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J F Ernst

Publications and source records attributed to J F Ernst.

At least 19 recordsLinked to original sources
PubMed2002

Sec20p-interacting proteins (Tip20p, Ufe1p) in the retrograde secretory pathway of the fungal pathogen Candida albicans.

Sec20p is an essential Type-II membrane protein of the human fungal pathogen Candida albicans, which is thought to be involved in mediating retrograde vesicle traffic from the Golgi to the endoplasmic reticulum (ER). Using an epitope-tagged Sec20p we obtained evidence for its localization in ER membranes, which is consistent with its proposed role in an ER-tSNARE complex. Two genes encoding potential interaction partners for Sec20p, Tip20p and Ufe1p, were identified in genomic sequences of C. albicans; these show 18% and 27% identity, respectively, to homologues in Saccharomyces cerevisiae. An interaction between the cytoplasmic domain of Sec20p and Tip20p was demonstrated by two-hybrid analysis; in addition, Tip20p was found to form homodimers. Interaction between Sec20p and Tip20p in vivo was verified by co-immunoprecipation experiments. CaUFE1, which encodes a potential ER-tSNARE, was able to complement a thermosensitive ufe1 mutation in S. cerevisiae, suggesting functional conservation between the two fungal proteins. Thus, although the sequences of some components of the ER-tSNARE complex have diverged considerably during evolution, it appears that they have retained similar functions in C. albicans and S. cerevisiae.

Amino Acid Sequence↗
PubMed2001

Human immunodeficiency virus type 1 Tat binds to Candida albicans, inducing hyphae but augmenting phagocytosis in vitro.

Tat, the human immunodeficiency virus type 1 (HIV-1) transactivating protein, binds through its RGD-motif to human integrin receptors. Candida albicans, the commonest cause of mucosal candidiasis in subjects infected with HIV-1, also possesses RGD-binding capacity. The present study reveals that Tat binds to C. albicans but not to C. tropicalis. Tat binding was markedly reduced by laminin and to a lesser extent by a complement C3 peptide containing the RGD motif, but not by a control peptide. The outgrowth of C. albicans was accelerated following binding of Tat, but phagocytosis of opsonized C. albicans was also increased after Tat binding. Thus, Tat binding promotes fungal virulence by inducing hyphae but may also reduce it by augmenting phagocytosis. The net effect of Tat in vivo is difficult to judge but in view of the many disease-promoting effects of Tat we propose that accelerating the formation of hyphae dominates over the augmentation of phagocytosis.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2001

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.

Alleles↗
PubMed2001

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.

Alanine↗
PubMed2001

Divergence of eukaryotic secretory components: the Candida albicans homolog of the Saccharomyces cerevisiae ++Sec20 protein is N terminally truncated, and its levels determine antifungal drug resistance and growth.

Sec20p is a component of the yeast Saccharomyces cerevisiae secretory pathway that does not have a close homolog in higher eukaryotic cells. To verify the function of Sec20p in other fungal species, we characterized the gene encoding a Sec20p homolog in the human fungal pathogen Candida albicans. The deduced protein has 27% identity with, but is missing about 100 N-terminal residues compared to S. cerevisiae Sec20p, which is part of the cytoplasmic tail interacting with the cytoplasmic protein Tip20p. Because a strain lacking both C. albicans SEC20 alleles could not be constructed, we placed SEC20 under transcriptional control of two regulatable promoters, MET3p and PCK1p. Repression of SEC20 expression in these strains prevented (MET3p-SEC20 allele) or retarded (PCK1p-SEC20 allele) growth and led to the appearance of extensive intracellular membranes, which frequently formed stacks. Reduced SEC20 expression in the PCK1p-SEC20 strain did not affect morphogenesis but led to a series of hypersensitivity phenotypes including supersensitivity to aminoglycoside antibiotics, to nystatin, to sodium dodecyl sulfate, and to cell wall inhibitors. These results demonstrate the occurrence and function of Sec20p in a fungal species other than S. cerevisiae, but the lack of the N-terminal domain and the apparent absence of a close TIP20 homolog in the C. albicans genome also indicate a considerable diversity in mechanisms of retrograde vesicle traffic in eukaryotes.

Amino Acid Sequence↗
PubMed2001

O-glycosylation.

O-Glycosylation in many fungal species is initiated in the endoplasmic reticulum by protein mannosyltransferases (Pmt-proteins), which transfer mannose to serine or threonine residues, and it is completed by mannosyltransferases (Mnt-proteins) in the Golgi. In this review, some recent results on O-glycosylation in the human fungal pathogen Candida albicans are discussed and compared to the corresponding knowledge in the non-pathogenic yeast Saccharomyces cerevisiae. The Pmt-family in C. albicans comprises five isoforms, of which Pmt1p and Pmt6p have been studied in detail. Surprisingly, O-glycosylation mediated by Pmt-proteins is required not only for the modification of several secreted and cell-wall proteins, but also affects yeast-hyphal morphogenesis (dimorphism) and resistance to several antifungal compounds. Furthermore, Pmt1- and Pmt6p-activities maximize adherence to host cells and determine or contribute to virulence in models of systemic infection. Thus, O-glycosylation processes directly and/or indirectly affect several virulence traits of C. albicans and can be considered as potential antifungal targets.

Antifungal Agents↗
PubMed2000

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.

Carrier Proteins↗
PubMed2000

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae.

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3delta background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using 55Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.

Bacterial Outer Membrane Proteins↗
PubMed2000

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.

Animals↗
PubMed1999

Identification of a fungal triacetylfusarinine C siderophore transport gene (TAF1) in Saccharomyces cerevisiae as a member of the major facilitator superfamily.

Transport proteins of microorganisms may either belong to the ATP-binding cassette (ABC) superfamily or to the major facilitator (MFS)-superfamily. MFS transporters are single-polypeptide membrane transporters that transport small molecules via uniport, symport or antiport mechanisms in response to a chemiosmotic gradient. Although Saccharomyces cerevisiae is a non-siderophore producer, various bacterial and fungal siderophores can be utilized as an iron source. From yeast genome sequencing data six genes of the unknown major facilitator (UMF) family were known of which YEL065w Sce was recently identified as a transporter for the bacterial siderophore ferrioxamine B (Sit1p). The present investigation shows that another UMF gene, YHL047c Sce, encodes a transporter for the fungal siderophore triacetylfusarinine C. The gene YHL047c Sce (designated TAF1) was disrupted using the kanMX disruption module in a fet3 background (strain DEY 1394 delta fet3), possessing a defect in the high affinity ferrous iron transport. Growth promotion assays and transport experiments with 55Fe-labelled triacetylfusarinine C showed a complete loss of iron utilization and uptake in the disrupted strain, indicating that TAF1 is the gene for the fungal triacetylfusarinine transport in Saccharomyces cerevisiae and possibly in other siderophore producing fungi.

ATP-Binding Cassette Transporters↗
PubMed1999

Genetic and biochemical characterization of phosphofructokinase from the opportunistic pathogenic yeast Candida albicans.

We have used the two PFK genes of Saccharomyces cerevisiae encoding the alpha and beta-subunit of the enzyme phosphofructokinase (Pfk) as heterologous probes to isolate fragments of the respective genes from the dimorphic pathogenic fungus Candida albicans. The complete coding sequences were obtained by combining sequences of chromosomal fragments and fragments obtained by inverse polymerase chain reaction (PCR). The CaPFK1 and CaPFK2 comprise open reading frames of 2961 bp and 2838 bp, respectively, encoding Pfk subunits with deduced molecular masses of 109 kDa and 104 kDa. The genes presumably evolved by a duplication event from a prokaryotic type ancestor, followed by another duplication. Heterologous expression in S. cerevisiae revealed that each gene alone was able to complement the glucose-negative phenotype of a pfk1 pfk2 double mutant. In vitro Pfk activity in S. cerevisiae was not only obtained after coexpression of both genes, but also in conjunction with the respective complementary subunits from S. cerevisiae. This indicates the formation of functional hetero-oligomers consisting of C. albicans and S. cerevisiae Pfk subunits. In C. albicans, specific Pfk activity was shown to decrease twofold upon induction of hyphal growth. CaPfk cross-reacts with a polyclonal antiserum raised against ScPfk and displays similar allosteric properties, i.e. inhibition by ATP and activation by AMP and fructose 2,6-bisphosphate.

Allosteric Regulation↗
PubMed1999

Chlamydospore formation in Candida albicans requires the Efg1p morphogenetic regulator.

Chlamydospore formation of the fungal pathogen Candida albicans was found to depend on the Efg1 protein, which regulates the yeast-hyphal transition. Isogenic mutants lacking EFG1 or encoding T206A and T206E variants did not differentiate chlamydospores, while cek1, cph1, or tpk2 mutations had no effect. Furthermore, filamentation of efg1 cph1 double mutants in microaerophilic conditions suggests a novel Efg1p/Cph1p-independent filamentation pathway in C. albicans.

Candida albicans↗
PubMed1999

Control of white-opaque phenotypic switching in Candida albicans by the Efg1p morphogenetic regulator.

Phenotypic switching in Candida albicans spontaneously generates different cellular morphologies and is manifested in strain WO-1 by the reversible switching between the white and opaque phenotypes. We present evidence that phenotypic switching is regulated by the Efg1 protein, which is known as an essential element of hyphal development (dimorphism). Firstly, EFG1 is expressed specifically in cells of the white but not the opaque phenotype. During mass conversion from the opaque to the white phenotype, the EFG1 transcript level correlates with competence of switching of opaque cells to the white form. Secondly, overexpression of EFG1 by a PCK1p-EFG1 fusion forces opaque-phase cells to switch to the white form with a high level of efficiency. Thirdly, low-level expression of EFG1 in strain CAI-8 generates a cellular phenotype similar to that of opaque cells in that cells bud as short rods, which cannot be induced to form hyphae in standard conditions; such cells (unlike authentic opaque cells) lack typical surface "pimples." Importantly, the opaque-specific OP4 transcript is induced in the opaque-like cells generated by strain CAI8 as a response to low-level expression of EFG1. The results suggest that high EFG1 expression levels induce and maintain the white cell form while low EFG1 expression levels induce and maintain the opaque cell form. It is proposed that changes in EFG1 expression determine or contribute to phenotypic switching events in C. albicans.

Candida albicans↗
PubMed1999

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.

Caco-2 Cells↗
PubMed1998

Overlapping coding regions and trancriptional units of two essential chromosomal genes (CCT8, TRP1)in the fungal pathogen Candida albicans.

Sequencing of the 3'-untranslated region of the CCT8 gene of the fungal pathogen Candida albicans revealed that the CCT8 coding region overlaps 13 bp with the coding region of the convergently orientated TRP1 gene. The same overlap was found in three strains with different genetic backgrounds. 3'-RACE was used to determine that the CCT8 and TRP1 transcripts extended significantly into the coding region of the adjacent gene, which also contained sequences encoding the poly(A) addition site. A strain retaining one wild-type CCT8/TRP1 locus on one chromosome and a deletion on the other homologous chromosome contained both CCT8 and TRP1 transcripts; this result indicates that both transcripts are synthesized from the same gene locus. The CCT8/TRP1 gene pair of C . albicans constitutes an extreme natural case of transcriptional overlap in a eukaryote. The results confirm that convergent overlapping transcription units are compatible with expression of the overlapping genes.

3' Untranslated Regions↗
PubMed1998

Multiple functions of Pmt1p-mediated protein O-mannosylation in the fungal pathogen Candida albicans.

Protein mannosylation by Pmt proteins initiates O-glycosylation in fungi. We have identified the PMT1 gene and analyzed the function of Pmt1p in the fungal human pathogen Candida albicans. Mutants defective in PMT1 alleles lacked Pmt in vitro enzymatic activity, showed reduced growth rates, and tended to form cellular aggregates. In addition, multiple specific deficiencies not known in Saccharomyces cerevisiae (including defective hyphal morphogenesis; supersensitivity to the antifungal agents hygromycin B, G418, clotrimazole, and calcofluor white; and reduced adherence to Caco-2 epithelial cells) were observed in pmt1 mutants. PMT1 deficiency also led to faster electrophoretic mobility of the Als1p cell wall protein and to elevated extracellular activities of chitinase. Homozygous pmt1 mutants were avirulent in a mouse model of systemic infection, while heterozygous PMT1/pmt1 strains showed reduced virulence. The results indicate that protein O-mannosylation by Pmt proteins occurs in different fungal species, where PMT1 deficiency can lead to defects in multiple cellular functions.

Alleles↗
PubMed1998

A small protein (Ags1p) and the Pho80p-Pho85p kinase complex contribute to aminoglycoside antibiotic resistance of the yeast Saccharomyces cerevisiae.

We identified the AGS1 and AGS3 genes by their ability to partially complement an ags mutant (RC1707) which is supersensitive to various aminoglycoside antibiotics (J. F. Ernst and R. K. Chan, J. Bacteriol. 163:8-14, 1985). AGS1 is located in proximity to the centromere of chromosome III and encodes a small protein of 88 amino acids. The size of the AGS1 transcript, which in wild-type cells is 1 kb, is reduced to 0.75 kb in mutant RC1707. Disruption of AGS1 rendered strains supersensitive to hygromycin B and increased their resistance to vanadate. In addition, ags1delta strains underglycosylated invertase but had normal carboxypeptidase Y glycosylation, suggesting that Ags1p is required for the elaboration of outer N-glycosyl chains. AGS3 was found to be identical to PHO80 (TUP7), which encodes a cyclin activating the Pho85p protein kinase. Deletion of either PHO80 or PHO85 led to aminoglycoside supersensitivity; pho80delta ags1delta strains showed an enhanced-sensitivity phenotype compared to single mutants. pho80 and pho85 mutants were rendered resistant by deletion of PHO4, indicating that activation of the Pho4p transcription factor is required for increased aminoglycoside sensitivity. Thus, both the Pho80p-Pho85p kinase complex (by Pho4p phosphorylation) and a novel component of the N glycosylation pathway contribute to basal levels of aminoglycoside resistance in Saccharomyces cerevisiae.

Amino Acid Sequence↗