Flavin mononucleotide-based fluorescent protein as an oxygen-independent reporter in Candida albicans and Saccharomyces cerevisiae.

Hypoxia is encountered frequently by pathogenic and apathogenic fungi. A codon-adapted gene encoding flavin mononucleotide-based fluorescent protein (CaFbFP) was expressed in Candida albicans and Saccharomyces cerevisiae. Both species produced CaFbFP and fluoresced even during hypoxia, suggesting that oxygen-independent CaFbFP is a useful, novel tool for monitoring hypoxic gene expression in fungi.

Large molecules as anti-adhesive compounds against pathogens.

Anti-adhesive compounds are potential prophylactic tools in alternative treatment regimes against bacterial infection, as bacterial adhesion is commonly mediated by carbohydrate-protein interactions between surface adhesions of microorganisms and the host cell. The use of exogenous polyvalent, high-molecular carbohydrates and tannin-like plant-derived compounds should antagonize the adhesive interaction. A range of carbohydrates and carbohydrate- and proanthocyanidin-enriched plant extracts were screened for potential anti-adhesive effects against Helicobacter pylori, Campylobacter jejuni, Porphyromonas gingivalis and Candida albicans in different in-situ assays on primary tissue. The adhesion of H. pylori on human stomach tissue was effectively blocked by glucuronic acid-enriched polysaccharides from immature okra fruits (Abelmoschus esculentus). These compounds also had strong in-vitro effects against C. jejuni (inhibition up to 80%), but were ineffective in an in-vivo study in infected chicken broilers due to metabolism in the gastrointestinal system. Polysaccharides from Glycyrrhizia glabra, also enriched with glucuronic acid, showed strong anti-adhesive properties against H. pylori and P. gingivalis (inhibition 60-70%). Pelargonium sidoides extract, containing mainly polymeric proanthocyanidins, was effective against H. pylori in a dose-dependent manner. Due to the multifunctional adhesive strategy of C. albicans, no effective compounds were detected against this yeast. Structure-activity relationships are presented and the potential in-vivo use of carbohydrate-based anti-adhesives is discussed.

CandidaDB: a genome database for Candida albicans pathogenomics.

CandidaDB is a database dedicated to the genome of the most prevalent systemic fungal pathogen of humans, Candida albicans. CandidaDB is based on an annotation of the Stanford Genome Technology Center C.albicans genome sequence data by the European Galar Fungail Consortium. CandidaDB Release 2.0 (June 2004) contains information pertaining to Assembly 19 of the genome of C.albicans strain SC5314. The current release contains 6244 annotated entries corresponding to 130 tRNA genes and 5917 protein-coding genes. For these, it provides tentative functional assignments along with numerous pre-run analyses that can assist the researcher in the evaluation of gene function for the purpose of specific or large-scale analysis. CandidaDB is based on GenoList, a generic relational data schema and a World Wide Web interface that has been adapted to the handling of eukaryotic genomes. The interface allows users to browse easily through genome data and retrieve information. CandidaDB also provides more elaborate tools, such as pattern searching, that are tightly connected to the overall browsing system. As the C.albicans genome is diploid and still incompletely assembled, CandidaDB provides tools to browse the genome by individual supercontigs and to examine information about allelic sequences obtained from complementary contigs. CandidaDB is accessible at http://genolist.pasteur.fr/CandidaDB.

Enzymatic release of iron from sideramines in fungi. NADH:sideramine oxidoreductase in Neurospora crassa.

Young mycelia of the fungus Neurospora crassa contain a soluble NADH-linked sideramine reductase, which may be responsible for liberating iron in vivo from accumulated sideramines during iron-deficient cultivation. The enzymes can be assayed using a soluble supernatant fraction, EDTA, and an atmosphere of pure nitrogen. The enzyme is stable without loss of activity up to 45 degrees C and has an optimum of activity at pH 7.0. Besides coprogen (Km = 100 micrometer, V=2.8 nmol/min per mg protein), some other ferrichrome-type compounds are reduced. However, ferrichrome, ferrirubin coprogen B and ferrioxamine are poor substrates. When the mucelia were grown in a medium containing 10(-5) M ferri iron, the activity of the reductase was found to be only 30% of that found under low iron conditions. The enzyme is inhibited by oxygen, SH-alkylating agents and partly by some detergents. Unlike the reductase of N. crassa, the corresponding enzyme from Aspergillus fumigatus revealed low reduction of coprogen and high reduction of ferrichrome, indicating genusdependent specificities of sideramine reduction enzymes in fungi. The participation of acids of the citric acid cycle as natural iron acceptors during strong iron deficiency is studied and confirmed by iron uptake measurements on isolated mitochondria.

Multiple base-pair mutations in yeast.

The nucleotide changes associated with both forward and reverse mutations at the CYC1 locus in the yeast Saccharomyces cerevisiae have been investigated by sequencing the mutated gene product, iso-1-cytochrome c and, more directly, by sequencing appropriate DNA segments. Although the majority of these mutations are the result of single base-pair changes, approximately 10% are the result of multiple mutations and these occur predominantly at certain sites and with certain patterns. Most multiple base-pair changes occur within 20 nucleotides of each other and are generally within six nucleotides. On the basis of the frequencies and patterns of mutations, these nucleotide changes are considered to have occurred as single, concerted events, rather than as multiple independent mutations. Analysis of these mutations indicates that multiple base-pair changes can arise by widely differing mechanisms. We have recognized the following classes of mutations: multiple base-pair changes that yield (1) direct repeats or (2) inverted repeats of local DNA sequences; (3) substitutions of two tandem base-pairs; (4) frameshift and contiguous single base-pair substitutions; and (5) recombination of the CYC1 gene with a non-allelic gene, resulting in alterations within contiguous segments that can be over 150 nucleotides in length. Some of the multiple base-pair changes do not fall into any of these categories. We suggest mechanisms to account for each of these five classes.

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.

DNA sequences of frameshift and other mutations induced by ICR-170 in yeast.

ICR-170-induced mutations in the CYC1 gene of the yeast Saccharomyces cerevisiae were investigated by genetic and DNA sequence analyses. Genetic analysis of 33 cyc1 mutations induced by ICR-170 and sequence analysis of eight representatives demonstrated that over one-third were frameshift mutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly, and a few of these were not frameshift mutations. The sequence results indicate that ICR-170 primarily induces G.C additions at sites containing monotonous runs of three G.C base pairs. However, some (Formula: see text) sites within the CYC1 gene were not mutated by ICR-170. Thus, ICR-170 is a relatively specific mutagen that preferentially acts on certain sites with monotonous runs of G.C base pairs.

Differential mismatch repair can explain the disproportionalities between physical distances and recombination frequencies of cyc1 mutations in yeast.

Recombination rates have been examined in two-point crosses of various defined cyc1 mutations that cause the loss or nonfunction of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. Recombinants arising by three different means were investigated, including X-ray induced mitotic recombination, spontaneous mitotic recombination, and meiotic recombination. Heteroallelic diploid strains were derived by crossing cyc1 mutants containing a series of alterations at or near the same site to cyc1 mutants containing alterations at various distances. Marked disproportionalities between physical distances and recombination frequencies were observed with certain cyc1 mutations, indicating that certain mismatched bases can significantly affect recombination. The marker effects were more pronounced when the two mutational sites of the heteroalleles were within about 20 base pairs, but separated by at least 4 base pairs. Two alleles, cyc1-163 and cyc1-166, which arose by G.C----C.G transversions at nucleotide positions 3 and 194, respectively, gave rise to especially high rates of recombination. Other mutations having different substitutions at the same nucleotide positions were not associated with abnormally high recombination frequencies. We suggest that these marker effects are due to the lack of repair of either G/G or C/C mismatched base pairs, while the other mismatched base pair of the heteroallele undergoes substantial repair. Furthermore, we suggest that diminished recombination frequencies are due to the concomitant repair of both mismatches within the same DNA tract.

Efficient intra- and extracellular production of human beta-1,4-galactosyltransferase in Saccharomyces cerevisiae is mediated by yeast secretion leaders.

We have compared the function of homologous and heterologous secretion leaders to mediate production of human beta-1,4-galactosyltransferase (Gal-Tf) in the yeast Saccharomyces cerevisiae. Although all genes encoding leader/Gal-Tf fusions were transcribed by strong yeast promoters, only low production levels were obtained for full-length Gal-Tf containing the human membrane-anchor region. In contrast, a gene fusion encoding the membrane-anchor region of the yeast alpha-1,2-mannosyltransferase (Mnt1) fused to soluble Gal-Tf yielded high mRNA and intracellular protein levels. Gal-Tf could also be produced extracellularly using a fusion of the pre-pro region of the yeast Mf alpha 1 precursor (MF alpha 1) to soluble Gal-Tf; a fusion containing only the pre-region of Mf alpha 1 was synthesized intracellularly, but did not lead to Gal-Tf activity in the culture medium. All yeast-produced Gal-Tf proteins were enzymatically active. These results demonstrate that yeast secretion leaders are advantageous to achieve efficient production of active Gal-Tf in S. cerevisiae.

Sequence and promoter regulation of the PCK1 gene encoding phosphoenolpyruvate carboxykinase of the fungal pathogen Candida albicans.

The PCK1 gene encoding PEP carboxykinase (Pck1) of the fungal pathogen Candida albicans was isolated and sequenced. The deduced Pck1 protein has high homology to ATP-dependent Pck1 proteins in other species, especially to Pck1 of Saccharomyces cerevisiae (70% homology), but not to GTP-dependent Pck1 proteins. PCK1 transcript levels were efficiently repressed by glucose and derepressed (induced) on gluconeogenetic carbon sources. PCK1 regulation occurs on the level of transcription, as demonstrated by a fusion of the PCK1 promoter to the LAC4 reporter gene, yielding derepressed/repressed expression ratios of > 100. Homologous sequences in the PCK1 promoters of C. albicans and S. cerevisiae were identified. The PCK1 promoter may be useful to efficiently regulate expression and thereby test the function of genes in C. albicans.

Identification and substrate specificity of a ferrichrome-type siderophore transporter (Arn1p) in Saccharomyces cerevisiae.

Genes encoding transporters for heterologous siderophores have been identified in Saccharomyces cerevisiae, of which SIT1, TAF1, and ENB1 encode the transporters for ferrioxamines, ferric triacetylfusarinine C and ferric enterobactin, respectively. In the present communication we have shown that a further gene encoding a member of the major facilitator superfamily, ARN1 (YHL040c), is involved in the transport of a specific class of ferrichromes, possessing anhydromevalonyl residues linked to N(delta)-ornithine (ARN). Ferrirubin and ferrirhodin, which both are produced by filamentous fungi, are the most common representatives of this class of ferrichromes. A strain possessing a disruption in the ARN1 gene was unable to transport ferrirubin, ferrirhodin and also ferrichrome A, indicating that the encoded transporter recognizes anhydromevalonyl and the structurally-related methylglutaconyl side-chains surrounding the iron center. Ferrichromes possessing short-chain ornithine-N(delta)-acetyl residues such as ferrichrome, ferricrocin and ferrichrysin, were excluded by the Arn1 transporter. Substitution of the iron-surrounding N-acyl chains of ferrichromes by propionyl residues had no effect, whereas substitution by butyryl residues led to recognition by the Arn1 transporter. This would indicate that a chain length of four C-atoms is sufficient to allow binding. Using different asperchromes (B1, D1) we also found that a minimal number of two anhydromevalonyl residues is sufficient for recognition by Arn1p. Contrary to the iron-surrounding N-acyl residues, the peptide backbone of ferrichromes was not an important determinant for the Arn1 transporter.

Caco-2 monolayer as a model for transepithelial migration of the fungal pathogen Candida albicans.

A model for transepithelial migration of human fungal pathogens was established, in which Candida albicans was shown to migrate across a monolayer of Caco-2 intestinal cells in a two-chamber system. Electron microscopy revealed typical stages of epithelial penetration by C. albicans including phagocytosis at the apical side, intra- and intercellular migration and exit on the basolateral side of the monolayer. Hyphal growth forms appeared particularly involved in penetration of the Caco-2 monolayer. The model was examined using defined C. albicans mutants defective in hyphal development (efg1/efg1) or growth (ura3/ura3). Transmigration of the efg1/efg1 mutant strain was reduced, while transmigration of the ura3/ura3 strain was blocked completely in the absence of uridine. Because these results parallel virulence characteristics of the mutants the Caco-2 monolayer system appears a useful model for the study of fungal-human host cell interactions.

Protein-O-mannosyltransferases in virulence and development.

Protein-O-mannosyltransferases (Pmt proteins) catalyse the addition of mannose to serine or threonine residues of secretory proteins. This modification was described first for yeast and later for other fungi, mammals, insects and recently also for bacteria. O-mannosylation depends on specific isoforms of the three Pmt1, 2 and 4 subfamilies. In fungi, O-mannosylation determines the structure and integrity of cell walls, as well as cellular differentiation and virulence. O-mannosylation of specific secretory proteins of the human fungal pathogen Candida albicans and of the bacterial pathogen Mycobacterium tuberculosis contributes significantly to virulence. In mammals and insects, Pmt proteins are essential for cellular differentiation and development, while lack of Pmt activity causes Walker-Warburg syndrome (muscular dystrophy) in humans. The susceptibility of human cells to certain viruses may also depend on O-mannosyl chains. This review focuses on the various roles of Pmt proteins in cellular differentiation, development and virulence.

Improved secretion of heterologous proteins by Saccharomyces cerevisiae: effects of promoter substitution in alpha-factor fusions.

The effects of promoter strength on secretion of a heterologous protein, somatomedin-C (SMC), by the yeast Saccharomyces cerevisiae were studied by using the promoters of the MF alpha 1, ACT, and CYC1 genes to control expression of alpha-factor/SMC gene fusions. When a low-copy centromere vector was used to carry the gene fusions in yeast transformants, the greatest secretion was obtained with the MF alpha 1 promoter construction and the least with the CYC1 promoter construction. Unexpectedly, using two types of multicopy vectors, the greatest secretion was obtained with the CYC1 promoter construction and the least with the MF alpha 1 promoter construction. The decrease in secretion by the strongest promoter construction (MF alpha 1 promoter) on multicopy vectors was associated with a decrease in SMC mRNA during growth, a decrease in vector copy number, a decrease in vector stability, and a decrease in transformation frequency. The results demonstrate that, unlike in intracellular expression, promoter strength is not simply related to secretion expression levels. Selection against oversecreting cells during growth may explain the reduced secretion efficiency of strong promoter constructions.

Efficient secretion and processing of heterologous proteins in Saccharomyces cerevisiae is mediated solely by the pre-segment of alpha-factor precursor.

A novel processing site was identified in fusions of the alpha-factor precursor of Saccharomyces cerevisiae following its 19 amino-terminal residues (pre-segment). Fusions of the pre-segment to heterologous proteins, including aminoglycoside phosphotransferase (APH) and human granulocyte-macrophage colony stimulating factor (hGM-CSF), were as efficiently secreted and processed as corresponding pre-pro fusions. Pre- and pre-pro fusions to hGM-CSF were identically N- and O-glycosylated. While pre-pro fusions to interleukin-1 beta were not cleaved, pre-fusions were correctly processed during secretion. The high secretion efficiency of pre-fusions suggests that the pro-segment of the alpha-factor precursor is not required for efficient secretion and processing of protein fusions.

Molecular genetics of pathogenic fungi: some recent developments and perspectives.

The diagnosis and the treatment of fungal diseases remains problematic in many cases. Difficulties in diagnosis are due (1) to the ubiquitous presence of fungal pathogens that may lead to false positive test results and (2) to difficulties in the evaluation of the aetiological significance of these pathogens. The relatively small number of effective antifungal agents reflects to a large extent on the fact that many aspects of fungal physiology and virulence are not well understood. The methods of molecular genetics provide effective tools for the diagnosis of mycoses and may also contribute to the identification of new targets for antifungals by genetic analyses of fungal virulence. During the last 3 years molecular genetic methods have been developed for the asexual pathogen Candida albicans that may be used for strain identification. This success indicates a general use of molecular genetics for the analysis of fungal pathogenesis.

Toxicity of a heterologous leucyl-tRNA (anticodon CAG) in the pathogen Candida albicans: in vivo evidence for non-standard decoding of CUG codons.

Plasmids containing derivatives of the Saccharomyces cerevisiae leucyl-tRNA (tRNA(3Leu)) gene that vary in anticodon sequence were constructed and transformed into the pathogen Candida albicans and S. cerevisiae. C. albicans could readily be transformed with plasmids encoding leucyl-tRNA genes with the anticodons CAA and UAA (recognizing the codons UUG and UUA) and expression of the heterologous tRNALeu could be demonstrated by Northern RNA blotting. In contrast, no transformants were obtained if the anticodons were UAG (codons recognized CUN, UUR) and CAG (codon CUG), indicating that the insertion of leucine at CUG codons is toxic for C. albicans. All tRNALeu-encoding plasmids transformed S. cerevisiae with equally high efficiencies. These results provide in vivo evidence that non-standard decoding of CUG codons is essential for the viability of C. albicans.

Iron regulation of triosephosphate isomerase transcript stability in the yeast Saccharomyces cerevisiae.

By differential hybridization we have identified cDNA clones that are derived from iron-regulated genes in Saccharomyces cerevisiae. Sequencing of seven cDNA clones revealed that five clones correspond to TPI1 encoding triosephosphate isomerase (Tpi1p) and one corresponds to TDH3 encoding glyceraldehyde-3-phosphate dehydrogenase (Tdh3p). During iron-limited growth mRNA levels for Tpi1p and Tdh3p were at least 3-fold lower than during iron-saturated growth; as shown with a hem1 mutant strain this regulation does not require haem synthesis. mRNA half-lives of TPI1 (TDH3) were 11.5 min (18 min) in low-iron medium and 30 min (32.5 min) in high-iron medium, indicating iron-regulation of transcript half-lives; the stabilities of the ACT1 and PDC1 transcripts were not influenced by iron. Increased glycerol production during growth in low-iron, as compared to high-iron medium, is consistent with a modification of the glycolytic flux during iron-limited growth in S. cerevisiae.