk1 and Tpk2). Each PKA subunit has distinct functions in C. albicans hyphal development, indicating even further adaptability of this otherwise highly conserved signaling system (Sonneborn et al 2000;Bockmuhl et al 2001;Cassola et al 2004). A major function of the C. albicans cAMP-PKA pathway is to downregulate the expression level of NRG1 (Lu et al 2011), the major repressor of hyphal morphogenesis (Braun et al 2001;Murad et al 2001).…”

The Flo8 Transcription Factor Is Essential for Hyphal Development and Virulence inCandida albicans

“…The G proteins act through PKA to induce morphogenesis in C. albicans. C. albicans PKA consists of one regulatory subunit, Bcy1, and two catalytic subunits, Tpk1 and Tpk2 (Sonneborn et al, 2000;Bockmuhl et al, 2001;Cassola et al, 2004). Tpk1 and Tpk2 have distinct functions in hyphal development because their mutants have differential effects in different media (Bockmuhl et al, 2001;Cassola et al, 2004).…”

“…C. albicans PKA consists of one regulatory subunit, Bcy1, and two catalytic subunits, Tpk1 and Tpk2 (Sonneborn et al, 2000;Bockmuhl et al, 2001;Cassola et al, 2004). Tpk1 and Tpk2 have distinct functions in hyphal development because their mutants have differential effects in different media (Bockmuhl et al, 2001;Cassola et al, 2004). One potential target of the cAMP/PKA pathway is Efg1, a basic helix-loop-helix protein similar to StuA of Aspergillus nidulans and Sok2 and Phd1 of S. cerevisiae (Stoldt et al, 1997).…”