Catalytic isoforms Tpk1 and Tpk2 of Candida albicans PKA have non-redundant roles in stress response, glycogen metabolism, pseudohyphal elongation, cell adherence and biofilm formation.

GIACOMETTI ROMINA; KRONBERG FLORENCIA; PASSERON SUSANA

Miami, Florida, USA

Conferencia; 10 th ASM Candida and Candidiasis Conference; 2010

AMERICAN SOCIETY FOR MICROBIOLOGY

Candida albicans undergoes a reversible morphological transition from single yeast cells to pseudohyphal and hyphal filaments. In this organism cAMP dependent protein kinase (PKA), coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1), mediates basic cellular processes, such as the yeast to hyphae transition and cell cycle regulation. In the present study, we investigated the role of C. albicans PKA in response to different stresses, in glycogen metabolism, pseudohyphal formation, and biofilm architecture. To elucidate potential functional differences we performed the study on several PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in different BCY1 genetic backgrounds. Interestingly, we showed that tpk1/tpk1 strains developed a lower tolerance to saline exposure, heat shock and to oxidative stress, while wild-type and tpk2/tpk2 mutants were resistant to these stresses, indicating that both isoforms play different roles in the stress response pathway. We also found that heterozygous and homozygous BCY1 mutants, irrespective of the TPK background were highly sensitive to heat treatment. Surprisingly, we observed that those strains devoid of one or both TPK1 alleles were defective in glycogen storage, while strains lacking Tpk2 accumulated higher levels of the polysaccharide, indicating that Tpk1 and Tpk2 have opposite roles in carbohydrate metabolism. A scheme of possible phosphorylation substrate preference for both isoforms is given. Although it is known that both Tpk isoforms play positive roles in vegetative growth and filamentation, little is known regarding their participation in pseudohyphal development. We observed that under hypha-only inducing conditions, all BCY1 heterozygous strains shifted growth toward pseudohyphal morphology; however the pseudohyphae/hyphae ratio was higher in strains devoid of TPK2, due to low Bcy1 expression level in these strains. Under pseudohypha-only inducing conditions, regardless of the BCY1 background, strains lacking TPK2 were prone to develop short and branched pseudohyphae. In tpk2 strains, confocal microscopy (CSLM) revealed that biofilm architecture was markedly less dense composed of short pseudohyphae and blastospores, with reduced adhesion ability to abiotic material, suggesting a significant defect in cell adherence. Complementation of the tpk2/tpk2 mutant with a wild-type copy of TPK2 restored all the altered functions. These results suggest a direct link between the Tpk2 isoform and the cytoskeleton that may be part of a conserved mechanism underlying polarized pseudohyphal morphogenesis. Our study constitutes a significant advance in revealing the distinct roles of catalytic subunits in the C. albicans stress response, pseudohyphal pathway and also in cell adherence, an important determinant of pathogenicity.