Biomedical subjects

Patrick Van Dijck

Publications and source records attributed to Patrick Van Dijck.

At least 19 recordsLinked to original sources

PubMed2025

Multi-Omics Analysis of Experimentally Evolved Candida auris Isolates Reveals Modulation of Sterols, Sphingolipids, and Oxidative Stress in Acquired Amphotericin B Resistance.

Clinical isolates of Candida auris show a high prevalence of resistance to Amphotericin B (AmB)-an uncommon trait in most Candida species. Alterations in ergosterol biosynthesis can contribute to acquired AmB resistance in C. auris laboratory strains but are rarely seen in clinical isolates. In this study, we experimentally evolved two drug-susceptible Clade II isolates of C. auris to develop AmB resistance. The evolved strains displayed a four to eight fold increase in MIC50 compared to the parental cells. We analyzed changes in their karyotype, genome, lipidome, and transcriptome associated with this acquired resistance. In one lineage, AOX2 was upregulated, and its deletion reversed the AmB resistance phenotype. The aox2Δ mutant also failed to evolve AmB resistance under experimental conditions. In the same lineage, restoring the UPC2 S332R and RTG3 S101T mutations to the wild-type allele restored AmB susceptibility. In another lineage, the ergosterol and sphingolipid pathways were observed to play a critical role, and upregulation of the ERG genes elevated the total sterol content, while significant downregulation of HSX11 (glucosylceramide synthase) resulted in lower levels of glucosylceramides. To our knowledge, this study is the first to show that AmB resistance in C. auris can be acquired through mechanisms both dependent on or independent of sterol content modulation, highlighting Aox2 and Upc2 as key regulators of amphotericin resistance.

Amphotericin B↗

PubMed2006

Isolation and characterization of brewer's yeast variants with improved fermentation performance under high-gravity conditions.

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22 degrees Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11 degrees C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.

Beer↗

PubMed2006

ABI4 mediates the effects of exogenous trehalose on Arabidopsis growth and starch breakdown.

The disaccharide trehalose has dramatic effects on plant metabolism, growth and development. Arabidopsis seedlings grown on trehalose-containing medium without sucrose display increased expression of the starch synthesis gene ApL3, hyper-accumulation of starch in the cotyledons and inhibition of root growth. Here we show that the ABI4 transcription factor mediates the effects of trehalose on starch metabolism and growth, independently of abscisic acid (ABA) synthesis and hexokinase (HXK1) signaling. Surprisingly, although the abi4 mutation partially rescued trehalose inhibition of root elongation, ApL3 expression levels were still enhanced. Gene expression analysis suggests that trehalose affects both starch synthesis and starch breakdown. The expression of genes involved in starch breakdown, such as SEX1 and the beta-amylase gene BMY8/BAM3, was strongly down-regulated in WT plants grown on trehalose but not in abi4 mutants. Addition of trehalose to liquid-grown WT seedlings also significantly reduced SEX1 expression after 6 h. Bypassing the need for starch breakdown by growth in continuous light or addition of sucrose rescued root growth on trehalose medium similar to the abi4 mutation. These results suggest that inhibition of starch mobilization rather than increased synthesis is involved in growth inhibition by exogenous trehalose. Trehalose also significantly enhanced ABI4 expression but reduced its sucrose induction, providing a possible molecular mechanism for the trehalose effect on plant gene expression and growth.

Arabidopsis↗

PubMed2006

Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate Charcot-Marie-Tooth neuropathy.

Charcot-Marie-Tooth (CMT) neuropathies are common disorders of the peripheral nervous system caused by demyelination or axonal degeneration, or a combination of both features. We previously assigned the locus for autosomal dominant intermediate CMT neuropathy type C (DI-CMTC) to chromosome 1p34-p35. Here we identify two heterozygous missense mutations (G41R and E196K) and one de novo deletion (153-156delVKQV) in tyrosyl-tRNA synthetase (YARS) in three unrelated families affected with DI-CMTC. Biochemical experiments and genetic complementation in yeast show partial loss of aminoacylation activity of the mutant proteins, and mutations in YARS, or in its yeast ortholog TYS1, reduce yeast growth. YARS localizes to axonal termini in differentiating primary motor neuron and neuroblastoma cultures. This specific distribution is significantly reduced in cells expressing mutant YARS proteins. YARS is the second aminoacyl-tRNA synthetase found to be involved in CMT, thereby linking protein-synthesizing complexes with neurodegeneration.

Amino Acid Sequence↗

PubMed2006

Why do microorganisms have aquaporins?

Aquaporins are channel proteins that enhance the permeability of cell membranes for water. They have been found in Bacteria, Archaea and Eukaryotes. However, their absence in many microorganisms suggests that aquaporins do not fulfill a broad role such as turgor regulation or osmoadaptation but, instead, fulfill a role that enables microorganisms to have specific lifestyles. The recent discovery that aquaporins enhance cellular tolerance against rapid freezing suggests that they have ecological relevance. We have identified several examples of large-scale freeze-thawing of microbes in nature and we also draw attention to alternative lifestyle-related functions for aquaporins, which will be a focus of future research.

Animals↗

PubMed2006

Water channels are important for osmotic adjustments of yeast cells at low temperature.

The importance of aquaporin expression in water permeability in Saccharomyces cerevisiae was assessed by measuring the osmotic water permeability coefficient (P(f)) and the activation energies (E(a)) from both hypo- and hypertonic experiments performed with whole protoplasts from four strains differing in aquaporin level of expression: parental, double-deleted and overexpressing AQY1 or AQY2. Double-deleted (lower P(f)) and AQY1-overexpressing strains (higher P(f)) presented linear Arrhenius plots with E(a) consistent with fluxes mainly through the lipids [16.3 kcal mol(-1) (68.2 kJ mol(-1))] and with a strong contribution of channels [9.6 kcal mol(-1) (40.2 kJ mol(-1))], respectively. The Arrhenius plots for the parental (swelling experiments) and overexpressing AQY2 strains (swelling and shrinking experiments) were not linear, presenting a break point with a change in slope around 23 degrees C. The E(a) values for these strains, calculated for temperatures ranging from 7 to 23 degrees C, were lower [9.5 kcal mol(-1) (39.7 kJ mol(-1))] than the values obtained from 23 to 38 degrees C [17 kcal mol(-1) (71.1 kJ mol(-1))]. This behaviour indicates that only in the lower temperature range did the water fluxes occur predominantly via the water channels. The permeabilities for each strain relative to the deletion strain show that an increase in permeability due to the presence of aquaporins was more relevant at low temperatures. Following our results, we propose that water channels play an important role for osmotic adjustment of yeast cells at low temperature.

Aquaporins↗

PubMed2005

The Saccharomyces cerevisiae EHT1 and EEB1 genes encode novel enzymes with medium-chain fatty acid ethyl ester synthesis and hydrolysis capacity.

Fatty acid ethyl esters are secondary metabolites produced by Saccharomyces cerevisiae and many other fungi. Their natural physiological role is not known but in fermentations of alcoholic beverages and other food products they play a key role as flavor compounds. Information about the metabolic pathways and enzymology of fatty acid ethyl ester biosynthesis, however, is very limited. In this work, we have investigated the role of a three-member S. cerevisiae gene family with moderately divergent sequences (YBR177c/EHT1, YPL095c/EEB1, and YMR210w). We demonstrate that two family members encode an acyl-coenzymeA:ethanol O-acyltransferase, an enzyme required for the synthesis of medium-chain fatty acid ethyl esters. Deletion of either one or both of these genes resulted in severely reduced medium-chain fatty acid ethyl ester production. Purified glutathione S-transferase-tagged Eht1 and Eeb1 proteins both exhibited acyl-coenzymeA:ethanol O-acyltransferase activity in vitro, as well as esterase activity. Overexpression of Eht1 and Eeb1 did not enhance medium-chain fatty acid ethyl ester content, which is probably due to the bifunctional synthesis and hydrolysis activity. Molecular modeling of Eht1 and Eeb1 revealed the presence of a alpha/beta-hydrolase fold, which is generally present in the substrate-binding site of esterase enzymes. Hence, our results identify Eht1 and Eeb1 as novel acyl-coenzymeA:ethanol O-acyltransferases/esterases, whereas the third family member, Ymr210w, does not seem to play an important role in medium-chain fatty acid ethyl ester formation.

Acyltransferases↗

PubMed2005

Trehalose-6-phosphate synthase as an intrinsic selection marker for plant transformation.

Insertion of foreign DNA into plant genomes occurs randomly and with low frequency. Hence, a selectable marker is generally required to identify transgenic plants. Until now, all selection systems have been based on the use of non-plant genes, derived from microorganisms and usually conferring antibiotic or herbicide resistance. The use of microorganism-derived genes however has raised biosafety concerns. We have developed a novel selection system based on enhancing the expression of a plant-intrinsic gene and the use of a harmless selection agent. Selection takes advantage of the reduced glucose sensitivity of seedlings with enhanced expression of AtTPS1, a gene encoding trehalose-6-P synthase. As a result, transformants can be identified as developing green seedlings amongst the background of small, pale non-transformed plantlets on high glucose medium. In addition, vegetative regeneration of tobacco leaf explants is very sensitive to high external glucose. Overexpression of AtTPS1 in tobacco allows selecting glucose insensitive transgenic shoots.

Arabidopsis↗

PubMed2005

The G protein-coupled receptor Gpr1 and the Galpha protein Gpa2 act through the cAMP-protein kinase A pathway to induce morphogenesis in Candida albicans.

We investigated the role in cell morphogenesis and pathogenicity of the Candida albicans GPR1 gene, encoding the G protein-coupled receptor Gpr1. Deletion of C. albicans GPR1 has only minor effects in liquid hypha-inducing media but results in strong defects in the yeast-to-hypha transition on solid hypha-inducing media. Addition of cAMP, expression of a constitutively active allele of the Galpha protein Gpa2 or of the catalytic protein kinase A subunit TPK1 restores the wild-type phenotype of the CaGPR1-deleted strain. Overexpression of HST7, encoding a component of the mitogen-activated protein kinase pathway, does not suppress the defect in filamentation. These results indicate that CaGpr1 functions upstream in the cAMP-protein kinase A (PKA) pathway. We also show that, in the presence of glucose, CaGpr1 is important for amino acid-induced transition from yeast to hyphal cells. Finally, as opposed to previous reports, we show that CaGpa2 acts downstream of CaGpr1 as activator of the cAMP-PKA pathway but that deletion of neither CaGpr1 nor CaGpa2 affects glucose-induced cAMP signaling. In contrast, the latter is abolished in strains lacking CaCdc25 or CaRas1, suggesting that the CaCdc25-CaRas1 rather than the CaGpr1-CaGpa2 module mediates glucose-induced cAMP signaling in C. albicans.

Animals↗

PubMed2005

Aquaporin expression and freeze tolerance in Candida albicans.

Aquaporins are members of the major intrinsic protein superfamily of integral membrane proteins which enable the transport of water, glycerol, and other solutes across membranes in various organisms. In microorganisms, the physiological role of aquaporins is not yet defined. We found a clear correlation between expression of the Candida albicans aquaporin-encoding gene AQY1 and freeze tolerance. A connection with the function for the aquaporin in the natural environment of C. albicans is, however, not obvious.

Aquaporins↗

PubMed2005

Heterologous aquaporin (AQY2-1) expression strongly enhances freeze tolerance of Schizosaccharomyces pombe.

Aquaporin membrane proteins enable the transport of water across membranes in various organisms. In yeast their expression has been shown to correlate strongly with freeze tolerance. When we analyzed the freeze tolerance of Schizosaccharomyces pombe, an organism whose genome sequence has revealed no genes encoding a bona fide water channel, we found very low intrinsic freeze tolerance compared to other yeast species with aquaporin-encoding genes. Deletion of Spac977.17, which encodes a putative glycerol facilitator, resulted in no significant differences in freeze tolerance with its corresponding wild-type strain in all growth conditions tested. However, when we expressed the Saccharomyces cerevisiae aquaporin-encoding gene AQY2-1 in S. pombe cells, we found that the relatively low freeze tolerance of S. pombe could be significantly enhanced. Therefore, (i) the absence of a bona fide water channel in S. pombe might provide in part an explanation for its overall low freeze tolerance compared to other yeast species, and (ii) aquaporin overexpression might be a tool to improve cryopreservation of many other cell types as well, as has recently been shown for mouse oocytes and fish embryos.

Aquaporins↗

PubMed2004

The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signaling.

In Arabidopsis (Arabidopsis thaliana), trehalose is present at almost undetectable levels, excluding its role as an osmoprotectant. Here, we report that overexpression of AtTPS1 in Arabidopsis using the 35S promoter led to a small increase in trehalose and trehalose-6-P levels. In spite of this, transgenic plants displayed a dehydration tolerance phenotype without any visible morphological alterations, except for delayed flowering. Moreover, seedlings overexpressing AtTPS1 exhibited glucose (Glc)- and abscisic acid (ABA)-insensitive phenotypes. Transgenic seedlings germinated on Glc were visibly larger with green well-expanded cotyledonary leaves and fully developed roots, in contrast with wild-type seedlings showing growth retardation and absence of photosynthetic tissue. An ABA dose-response experiment revealed a higher germination rate for transgenic plants overexpressing AtTPS1 showing insensitive germination kinetics at 2.5 mum ABA. Interestingly, germination in the presence of Glc did not trigger an increase in ABA content in plants overexpressing AtTPS1. Expression analysis by quantitative reverse transcription-PCR in transgenic plants showed up-regulation of the ABI4 and CAB1 genes. In the presence of Glc, CAB1 expression remained high, whereas ABI4, HXK1, and ApL3 levels were down-regulated in the AtTPS1-overexpressing lines. Analysis of AtTPS1 expression in HXK1-antisense or HXK1-sense transgenic lines suggests the possible involvement of AtTPS1 in the hexokinase-dependent Glc-signaling pathway. These data strongly suggest that AtTPS1 has a pivotal role in the regulation of Glc and ABA signaling during vegetative development.

Abscisic Acid↗

PubMed2004

Glucose and sucrose act as agonist and mannose as antagonist ligands of the G protein-coupled receptor Gpr1 in the yeast Saccharomyces cerevisiae.

Several examples of G protein-coupled receptors have recently been suggested to respond to common sugars in millimolar concentrations. This low affinity has made it difficult to demonstrate direct receptor-ligand interaction. In the yeast Saccharomyces cerevisiae, rapid activation of the cAMP pathway by glucose and sucrose requires the GPCR Gpr1. Our results obtained by cysteine scanning mutagenesis and SCAM (substituted cysteine accessibility method) of residues in TMD VI provide strong evidence that glucose and sucrose directly interact as ligands with Gpr1. The affinity for sucrose is much higher. Structurally similar sugars such as galactose, mannose, and fructose do not act as agonists, but mannose acts as an antagonist for both sucrose and glucose. These results support the idea that Gpr1 directly senses sugars and that sugars can effectively bind GPCRs with a low affinity in a binding pocket formed by the transmembrane domains. The ligand repertoire of GPCRs can thus be extended to common sugars in millimolar concentrations.

Cyclic AMP↗

PubMed2004

The high general stress resistance of the Saccharomyces cerevisiae fil1 adenylate cyclase mutant (Cyr1Lys1682) is only partially dependent on trehalose, Hsp104 and overexpression of Msn2/4-regulated genes.

The initiation of fermentation in the yeast Saccharomyces cerevisiae is associated with a rapid drop in general stress resistance. Previously we identified a mutant which is deficient in fermentation-induced loss of stress resistance (fil1), as a partially inactivating mutant in adenylate cyclase. We have now investigated possible causes of its high stress resistance. Deletion of the TPS1 gene, encoding the first enzyme in the biosynthesis of trehalose, or the heat shock protein gene HSP104 only resulted in a minor effect on heat stress resistance compared with deletion of these genes in a wild-type background. A strain with a deletion of both genes still showed a higher stress resistance in the fil1 background compared to the corresponding wild-type background. Deletion of the transcription factor genes MSN2 and MSN4, which are required for the expression of STRE-regulated genes, resulted in a dramatic drop in heat resistance in the wild-type background but had much less effect in the fil1 mutant. The fil1 msn2Deltamsn4Delta strain remained more heat-resistant than a wild-type strain. A strain in which all four genes, TPS1, HSP104, MSN2 and MSN4, are deleted was very sensitive to heat stress and also to oxidative and salt stress. Presence of the fil1 mutation in such a strain, however, still clearly enhanced heat, oxidative and salt stress resistance. These results indicate that, in addition to trehalose, Hsp104 and the Msn2/4-controlled genes, other factors exist in S. cerevisiae that can, significantly and independently of the known factors, enhance general stress resistance. The mutants described in this work provide a tool to identify these novel components.

Adenylyl Cyclases↗

PubMed2004

Glucose and sucrose: hazardous fast-food for industrial yeast?

Yeast cells often encounter a mixture of different carbohydrates in industrial processes. However, glucose and sucrose are always consumed first. The presence of these sugars causes repression of gluconeogenesis, the glyoxylate cycle, respiration and the uptake of less-preferred carbohydrates. Glucose and sucrose also trigger unexpected, hormone-like effects, including the activation of cellular growth, the mobilization of storage compounds and the diminution of cellular stress resistance. In an industrial context, these effects lead to several yeast-related problems, such as slow or incomplete fermentation, 'off flavors' and poor maintenance of yeast vitality. Recent studies indicate that the use of mutants with altered responses to carbohydrates can significantly increase productivity. Alternatively, avoiding unnecessary exposure to glucose and sucrose could also improve the performance of industrial yeasts.

Gene Expression Regulation, Fungal↗

PubMed2004

Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions.

Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.

Aquaporins↗

PubMed2004

New selection marker for plant transformation.

A number of systems to insert foreign DNA into a plant genome have been developed so far. However, only a small percentage of transgenic plants are obtained using any of these methods. Stable transgenic plants are selected by co-introduction of a selectable marker gene, which in most cases are genes that confer resistance against antibiotics or herbicides. In this chapter we describe a new method for selection of transgenic plants after transformation. The selection agent used is the nontoxic and common sugar glucose. Wild-type Arabidopsis thaliana plantlets that have been germinated on glucose have small white cotyledons and remain petite because the external sugar switches off the photosynthetic mechanism. The selectable marker gene encodes the essential trehalose-6-phophate synthase, AtTPS1, that catalyzes the first reaction of the two-step trehalose synthesis. Upon ectopic expression of AtTPS1 driven by the 35S promoter, transformed Arabidopsis thaliana plants became insensitive to glucose in comparison to wild-type plants. After transformation using AtTPS1 as a selection marker and 6% glucose as selection agent it is possible to single out the green and normal sized transgenic plants amid the nontransformed plantlets.

Arabidopsis↗

PubMed2003

Multi-level response of the yeast genome to glucose.

The yeast Saccharomyces cerevisiae shows a great variety of cellular responses to glucose via several glucose-sensing and signaling pathways. Recent microarray analysis has revealed multiple levels of genomic sensitivity to glucose and highlighted the power of genome-wide analysis to detect cellular responses to minute environmental changes.

Gene Expression Regulation, Fungal↗