Candida albicans Tpk1p and Tpk2p isoforms differentially regulate pseudohyphal development, biofilm structure, cell aggregation and adhesins expression

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-hypha transition and cell cycle regulation. It is known that both Tpk isoforms play positive roles in vegetative growth and filamentation, although distinct roles have been found in virulence, stress response and glycogen storage. However, little is known regarding the participation of Tpk1p and/or Tpk2p in pseudohyphal development. This point was addressed using several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in different BCY1 genetic backgrounds. We observed that under hypha-only inducing conditions, all BCY1 heterozygous strains shifted growth toward pseudohyphal morphology; however, the pseudohypha:hypha ratio was higher in strains devoid of TPK2. Under pseudohypha-only inducing conditions, strains lacking TPK2 were prone to develop short and branched pseudohyphae. In tpk2 Δ/tpk2 Δ strains, 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. Immunolabelling assays showed a decreased expression of adhesins Als1p and Als3p only in the tpk2 Δ/tpk2 Δ strain. Complementation of this mutant with a wild-type copy of TPK2 restored all the altered functions: pseudohyphae elongation, biofilm composition, cell aggregation and adhesins expression. Our study suggests that the Tpk2p isoform may be part of a mechanism underlying not only polarized pseudohyphal morphogenesis but also cell adherence.     

Catalytic isoforms Tpk1 and Tpk2 of Candida albicans PKA have non‐redundant roles in stress response and glycogen storage

Candida albicans cAMP‐dependent protein kinase (PKA) is coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1). In this organism the cAMP/PKA signalling pathway 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 saline, heat and oxidative stresses as well as in glycogen storage. To fine‐tune the analysis, we performed the studies on several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in a different BCY1 genetic background. We observed that tpk1Δ/tpk1Δ strains developed a lower tolerance to saline exposure, heat shock and oxidative stress, while wild‐type and tpk2Δ/tpk2Δ mutants were resistant to these stresses, indicating that both isoforms play different roles in the stress response pathway. We also found that regardless of the TPK background, heterozygous and homozygous BCY1 mutants 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. Copyright © 2009 John Wiley & Sons, Ltd.     

The level of cAMP-dependent protein kinase A activity strongly affects osmotolerance and osmo-instigated gene expression changes in Saccharomyces cerevisiae

The influence of cAMP-dependent protein kinase (PKA) on protein expression during exponential growth under osmotic stress was studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The responses of isogenic strains (tpk2Deltatpk3Delta) with either constitutively low (tpk1(w1)), regulated (TPK1) or constitutively high (TPK1bcy1Delta) PKA activity were compared. The activity of cAMP-dependent protein kinase (PKA) was shown to be a major determinant of osmotic shock tolerance. Proteins with increased expression during growth under sodium chloride stress could be grouped into three classes with respect to PKA activity, with the glycerol metabolic proteins GPD1, GPP2 and DAK1 standing out as independent of PKA. The other osmotically induced proteins displayed a variable dependence on PKA activity; fully PKA-dependent genes were TPS1 and GCY1, partly PKA-dependent genes were ENO1, TDH1, ALD3 and CTT1. The proteins repressed by osmotic stress also fell into distinct classes of PKA-dependency. Ymr116c was PKA-independent, while Pgi1p, Sam1p, Gdh1p and Vma1p were fully PKA-dependent. Hxk2p, Pdc1p, Ssb1p, Met6p, Atp2p and Hsp60p displayed a partially PKA-dependent repression. The promotors of all induced PKA-dependent genes have STRE sites in their promotors suggestive of a mechanism acting via Msn2/4p. The mechanisms governing the expression of the other classes are unknown. From the protein expression data we conclude that a low PKA activity causes a protein expression resembling that of osmotically stressed cells, and furthermore makes cells tolerant to this type of stress.     

Functional analysis of six ORFs from Saccharomyces cerevisiae chromosome IV: two-spored asci produced by disruptant of YDR027c and strain-dependent DNA heterogeneity around YDR036c.

Six S. cerevisiae FY1679 heterozygous deletion mutants were made by replacing six open reading frames (ORFs) of the chromosome IV right arm with kanMX4 selection marker. Haploid and homozygous diploid deletion mutants were obtained from sporulation, dissection and mating experiments. No essential genes were found. The basic phenotypic analysis showed that the haploid and homozygous deletants for the ORF YDR027c (LUV1, VSP54 or RKI1) grew slowly. The diploid homozygous deletants for this ORF had a low frequency of sporulation. They produced asci with no more than one or two haploid spores and the majority of these spores formed were not viable. The deletion of the other ORFs, YDR022c (CIS1), YDR030c (RAD28), YDR032c (PST2), YDR033w (MRH1) and YDR036c, did not change the phenotypes tested in strain FY1679 or the first four ORFs in strain CEN.PK2. This work showed some differences in the DNA sequences between FY1679 and CEN.PK2: the regions immediately 1 kb upstream from YDR036c in these two strains are too different to hybridize properly, preventing deletion of YDR036c in the CEN.PK2 background by recombination with a disruption cassette designed for FY1679. In addition, there are different sets of transposable elements on the other side of the ORF, the differences starting at about 3.5 kb downstream from YDR036c.     

Characterization of the adr1-1 nonsense mutation identifies the translational start of the yeast transcriptional activator ADR1

We have characterized a nonsense mutation in the ADR1 gene that identifies the translational start of the ADR1 protein. The ADR1 gene of Saccharomyces cerevisiae is required for synthesis of the glucose-repressible alcohol dehydrogenase (ADH2). The adr1-1 mutation, which inhibits ADH2 expression, was identified as a C to G transversion at base pair +32. This alteration would result in a UGA nonsense codon in place of a serine codon that would lead to termination of the ADR1 polypeptide after the 10th amino acid. The effect of the adr1-1 mutation was partially reversed by UGA-tRNA suppressors, indicating that the adr1-1 mutation affects ADR1 expression at the translational level. These observations establish that the first available AUG in the ADR1 sequence is used as the translational start site of ADR1. Tyrosine or leucine UGA-tRNA-suppressors resulted in levels of adr1-1 activity similar to that found for a serine UGA-tRNA-suppressor, suggesting that serine residue-11 is not essential to ADR1 function. Northern analyses showed that the 5.1 kb ADR1 mRNA was two- to three-fold more abundant when isolated from a strain carrying the ADR1 allele than from an isogenic strain containing the adr1-1 allele. These data confirm that the 5.1 kb mRNA is the ADR1 mRNA and suggest that inhibition of adr1-1 mRNA translation results in more rapid degradation of the adr1-1 mRNA.     

Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro

The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cells in vitro. Bovine granulosa cells from small follicles (2-4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levels and oestradiol accumulation (P < 0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance in insulin- and FSH-stimulated cells. P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway.     

Isolation and characterization of sake yeast mutants deficient in gamma-aminobutyric acid utilization in sake brewing

Sake yeasts take up gamma-aminobutyric acid (GABA) derived from rice-koji in the primary stage of sake brewing. The GABA content in sake brewed with the UGA1 disruptant, which lacked GABA transaminase, was higher than that brewed with the wild-type strain K701. The UGA1 disruptant derived from sake yeast could not grow on a medium with GABA as the sole nitrogen source. We have isolated the sake yeast mutants of K701 that were unable to grow on a medium containing GABA as the sole nitrogen source. The growth defect of GAB7-1 and GAB7-2 mutants on GABA plates was complemented by UGA1, which encodes GABA transaminase, and UGA2, which encodes succinic semialdehyde dehydrogenase (SSADH), respectively. DNA sequence analysis revealed that GAB7-1 had a homozygous nonsense mutation in UGA1 and GAB7-2 had a heterozygous mutation (G247D) in UGA2. The GABA transaminase activity of GAB7-1 and the SSADH activity of GAB7-2 were markedly lower than those of K701. These GAB mutants displayed a higher intracellular GABA content. The GABA contents in sake brewed with the mutants GAB7-1 and GAB7-2 were 2.0 and 2.1 times higher, respectively, than that brewed with the wild-type strain K701. These results suggest that the reduced function of the GABA utilization pathway increases the GABA content in sake.     

The cyclic AMP/PKA signal pathway is required for initiation of spore germination in Schizosaccharomyces pombe

Spore germination, a transition from the quiescent G0 phase to the proliferation cycle, is triggered by glucose in Schizosaccharomyces pombe. The role of cAMP/protein kinase A (PKA) signalling in germination is investigated. Gene disruption of cyr1+, pka1+ and gpa2+ encoding adenylate cyclase, PKA and the alpha-subunit of a trimeric GTP-binding protein, respectively, reduced the colony-forming efficiency of spores in minimal medium. Isolated spores of these null mutants did not germinate in minimal medium for up to 12 h, at which time wild-type spores had completed germination and formed germ projections. In wild-type spores, cortical actin patches randomly distributed in the early stage of outgrowth and then localized to one side of spores before the formation of projections. In contrast, the mutant spores exhibited no actin patches, but the cell surface was predominantly stained, like ungerminated spores of wild-type. Flow fluorocytometric analysis of propidium iodide-stained spores revealed a distinct 1C DNA peak after germination was completed. The fluorescent profile of the mutant spores, however, did not change during 12 h incubation in the minimal medium. These observations indicate that spores harbouring either cyr1Delta, pka1Delta or gpa2Delta are hardly triggered to germination. When wild-type spores were exposed to glucose, the intracellular cAMP level transiently increased in a few minutes, but gpa2Delta spores did not respond to glucose. We conclude that S. pombe spores initiate germination in response to glucose through the cyclic AMP-PKA pathway.     

Hyperthermotolerant fission yeast mutations, sow1 and sow2, suppress the cell cycle defect and stress sensitivity of MAP kinase kinase wis1Delta

Wis1 is a mitogen-activated protein kinase kinase (MAPKK) that regulates mitosis and mediates stress responses in the fission yeast, Schizosaccharomyces pombe. wis1Delta strains are viable but stress-sensitive and show a mitotic delay. At high temperatures, wis1Delta cells cease division but cellular growth continues. Mutations that suppress the heat sensitivity of a wis1Delta strain were isolated and map to two apparently novel loci, sow1 (for suppressor of wis1Delta) and sow2. In addition to suppressing wis1Delta heat sensitivity, sow1 and sow2 can suppress wis1Delta osmosensitivity and cell cycle defects. sow1 and sow2 mutants in a wis1+ background were able to grow at higher temperatures than wild-type and sow1 showed a mitotic advance. The sow genes may therefore define a novel connection between stress tolerance and cell cycle control.     

Function of Candida glabrata ABC transporter gene,PDH1

The rapid increase in azole resistance during treatment of patients infected with Candida glabrata may be due to increased azole efflux mediated by ABC transporters, as occurs with increased expression of PDR5 in Saccharomyces cerevisiae. Two known C. glabrata homologues of PDR5 influencing azole susceptibility are PDH1 (CgCDR2) and CgCDR1. Disruption of PDH1 in a cgcdr1::ura3 strain increased susceptibility to rhodamine 6G, cycloheximide and chloramphenicol, and also increased rhodamine 6G accumulation, all properties of pdr5 null mutants. Overexpression of PDH1 in S. cerevisiae complemented the pdr5 mutation by reversing susceptibility to rhodamine 6G, chloramphenicol and cycloheximide, as well as by decreasing rhodamine 6G intracellular concentration. Expression of PDH1 in a C. glabrata cgcdr1::ura3 pdh1Delta::ura3 mutant using a multicopy plasmid almost completely restored the wild-type phenotype, showing that PDH1 at higher levels of expression can replace CgCDR1. Because PDH1 and CgCDR1 have both been reported to have upstream sequences similar to the Pdr1p- and Pdr3p-binding elements of PDR5, we sought similarities in regulation between the three genes. Abundance of PDH1 and CgCDR1 mRNA in C. glabrata was increased by rhodamine 6G, cycloheximide and oligomycin, properties in common with PDR5. PDH1, CgCDR1 and PDR5 have striking similarities in function and regulation.     

Isolation and characterization of Saccharomyces cerevisiae mutants with derepressed thiamine gene expression

Using a THI4-lacZ reporter gene, mutant strains have been isolated that display constitutive expression of thiamine genes in the presence of normally repressing levels of exogenous thiamine. In total, eight strains were isolated in which this derepressed expression on thiamine (Det(-)) phenotype was the result of single gene mutations. The Det(-) mutations of three of these strains were partially dominant in a heterozygous diploid configuration, whereas the other five were recessive. The partially dominant mutants DET1, DET12 and DET13, and the recessive mutant det2, all showed derepressed THI4-lacZ expression levels comparable to those of a fully induced normal strain. Use of other promoter-lacZ gene fusions revealed that these four mutants were pleiotropic; expression levels of all thiamine-regulated genes tested were also derepressed. Genetic analysis of the four mutants suggested that det2 and DET13 were allelic, whereas the others were at different loci; these four mutations therefore represent three different genes. None of the mutations were allelic with THI80, mutations of which have previously been shown to confer derepression on thiamine-regulated genes. Also, intracellular thiamine levels were close to normal and none of the four mutants excreted thiamine into the growth medium. All mutant strains were found to be prototrophic for thiamine and none of those tested were compromised for thiamine uptake. It is possible that some may be alleles of, or interact with, the activator gene THI3. Taken together, these results imply that DET1, det2, DET12 and DET13 represent new genes encoding negative regulators of thiamine-repressed genes.     

Identification and characterization of Saccharomyces cerevisiae mutants defective in fluid-phase endocytosis

A mutant library generated by the European Functional Analysis Network (EUROFAN) was screened for strains defective in fluid-phase endocytosis. Accumulation of Lucifer yellow in the vacuole was used as a marker for efficient endocytosis. Fourteen mutants, including ede1Delta, rcy1Delta, sys1Delta and tlg2Delta, previously described to be involved in membrane trafficking, were identified in this screen. alpha-Factor uptake, endocytosis of FM4-64, carboxypeptidase Y secretion, vacuolar morphology, and a vma2 synthetic growth defect were used as criteria to characterize the endocytic defect of the mutant strains obtained. Accordingly, eight mutant strains have endocytic phenotypes in addition to their defect in Lucifer yellow accumulation. These fluid-phase endocytosis mutants are defective at different steps of the endocytic pathway. Interestingly, only two mutants were defective for internalization, two for vacuolar protein sorting and four mutants had aberrant vacuolar morphologies. Some of the mutants identified in this screen that sort carboxypeptidase Y correctly may affect endocytosis at an early post-internalization step before the intersection of the endocytic with the vacuolar protein-sorting pathway.     

CaALK8, an alkane assimilating cytochrome P450, confers multidrug resistance when expressed in a hypersensitive strain of Candida albicans

We report the isolation of a novel C. albicans gene designated CaALK8, by its ability to complement drug hypersensitivity of a pdr5 (ABC: ATP-binding cassette drug extrusion pump) null mutant of S. cerevisiae (JG436). CaALK8 in JG436 conferred resistance to drugs such as cycloheximide (CYH), fluconazole (FCZ), O-phenanthroline (PHE) and 4-nitroquinoline oxide (NQO). The gene was so designated because its sequence was identical to a partial sequence entry named as ALK8 in the Candida database (http://alces.med.umn.edu/candida.html). CaALK8 encodes for a putative 515 amino acid protein highly homologous to alkane-inducible cytochromes P450 (CYP52 gene family) of C. maltosa and C. tropicalis. The ability of CaALK8 to confer drug resistance was also established by its expression in another drug-hypersensitive strain of S. cerevisiae (AD 1234568), which was deleted in seven ABC efflux pumps. The homozygous disruption of CaALK8 in a wild-type C. albicans strain (CAI4) did not result in altered drug susceptibilities. The overexpression of CaALK8 in CAI4 resulted in only FCZ resistance. However, a distinct MDR phenotype was evident when CaALK8 was overexpressed in a drug-hypersensitive C. albicans strain disrupted in both CDR1 and CDR2 (ABC drug extrusion pumps of C. albicans). Alk8p, similar to other Alk proteins from C. maltosa and C. tropicalis, could hydroxylate alkanes and fatty acids. In this study we demonstrate that several drugs could compete with the hydroxylation activity by directly interacting with CaAlk8p. Taken together, our results suggest that a member of the CYP52 gene family could mediate MDR in C. albicans, although it does not seem to be involved in the development of azole resistance in clinical isolates. The nucleotide sequence reported in this paper has been submitted to GenBank under Accession No. Y14766.     

Effects of luteinizing hormone on peroxisome proliferator-activated receptor gamma in the rat ovary before and after the gonadotropin surge

We have shown previously that mRNA for peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in granulosa cells and downregulated by the luteinizing hormone (LH) surge. The current studies were undertaken to test the hypothesis that LH stimulates a decrease in the expression of PPARgamma, as well as its activity, in granulosa cells. Ovaries were collected from immature rats 0 and 48 h after they received pregnant mares' serum gonadotropin (PMSG), and 4 and 24 h after administration of human chorionic gonadotropin (hCG), and used for protein isolation or processed for immunolocalization of PPARgamma. The amount of phosphorylated PPARgamma was measured by immunoblot analysis to determine how LH affects the phosphorylation status, and therefore the activity, of PPARgamma. Granulosa cells were also collected from immature rats 48 h after PMSG. Cells were cultured with LH in the absence and presence of H89 and cycloheximide to investigate the role of PKA and protein synthesis in the LH-mediated decline in mRNA for PPARgamma respectively. Protein corresponding to PPARgamma was localized to nuclei of granulosa cells 0 and 48 h after PMSG. Expression was greatly reduced by 4 h after hCG, with expression in mural granulosa cells lost before that in cumulus cells. The amount of phosphorylated PPARgamma did not change during the periovulatory period. Blocking PKA activity had no effect on levels of mRNA for PPARgamma. However, levels of mRNA for PPARgamma were significantly increased in cells treated with cycloheximide (P < 0.05, ANOVA followed by Tukey's HSD). These data suggest that PPARgamma is tightly regulated in the ovary and that its expression is the primary mechanism by which LH influences the activity of PPARgamma. In addition, protein synthesis may be involved in modulating levels of PPARgamma in granulosa cells.