A potential phosphorylation site for an A-type kinase in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans

Efg1p in the human fungal pathogen Candida albicans is a member of the conserved APSES class of proteins regulating morphogenetic processes in fungi. We have analyzed the importance for hyphal morphogenesis of a putative phosphorylation site for protein kinase A (PKA), threonine-206, within an Efg1p domain highly conserved among APSES proteins. Alanine substitution of T206, but not of the adjacent T207 and T208 residues, led to a block of hypha formation on solid and in liquid media, while a T206E exchange caused hyperfilamentation. The extent of the morphogenetic defect caused by the T206A mutation depended on hypha-induction conditions. Extragenous suppression of mutations in signaling components, including tpk2 and cek1 mutations, was achieved by wild-type- and T206E-, but not by the T206A-variant-encoding allele of EFG1. All muteins tested were produced at equal levels and at high production levels supported pseudohyphal formation. The results are consistent with a role of Efg1p as a central downstream component of a PKA-signaling pathway including Tpk2p or other PKA isoforms. Threonine-206 of Efg1p is essential as a putative phosphorylation target to promote hyphal induction by a subset of environmental cues.     

Candida albicans Int1p interacts with the septin ring in yeast and hyphal cells.

The ability to switch between yeast and hyphal morphologies is an important virulence factor for the opportunistic pathogen Candida albicans. Although the kinetics of appearance of the filamentous ring that forms at the incipient septum differ in yeast and cells forming hyphae (germ tubes) (), the molecular mechanisms that regulate this difference are not known. Int1p, a C. albicans gene product with similarity in its C terminus to Saccharomyces cerevisiae Bud4p, has a role in hyphal morphogenesis. Here we report that in S. cerevisiae, Int1p expression results in the growth of highly polarized cells with delocalized chitin and defects in cytokinesis and bud-site selection patterns, phenotypes that are also seen in S. cerevisiae septin mutant strains. Expression of high levels of Int1p in S. cerevisiae generated elaborate spiral-like structures at the periphery of the polarized cells that contained septins and Int1p. In addition, Int1p coimmunoprecipitated with the Cdc11p and Cdc12p septins, and Cdc12p is required for the establishment and maintenance of these Int1p/septin spirals. Although Swe1p kinase contributes to INT1-induced filamentous growth in S. cerevisiae, it is not required for the formation of ectopic Int1p/septin structures. In C. albicans, Int1p was important for the axial budding pattern and colocalized with Cdc3p septin in a ring at the mother-bud neck of yeast and pseudohyphal cells. Under conditions that induce hyphae, both Cdc3p and Int1p localized to a ring distal to the junction of the mother cell and germ tube. Thus, placement of the Int1p/septin ring with respect to the mother-daughter cell junction distinguishes yeast/pseudohyphal growth from hyphal growth in C. albicans.     

Distinct and redundant roles of the two protein kinase A isoforms Tpk1p and Tpk2p in morphogenesis and growth of Candida albicans

TPK1 and TPK2 encode both isoforms of protein kinase A (PKA) catalytic subunits in Candida albicans. Mutants lacking both TPK1 alleles showed defective hyphal morphogenesis on solid inducing media, whereas in liquid hypha, formation was affected slightly. In contrast, tpk2 mutants were only partially morphogenesis defective on solid media, whereas a strong block was observed in liquid. In addition, the yeast forms of tpk2-- but not tpk1-- mutants were completely deficient in invading agar. Because Tpk1p and Tpk2p differ in their N-terminal domains of approximately 80--90 amino acids, while the catalytic portions are highly homologous, the functions of hybrid Tpk proteins with exchanged N-terminal domains were tested. The results demonstrate that the catalytic portions mediate Tpk protein specificities with regard to filamentation, whereas agar invasion is mediated by the N-terminal domain of Tpk2p. Homozygous tpk1 and tpk2 mutants grew normally; however, a tpk2 mutant strain containing a single regulatable TPK1 allele (PCK1p-TPK1) at low expression levels was severely growth defective. It was completely blocked in hyphal morphogenesis and was stress resistant to high osmolarities or temperatures. Thus, both Tpk isoforms in C. albicans share growth functions but, unlike Saccharomyces cerevisiae isoforms, they have positive, specific roles in filament formation in different environments.     

A screen in Saccharomyces cerevisiae identified CaMCM1, an essential gene in Candida albicans crucial for morphogenesis

Morphogenesis in Saccharomyces cerevisiae and the pathogenic yeast Candida albicans is governed in part by the same molecular circuits. In S. cerevisiae, FLO11/MUC1 expression has been shown to be modulated by multiple signalling pathways required for pseudohyphal development. We have established a screen in S. cerevisiae to identify regulators of fungal development in C. albicans based on FLO11::lacZ expression as a reporter. This screen identified both known components of the mitogen-activated protein kinase (MAPK) cascade and the cAMP cascade that are important for hyphal development in C. albicans, as well as genes not yet known to be involved in morphogenesis. The Candida homologue of MCM1 is one of the novel factors identified in this screen as being important for morphogenesis. CaMcm1p levels do not vary significantly in different cell types and respond to an autoregulatory feedback mechanism, arguing that CaMcm1p activity is regulated by post-translational modifications. Both overexpression and repression of this essential gene led to the induction of hyphae. Moreover, we found that the expression of HWP1, a hyphae-specific gene, was induced by repression of CaMCM1. The changes in morphology and HWP1 expression were not the result of a change in expression levels of NRG1 or TUP1, known repressors of hyphal development. Thus, CaMcm1p is a component of a hitherto unknown regulatory mechanism of hyphal growth.     

Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans

External signals induce the switch from a yeast to a hyphal growth form in the fungal pathogen Candida albicans. We demonstrate here that the catalytic subunit of a protein kinase A (PKA) isoform encoded by TPK2 is required for internal signalling leading to hyphal differentiation. TPK2 complements the growth defect of a Saccharomyces cerevisiae tpk1-3 mutant and Tpk2p is able to phosphorylate an established PKA-acceptor peptide (kemptide). Deletion of TPK2 blocks morphogenesis and partially reduces virulence, whereas TPK2 overexpression induces hyphal formation and stimulates agar invasion. The defective tpk2 phenotype is suppressed by overproduction of known signalling components, including Efg1p and Cek1p, whereas TPK2 overexpression reconstitutes the cek1 but not the efg1 phenotype. The results indicate that PKA activity of Tpk2p is an important contributing factor in regulating dimorphism of C. albicans.     

The Candida albicans HYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall proteins.

A hyphally regulated gene (HYR1) from the dimorphic human pathogenic fungus Candida albicans was isolated and characterized. Northern (RNA) analyses showed that the HYR1 mRNA was induced specifically in response to hyphal development when morphogenesis was stimulated by serum addition and temperature elevation, increases in both culture pH and temperature, or N-acetylglucosamine addition. The HYR1 gene sequence revealed a 937-codon open reading frame capable of encoding a protein with an N-terminal signal sequence, a C-terminal glycosylphosphatidylinositol-anchoring domain, 17 potential N glycosylation sites, and a large domain rich in serine and threonine (51% of 230 residues). These features are observed in many yeast cell wall proteins, but no homologs are present in the databases. In addition, Hyr1p contained a second domain rich in glycine, serine, and asparagine (79% of 239 residues). The HYR1 locus in C. albicans CAI4 was disrupted by "Ura-blasting," but the resulting homozygous delta hyr1/delta hyr1 null mutant displayed no obvious morphological phenotype. The growth rates for yeast cells and hyphae and the kinetics of germ tube formation in the null mutant were unaffected. Aberrant expression of HYR1 in yeast cells, when an ADH1-HYR1 fusion was used, did not stimulate hyphal formation in C. albicans or pseudohyphal growth in Saccharomyces cerevisiae. HYR1 appears to encode a nonessential component of the hyphal cell wall.     

Distinct roles of two ceramide synthases, CaLag1p and CaLac1p, in the morphogenesis of Candida albicans

Lag1p and Lac1p catalyse ceramide synthesis in Saccharomyces cerevisiae. This study shows that Lag1 family proteins are generally required for polarized growth in hemiascomycetous yeast. However, in contrast to S. cerevisiae where these proteins are functionally redundant, C. albicans Lag1p (CaLag1p) and Lac1p (CaLac1p) are functionally distinct. Lack of CaLag1p, but not CaLac1p, caused severe defects in the growth and hyphal morphogenesis of C. albicans. Deletion of CaLAG1 decreased expression of the hypha-specific HWP1 and ECE1 genes. Moreover, overexpression of CaLAG1 induced pseudohyphal growth in this organism under non-hypha-inducing conditions, suggesting that CaLag1p is necessary for relaying signals to induce hypha-specific gene expression. Analysis of ceramide and sphingolipid composition revealed that CaLag1p predominantly synthesizes ceramides with C24:0/C26:0 fatty acid moieties, which are involved in generating inositol-containing sphingolipids, whereas CaLac1p produces ceramides with C18:0 fatty acid moieties, which are precursors for glucosylsphingolipids. Thus, our study demonstrates that CaLag1p and CaLac1p have distinct substrate specificities and physiological roles in C. albicans.