Repression of hyphal proteinase expression by the mitogen-activated protein (MAP) kinase phosphatase Cpp1p of Candida albicans is independent of the MAP kinase Cek1p.

Cpp1p is a putative mitogen-activated protein (MAP) kinase phosphatase that suppresses Candida albicans hyphal formation at 25 degrees C through its probable substrate, the Cek1p filamentation MAP kinase. Here we report that expression of the serum-induced genes SAP4-6 and HYR1 increased several fold in hyphal forms of a cpp1/cpp1 null mutant, while the rate and extent of hyphal development up to 5 h were normal. Therefore, we provide evidence that Cpp1p represses hyphal gene expression by acting through a Cek1p-independent mechanism. SAP4-6 and HYR1 transcripts were undetectable in a null mutant of another key regulator of filamentation, Efg1p; thus, Efg1p and Cpp1p oppose each other during the expression of these genes in hyphal forms.

Candida glabrata displays pseudohyphal growth.

The ability to undergo morphological change has been reported as an advantageous trait in fungal pathogenesis. Here we demonstrate that Candida glabrata ATCC2001, like diploid Saccharomyces cerevisiae strains, forms elongated chains of pseudohyphal cells on solid nitrogen starvation media (SLAD). Constrictions were apparent between adjoining cells; no parallel-sided hyphae were seen and pseudohyphae invaded the agar. When SLAD was supplemented with ammonium sulfate both C. glabrata and diploid S. cerevisiae strains lost their ability to undergo pseudohyphal growth. However, on this media C. glabrata yeast cells invaded the agar in a similar fashion to the invasive growth mode exhibited by haploid strains of S. cerevisiae cultured on rich media such as YPD. C. glabrata was not capable of invading YPD demonstrating that the process of filamentation is distinct in these two fungi. To our knowledge this is the first report to demonstrate that C. glabrata can undergo morphological change and grow as an invasive filamentous organism.

Differences in virulence between two Candida albicans strains in experimental keratitis.

PURPOSE: To study the differences in disease caused by two wild-type strains of Candida albicans in a model of contact lens-facilitated keratitis in rabbits. METHODS: Two strains, SC5314 and VE175, were examined. Standardized inocula were placed on the debrided corneal surface of one eye in Dutch belted rabbits and covered with a contact lens. A temporary tarsorrhaphy was opened after 24 hours with removal of the contact lens. Six days later, corneas were photographed and animals killed. Corneas were bisected with one half for quantitative isolate recovery and the other for stromal penetration by hyphae. RESULTS: Strain SC5314 was significantly more virulent. The mean hyphal penetration into the cornea was 24.4% +/- 8.5% of the corneal thickness, and in three of six corneas hyphae penetrated through the entire cornea. In contrast, for VE175, the mean hyphal penetration was 2.6% +/- 1.2%. The difference between these two strains was statistically significant (P = 0.0297). Hyphae did not penetrate into the deep layers of the cornea in any of the six rabbits infected with VE175. The grading of clinical disease was consistent with histology, in that strain SC5314 caused more severe infection than VE175 and the difference was statistically significant (P = 0.0048). There was no difference in isolate recovery. CONCLUSIONS: Wild-type strains of C. albicans can differ significantly in virulence as measured by depth of fungal invasion into corneas and clinical evaluation of infection. Further characterization of the intrinsic genetic differences between such strains may help identify factors responsible for fungal virulence.

Mitogen-activated protein kinase-defective Candida albicans is avirulent in a novel model of localized murine candidiasis.

Candida albicans strains with a deletion of the mitogen-activated protein kinase CEK1 gene are defective in the yeast to hyphal transition on solid surfaces in vitro. The virulence of a cek1 delta/cek1 delta null mutant strain was compared with its wild-type parent strain (WT) in a novel model of localized candidiasis. The mammary glands of lactating mice (at day 5 postpartum) were infected for 2, 4 and 6 days with 50 microliter suspension containing 1 x 10(5), 1 x 10(6) and 1 x 10(7) blastopores before death. Infected and non-infected control glands were evaluated pathologically. All animals infected with cek1 delta/cek1 delta null mutant strains showed no lesions while 65% of animals infected with the WT strain had severe lesions characterized by widespread heterophilic infiltration, necrosis, and abscess formation. As an additional control, animals infected with the disrupted strain complemented with the WT CEK1, on a replicating plasmid, also showed severe pathological changes similar to the WT strain. These results clearly demonstrate that the CEK1 gene codes for a virulence determinant of C. albicans and that the mouse mastitis model is well suited for the discriminative study of the pathogenicity of different C. albicans strains.

Roles of the Candida albicans mitogen-activated protein kinase homolog, Cek1p, in hyphal development and systemic candidiasis.

Extracellular signal-regulated protein kinase (ERK, or mitogen-activated protein kinase [MAPK]) regulatory cascades in fungi turn on transcription factors that control developmental processes, stress responses, and cell wall integrity. CEK1 encodes a Candida albicans MAPK homolog (Cek1p), isolated by its ability to interfere with the Saccharomyces cerevisiae MAPK mating pathway. C. albicans cells with a deletion of the CEK1 gene are defective in shifting from a unicellular budding colonial growth mode to an agar-invasive hyphal growth mode when nutrients become limiting on solid medium with mannitol as a carbon source or on glucose when nitrogen is severely limited. The same phenotype is seen in C. albicans mutants in which the homologs (CST20, HST7, and CPH1) of the S. cerevisiae STE20, STE7, and STE12 genes are disrupted. In S. cerevisiae, the products of these genes function as part of a MAPK cascade required for mating and invasiveness of haploid cells and for pseudohyphal development of diploid cells. Epistasis studies revealed that the C. albicans CST20, HST7, CEK1, and CPH1 gene products lie in an equivalent, canonical, MAPK cascade. While Cek1p acts as part of the MAPK cascade involved in starvation-specific hyphal development, it may also play independent roles in C. albicans. In contrast to disruptions of the HST7 and CPH1 genes, disruption of the CEK1 gene adversely affects the growth of serum-induced mycelial colonies and attenuates virulence in a mouse model for systemic candidiasis.

Reduced pathogenicity of a Candida albicans MAP kinase phosphatase (CPP1) mutant in the murine mastitis model.

Candida albicans strains with a deletion of the mitogen-activated protein kinase tyrosine phosphatase gene (CPP1) are derepressed in the yeast-to-hyphal transition on solid surfaces in vitro at ambient temperatures and this gene is therefore required for repression of the yeast-to-hyphal switch. The pathology caused by a CPP1 null mutant strain was compared with that of the null mutant into which the wild-type CPP1 gene was introduced by homologous recombination and with the wild-type parent strain in a murine mycotic mastitis model. The mammary glands of lactating mice (at day 5 postpartum) were infected for 2, 4 and 6 days with 1 x 10(5), 1 x 10(6) and 1 x 10(7) cell-forming units before euthanasia. Infected and non-infected control glands were evaluated histopathologically. The null mutant strains showed less severe pathology than the two control strains. The Cpplp tyrosine phosphatase may thus be considered a virulence determinant during localized infection in C. albicans.

Derepressed hyphal growth and reduced virulence in a VH1 family-related protein phosphatase mutant of the human pathogen Candida albicans.

Mitogen-activated protein (MAP) kinases are pivotal components of eukaryotic signaling cascades. Phosphorylation of tyrosine and threonine residues activates MAP kinases, but either dual-specificity or monospecificity phosphatases can inactivate them. The Candida albicans CPP1 gene, a structural member of the VH1 family of dual- specificity phosphatases, was previously cloned by its ability to block the pheromone response MAP kinase cascade in Saccharomyces cerevisiae. Cpp1p inactivated mammalian MAP kinases in vitro and acted as a tyrosine-specific enzyme. In C. albicans a MAP kinase cascade can trigger the transition from the budding yeast form to a more invasive filamentous form. Disruption of the CPP1 gene in C. albicans derepressed the yeast to hyphal transition at ambient temperatures, on solid surfaces. A hyphal growth rate defect under physiological conditions in vitro was also observed and could explain a reduction in virulence associated with reduced fungal burden in the kidneys seen in a systemic mouse model. A hyper-hyphal pathway may thus have some detrimental effects on C. albicans cells. Disruption of the MAP kinase homologue CEK1 suppressed the morphological effects of the CPP1 disruption in C. albicans. The results presented here demonstrate the biological importance of a tyrosine phosphatase in cell-fate decisions and virulence in C. albicans.

Isoleucyl-tRNA synthetase from the ciliated protozoan Tetrahymena thermophila. DNA sequence, gene regulation, and leucine zipper motifs.

We have determined the nucleotide sequence of a protozoan aminoacyl-tRNA synthetase. The isoleucyl-tRNA synthetase (ileRS) gene [ilsA; formerly cupC, Martindale, D. W., Martindale, H. M., and Bruns, P. J. (1986) Nucleic Acids Res. 14, 1341-1354] from the ciliate Tetrahymena thermophila was sequenced and found to have eight introns, four transcription start sites, and a putative polypeptide of 1081 amino acids. A polypeptide 20 amino acids longer could be made if a transcribed in-frame ATG close to the start sites and with suboptimal sequence context is used. This gene was identified through hybridization and amino acid sequence similarity to the previously cloned and sequenced ileRS (cytoplasmic) gene from Saccharomyces cerevisiae [Englisch, U., Englisch, S., Markmeyer, P., Schischkoff, J., Sternbach, H., Kratzin, H., and Cramer, F. (1987) Biol. Chem. Hoppe-Seyler 368, 971-979; Martindale, D. W., Gu, Z. M., and Csank, C. (1989) Curr. Genet. 15, 99-106] with which it shares 47% of its amino acids. We also compared it to ileRS genes from E. coli and an archaebacterium. Two leucine zippers motifs were identified in the carboxyl-terminal domain of the polypeptide; one of these motifs is in the same area as the zinc finger motif found in the E. coli enzyme. The transcription pattern of the ilsA gene was monitored under various culture conditions and parallels changes in protein synthesis.

Nuclear pre-mRNA introns: analysis and comparison of intron sequences from Tetrahymena thermophila and other eukaryotes.

We have sequenced 14 introns from the ciliate Tetrahymena thermophila and include these in an analysis of the 27 intron sequences available from seven T. thermophila protein-encoding genes. Consensus 5' and 3' splice junctions were determined and found to resemble the junctions of other nuclear pre-mRNA introns. Unique features are noted and discussed. Overall the introns have a mean A + T content of 85% (21% higher than neighbouring exons) with smaller introns tending towards a higher A + T content. Approximately half of the introns are less than 100 bp. Introns from other organisms (approximately 30 of each) were also examined. The introns of Dictyostelium discoideum, Caenorhabditis elegans and Drosophila melanogaster, like those of T. thermophila, have a much higher mean A + T content than their neighbouring exons (greater than 20%). Introns from plants, Neurospora crassa and Schizosaccharomyces pombe also have a significantly higher A + T content (10%-20%). Since a high A + T content is required for intron splicing in plants (58), the elevated A + T content in the introns of these other organisms may also be functionally significant. The introns of yeast (Saccharomyces cerevisiae) and mammals (humans) appear to lack this trait and thus in some aspects may be atypical. The polypyrimidine tract, so distinctive of vertebrate introns, is not a trait of the introns in the non-vertebrate organisms examined in this study.

Isolation and complete sequence of the yeast isoleucyl-tRNA synthetase gene (ILS1).

The isoleucyl-tRNA synthetase gene (ILS1) from the yeast Saccharomyces cerevisiae was cloned and sequenced. This gene was initially cloned because it cross-hybridizated to what is now presumed to be the isoleucyl-tRNA synthetase gene (cupC) from the protozoan Tetrahymena thermophila. The ILS1 gene was determined to be 1,072 amino acids in length. A comparison with a recently published sequence of ILS1 from another laboratory (Englisch et al. 1987) was made and differences noted. Two promoter elements were detected, one for general amino acid control and one for constitutive transcription. A heat shock protein (hsp70) gene (probably SSA3) was found 237 bp upstream from the ILS1 translation start site. The ILS1 amino acid sequence was compared to isoleucyl-tRNA synthetases from other organisms, as well as to valyl-, leucyl- and methionyl-tRNA synthetases. Regions of conservation between these enzymes were found.