0000000000181053
AUTHOR
Eulogio Valentín
Identification of Candida auris and related species by multiplex PCR based on unique GPI protein‐encoding genes
Background The pathogen Candida auris is rapidly gaining clinical importance because of its resistance to antifungal treatments and its persistence in hospital environments. Early and accurate diagnosis of C. auris infections is crucial, however, the fungus has often been misidentified by commercial systems. Objectives To develop conventional and real-time PCR methods for accurate and rapid identification of C. auris and its discrimination from closely related species by exploiting the uniqueness of certain glycosylphosphatidylinositol-modified protein-encoding genes. Methods Species-specific primers for two unique putative GPI protein-encoding genes per species were designed for C. auris, …
Homozygous deletion of ATC1 and NTC1 genes in Candida parapsilosis abolishes trehalase activity and affects cell growth, sugar metabolism, stress resistance, infectivity and biofilm formation
A double homozygous atc1Δ/atc1Δ/ntc1Δ/ntc1Δ mutant (atc1Δ/ntc1Δ KO) was constructed in the pathogen opportunistic yeast Candida parapsilosis by disruption of the two chromosomal alleles coding for NTC1 gene (encoding a neutral trehalase) in a Cpatc1Δ/atc1Δ background (atc1Δ KO strain, deficient in acid trehalase). The Cpatc1Δ/ntc1Δ KO mutant failed to counteract the inability of Cpatc1Δ cells to metabolize exogenous trehalose and showed a similar growth pattern on several monosaccharides and disaccharides. However, upon prolonged incubation in either rich medium (YPD) or nutrient-starved medium the viability of Cpatc1Δ cells exhibited a sensitive phenotype, which was augmented by further Cp…
Phenotype traits associated with different alleles at the RPS5 locus in Saccharomyces cerevisiae
The RPS5 gene has been characterised through its ability to reduce invertase production by the SUC5 gene. In this paper we show that RPS5 acts by maintaining low levels of SUC5 mRNA. We also show that RPS5 acts on the SUC1 and SUC4 genes but not on SUC2 and SUC3, which are members of the SUC family. RPS5 also shows a pleiotropic effect on the amount of mitochondrial cytochromes.
CandidaDB: a genome database for Candida albicans pathogenomics.
CandidaDB is accessible at http://genolist.pasteur.fr/CandidaDB.; International audience; 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 th…
Molecular cloning of the RPS0 gene from Candida tropicalis.
The Saccharomyces cerevisiae RPS0 A and B genes encode proteins essential for maturation of the 40S ribosomal subunit precursors. We have isolated a homologue of the RPS0 gene from Candida tropicalis, which we named CtRPS0. The C. tropicalisRPS0 encodes a protein of 261 amino acid residues with a predicted molecular weight of 28.65 kDa and an isoelectric point of 4.79. CtRps0p displays significant amino acid sequence homology with Rps0p from C. albicans, S. cerevisiae, Neurospora crassa, Schizosaccharomyces pombe, Pneumocystis carinii and higher organisms, such as human, mouse and rat. CtRPS0 on a high copy number vector can complement the lethal phenotype linked to the disruption of both R…
Deletion of GLX3 in Candida albicans affects temperature tolerance, biofilm formation and virulence.
Candida albicans is a predominant cause of fungal infections in mucosal tissues as well as life-threatening bloodstream infections in immunocompromised patients. Within the human body, C. albicans is mostly embedded in biofilms, which provides increased resistance to antifungal drugs. The glyoxalase Glx3 is an abundant proteomic component of the biofilm extracellular matrix. Here, we document phenotypic studies of a glx3Δ null mutant concerning its role in biofilm formation, filamentation, antifungal drug resistance, cell wall integrity and virulence. First, consistent with its function as glyoxalase, the glx3 null mutant showed impaired growth on media containing glycerol as the carbon sou…
Identification ofCandida albicanswall mannoproteins covalently linked by disulphide and/or alkali-sensitive bridges
This paper describes the results obtained by analysing the human pathogen Candida albicans cell wall subproteome by mass spectrometry, using extraction procedures aimed at releasing proteins bound by disulphide bridges (RAE-CWP) or alkali-labile ester linkages (ALS-CWP). Ten of the total proteins released from the wall by β-ME and/or NaOH contained a potential signal peptide, lacked a GPI cell wall hydrophobic C-terminal domain and were identified as true wall proteins by in silico analysis, whereas four additional proteins were identified as bound to the plasma membrane. The results surprisingly demonstrated that, in addition to the expected RAE-CWP and ALS-CWP proteins, 16 GPI proteins we…
Molecular typing of clinical Candida strains using random amplified polymorphic DNA and contour-clamped homogenous electric fields electrophoresis.
Aims: This report describes an investigation into the genetic profiles of 38 Candida albicans and 19 Candida glabrata strains collected from a dental hospital of Monastir (Tunisia) and the Laboratory of Parasitology, Farhat Hached Hospital of Sousse (Tunisia), using two typing methods: random amplified polymorphic DNA (RAPD) and contour-clamped homogenous electric fields (CHEF). Methods and Results: The two methods (RAPD and CHEF electrophoresis) were able to identify clonal-related isolates from different patients. RAPD method using two primers (CA1 and CA2) exhibited the highest discriminatory power by discriminating 22 genotypes for C. albicans with CA1 oligonucleotides and 19 genotype…
Null mutants of Candida albicans for cell-wall-related genes form fragile biofilms that display an almost identical extracellular matrix proteome.
By two-dimensional gel electrophoresis (2-DE) and mass spectrometry, we have characterized the polypeptide species present in extracts obtained by 60% ethanol treatment of whole mature (48 h) biofilms formed by a reference strain (CAI4- URA3 ) and four Candida albicans null mutants for cell-wall-related genes ( ALG5, CSA1, MNN9 and PGA10) . Null mutants form fragile biofilms that appeared partially split and weakly attached to the substratum contrary to those produced by the reference strain. An almost identical, electrophoretic profile consisting of about 276 spots was visualized in all extracts examined. Proteomic analysis led to the identification of 131 polypeptides, corresponding to 86…
5 The Ascomycetous Cell Wall: From a Proteomic Perspective
Cell walls are essential organelles for fungi; they define cell shape during growth and provide osmotic integrity and protection against harmful influences in the growth environment. Fungal walls also play an important role in developing fungal infections as they form the first contact between the pathogen and the host immune system. In many ascomycetes, the cell wall consists of a polysaccharide matrix surrounded by a layer of covalently bound glycoproteins. With the complete genome sequences being available for many species, cell wall research in recent years has largely focused on identifying and elucidating the functions of cell wall proteins. In this chapter, we discuss, with a main fo…
Incorporation of mannoproteins into the walls of aculeacin A-treated yeast cells
Inhibition of the synthesis of alkali-insoluble glucan by aculeacin A in Saccharomyces cerevisiae cells caused a decrease in the incorporation of a high molecular weight heterogeneous mannoprotein material and of a 33,000 mannoprotein into the wall network. This was concomitant with the excretion of the latter molecule into the growth medium. Regenerating yeast protoplasts liberated considerable amounts of the heterogeneous material to the medium independently of the presence of aculeacin. The protoplast walls did lack this component and contained only minor amounts of the 33,000 molecule, which was also completely absent from walls of aculeacin-treated protoplasts. Considerable levels of t…
A Candida albicans 37 kDa polypeptide with homology to the laminin receptor is a component of the translational machinery.
A cDNA encoding a 37 kDa protein was isolated from an expression library using antibodies raised against mycelial cell walls fromCandida albicans.The 37 kDa protein has over 60% sequence identity with the 37 kDa laminin-binding protein (LBP) from humans and over 80% identity with the Yst proteins ofSaccharomyces cerevisiae. TheC. albicansprotein was named CaYst1. It was found in membrane and ribosome fractions but surprisingly, was not found in cell walls. Unlike the human LBP, CaYst1p does not bind laminin. These data indicate that CaYst1p is not a cell-surface receptor for laminin as has been proposed for the human LBP. Instead, like theS. cerevisiaeYst proteins, it appears to be a riboso…
On the biochemical classification of yeast trehalases: Candida albicans contains two enzymes with mixed features of neutral and acid trehalase activities
Abstract Two enzymes endowed with trehalase activity are present in Candida albicans . The cytosolic trehalase (Ntc1p), displayed high activity in exponential phase regardless of the carbon source (glucose, trehalose or glycerol). Ntc1p activity was similar in neutral (pH 7.1) or acid (pH 4.5) conditions, strongly inhibited by ATP, weakly stimulated by divalent cations (Ca 2+ or Mn 2+ ) and unaffected in the presence of cyclic AMP. The Ntc1p activity decreased in stationary phase, except in glycerol-grown cultures, but the catalytic properties did not change. In turn, the cell wall-linked trehalase (Atc1p) showed elevated activity in resting cells or in cultures growing on trehalose or glyc…
A novel cell wall protein specific to the mycelial form of Yarrowia lipolytica.
A cDNA clone specifying a cell wall protein was isolated from a Yarrowia lipolytica cDNA library. The cDNA library was constructed in the expression vector lambda gt 11, with the RNA isolated from actively growing mycelial cells. The deduced amino acid sequence shows that the encoded protein contains an N-terminal hydrophobic signal peptide. We have designated this protein YWP1 for Yarrowia lipolytica cell Wall Protein. Northern hybridization identified YWP1 transcript only when Y. lipolytica was growing in the mycelial form. The encoded protein seems to be covalently bound to the glucan cell wall since it is not released from the cell walls by sodium dodecyl sulphate extraction, but it is …
Time-kill assays of amphotericin B plus anidulafungin against Candida tropicalis biofilms formed on two different biomaterials.
Purpose: To determine the fungicidal activity by time-killing assays of amphotericin B (AMB) combined with anidulafungin (ANF) against biofilms of 2 clinical isolates of Candida tropicalis and the reference strain ATCC® 750, developed on polytetrafluoroethylene (PTFE) and titanium, using the CDC Biofilm Reactor (CBR) as an in vitro model. Methods: Biofilms were developed for 24 hours on the disk surfaces and then exposed to AMB (40 mg/L), ANF (8 mg/L), alone and combined. At predetermined time points after drug exposure, biofilms were removed from the disk surface by vortexing-sonication to quantify viable biofilm cells. Results: Drug activity was dependent on strain and time. After exposur…
Cell wall mannoproteins during the population growth phases in Saccharomyces cerevisiae.
Mannoproteins from cell walls of Saccharomyces cerevisiae synthesized at successive stages of the population growth cycle have been solubilized with Zymolyase and subsequently analyzed. The major change along the population cycle concerned a large size mannoprotein material; the size of the newly-synthesized molecules varied from 120,000–500,000 (mean of about 200,000) at early exponential phase to 250,000–350,000 (mean of about 300,000) at late exponential phase. These differences are due to modifications in the amount of N-glycosidically linked mannose residues, since the size of the peptide moiety was 90,000–100,000 at all growth stages and the level of O-glycosylation changed only sligh…
Identification and study of a Candida albicans protein homologous to Saccharomyces cerevisiae Ssr1p, an internal cell-wall protein
After screening of aCandida albicansgenome database, the product of an ORF (IPF 3054) that has 62 % homology withSaccharomyces cerevisiaeSsr1p, an internal cell-wall protein, was identified and named CaSsr1p. The deduced amino acid sequence shows that CaSsr1p contains an N-terminal hydrophobic signal peptide, is rich in Ser and Thr amino acids and has a potential glycosylphosphatidylinositol-attachment signal. CaSsr1p is released following degradation of isolated cell walls by zymolyase (mainly a 1,3-β-glucanase) and therefore seems to be covalently linked to theβ-glucan of the cell walls. Both disruption and overexpression of theCaSSR1gene caused an increased sensitivity to calcofluor whit…
Effect of α-factor on individual wall mannoproteins fromSaccharomyces cerevisiae acells
Treatment of Saccharomyces cerevisiae a cells with α-factor partially inhibits mannosylation of the high Mr mannoproteins, although there is an increase in the total amount of these molecules present in the wall. They show a similar mobility in SDS-acrylamide gels to those from untreated mnn2 cells. No other significant effects on wall mannoproteins have been observed, except a decrease in the amount of the 29 kDa species.
Pga13 in Candida albicans is localized in the cell wall and influences cell surface properties, morphogenesis and virulence.
The fungal cell wall is an essential organelle required for maintaining cell integrity and also plays an important role in the primary interactions between pathogenic fungi and their hosts. PGA13 encodes a GPI protein in the human pathogen Candida albicans, which is highly up-regulated during cell wall regeneration in protoplasts. The Pga13 protein contains a unique tandem repeat, which is present five times and is characterized by conserved spacing between the four cysteine residues. Furthermore, the mature protein contains 38% serine and threonine residues, and therefore probably is a highly glycosylated cell wall protein. Consistent with this, a chimeric Pga13-V5 protein could be localiz…
Cloning of cDNAs coding forCandida albicanscell surface proteins
Two cDNA libraries were constructed from mRNAs obtained from yeast cells and germ-tubes of Candida albicans in lambda gt11. Immunoscreening with polyclonal antibodies raised against cell wall components allowed the detection of 29 positive clones. Two of these clones were selected for their specific reactivity with antisera either from yeast (clone 11Y) or germ-tubes (clone 24M). cDNA fragments were isolated by the digestion of lambda DNA with EcoRI. Southern blot analysis with these fragments as probes demonstrated homology with C. albicans DNA, and by Northern analysis two mRNAs transcripts were detected with sizes of approximately 1·5 kb for 11Y and 1·1 kb for 24M. Both transcripts were …
TheGCA1gene encodes a glycosidase-like protein in the cell wall ofCandida albicans
Candida albicans Gca1p is a putative glucoamylase enzyme which contains 946 amino acids, 11 putative sites for N -glycosylation and 9 for O -glycosylation. Gca1p was identified in β-mercaptoethanol extracts from isolated cell walls of strain C. albicans SC5314 and it is involved in carbohydrate metabolism. The significance and the role of this protein within the cell wall structure were studied in the corresponding mutants. The homozygous mutant showed that GCA1 was not an essential gene for cell viability. Subsequent phenotypic analysis performed in the mutants obtained did not show significant difference in the behavior of mutant when compared with the wild strain SC5314. Zymoliase, Calco…
Genomic response programs of Saccharomyces cerevisiae following protoplasting and regeneration.
Abstract Global transcription profiling during regeneration of Saccharomyces cerevisiae protoplasts was explored. DNA microarrays measured the expression of 6388 genes and wall removal resulted initially in over-expression of 861 genes that decayed later on, a behaviour expected from a transient stress response. Kinetics of expression divided the genes into 25 clusters. Transcription of the genes from clusters 14–25 was initially up-regulated, suggesting that the grouped genes permitted cell adaptation to the removal of the wall. Clustering of genes involved in “wall structure and biosynthesis” showed that most of them had initially low levels of expression that increased along the process.…
Functional analysis of the cysteine residues and the repetitive sequence ofSaccharomyces cerevisiaePir4/Cis3: the repetitive sequence is needed for binding to the cell wall β-1,3-glucan
Identification of PIR/CIS3 gene was carried out by amino-terminal sequencing of a protein band released by β-mercaptoethanol (β-ME) from S. cerevisiae mnn9 cell walls. The protein was released also by digestion with β-1,3-glucanases (laminarinase or zymolyase) or by mild alkaline solutions. Deletion of the two carboxyterminal Cys residues (Cys214-12aa-Cys227-COOH), reduced but did not eliminate incorporation of Pir4 (protein with internal repeats) by disulphide bridges. Similarly, site-directed mutation of two other cysteine amino acids (Cys130Ser or Cys197Ser) failed to block incorporation of Pir4; the second mutation produced the appearance of Kex2-unprocessed Pir4. Therefore, it seems th…
Yarrowia lipolytica cell wall architecture: interaction of Ywp1, a mycelial protein, with other wall components and the effect of its depletion
Linkages of Ywp1 to other components of the Yarrowia lipolytica mycelial cell wall were studied by extraction with beta-mercaptoethanol and zymolyase (a beta-glucanase complex) and by the use of rabbit polyclonal antibody preparation raised against Ywp1. Ywp1 complexed with an N-glycosylated cell wall protein(s) to form supramolecular complexes through disulphide bridges (extractable with beta-mercaptoethanol) or bonded to beta-1,3-glucan (extractable with zymolyase). The lack of a specific morphological phenotype when YWP1 was knocked out by gene disruption might indicate that other proteins present in the cell wall of Y. lipolytica compensated for its loss. In this mutant, the electrophor…
Comparative genomics of yeast species: new insights into their biology
The genomes of two hemiascomycetous yeasts (Saccharomyces cerevisiae and Candida albicans) and one archiascomycete (Schizosaccharomyces pombe) have been completely sequenced and the genes have been annotated. In addition, the genomes of 13 more Hemiascomycetes have been partially sequenced. The amount of data thus obtained provides information on the evolutionary relationships between yeast species. In addition, the differential genetic characteristics of the microorganisms explain a number of distinctive biological traits. Gene order conservation is observed between phylogenetically close species and is lost in distantly related species, probably due to rearrangements of short regions of D…
Disruption of the Candida albicans ATC1 gene encoding a cell-linked acid trehalase decreases hypha formation and infectivity without affecting resistance to oxidative stress.
In Candida albicans, the ATC1 gene, encoding a cell wall-associated acid trehalase, has been considered as a potentially interesting target in the search for new antifungal compounds. A phenotypic characterization of the double disruptant atc1Delta/atc1Delta mutant showed that it was unable to grow on exogenous trehalose as sole carbon source. Unlike actively growing cells from the parental strain (CAI4), the atc1Delta null mutant displayed higher resistance to environmental insults, such as heat shock (42 degrees C) or saline exposure (0.5 M NaCl), and to both mild and severe oxidative stress (5 and 50 mM H(2)O(2)), which are relevant during in vivo infections. Parallel measurements of int…
Expression ofYWP1,a Gene That Encodes a SpecificYarrowia lipolyticaMycelial Cell Wall Protein, inSaccharomyces cerevisiae
Abstract The YWP1 gene encoding a specific mycelial cell wall protein of Yarrowia lipolytica has been cloned and expressed in Saccharomyces cerevisiae using different episomal plasmids. Because the plasmids pYAE35BB and pYAE35ES carrying the YWP1 gene (including the 5′ noncoding promoter sequences) failed to express it, the YWP1 gene was cloned under the control of GAL/CYC or ACT S. cerevisiae promoters. A main band with an apparent molecular mass of 70 kDa was detected by immunoblotting in the cell wall fraction of transformants. Ywp1 processing and incorporation to the cell wall were similar in both Y. lipolytica and S. cerevisiae but not in its final localization in the cell wall. In Y. …
Anchorage of Candida albicans Ssr1 to the cell wall, and transcript profiling of the null mutant.
Incorporation into the wall of Candida albicans Ssr1, a GPI-dependent protein, was investigated by construction of different truncated genes for which the three potential omega sites (S199, S215 and G216) and the corresponding omega+1 and omega+2 were eliminated or modified. Cells of the C. albicans ssr1Delta mutant were transformed with pADH-pl harboring the truncated versions of CaSSR1, pADH-DeltaCaSSR1t(217-234) (lacking a C-terminal hydrophobic stretch of 18 aa including the putative omega+2 and omega+1, omega+2 of S215 and G216) or pADH-DeltaCaSSR1t(199-201) (lacking three serine residues), and their walls were analyzed for the protein. Results suggested that the three serine residues …
Cloning and characterization of the phenylalanyl-tRNA synthetase β subunit gene fromCandida albicans
A Candida albicans expression library was constructed from RNA isolated from regenerating protoplasts. A 1.4-kb cDNA clone was used to isolate a genomic fragment. Sequence analysis revealed an open reading frame of 593 amino acids with an overall identity of 63.6% with the phenylalanyl-tRNA synthetase beta subunit (FRS1) of Saccharomyces cerevisiae. We named it CaFRS1. It is located in a single copy in chromosome R, SfiI fragment M. Its expression showed a decrease during the cell wall regeneration process in protoplasts of both yeast and mycelial cells of C. albicans, suggesting its requirement thereof in initial steps of the cell wall synthesis.
Characterization of aCandida albicansgene encoding a putative transcriptional factor required for cell wall integrity
After screening a Candida albicans genome database the product of an open reading frame (ORF) (CA2880) with 49% homology to the product of Saccharomyces cerevisiae YPL133c, a putative transcriptional factor, was identified. The disruption of the C. albicans gene leads to a major sensitivity to calcofluor white and Congo red, a minor sensitivity to sodium dodecyl sulfate, a major resistance to zymolyase, and an alteration of the chemical composition of the cell wall. For these reasons we called it CaCWT1 (for C. albicans cell wall transcription factor). CaCwt1p contains a putative Zn(II) Cys(6) DNA binding domain characteristic of some transcriptional factors and a PAS domain. The CaCWT1 gen…
Global transcriptional profiling ofCandida albicans cwt1 null mutant
CaCwt1p is a Candida albicans putative transcriptional factor homologue to Rds2p in Saccharomyces cerevisiae. The lack of this protein in S. cerevisiae leads to a pleiotropic resistance to drugs and defects in cell wall architecture that are also detectable in C. albicans. It is also known that CaCwt1p is mainly expressed in the stationary growth phase of this fungus. In order to elucidate the role of CWT1, transcriptome analysis of the mutant strain was performed in exponential and stationary growth phases. A total of 460 genes were found to be up- or downregulated in the mutant strain growing exponentially, and 666 genes presented a misregulation when cwt1 cells reached the stationary pha…
Dosage-dependent roles of the Cwt1 transcription factor for cell wall architecture, morphogenesis, drug sensitivity and virulence in Candida albicans.
The Cwt1 transcription factor is involved in cell wall architecture of the human fungal pathogen Candida albicans. We demonstrate here that deficiency of Cwt1 leads to decreased β1,6-glucan in the cell wall, while mannoproteins are increased in the cell wall of exponentially growing cells and are released into the medium of stationary phase cells. Hyphal morphogenesis of cwt1 mutants is reduced on the surfaces of some inducing media. Unexpectedly, the CWT1/cwt1 heterozygous strains shows some stronger in vitro phenotypes compared to the homozygous mutant. The heterozygous but not the homozygous strain is also strongly impaired for its virulence in a mouse model of systemic infection. We sug…
Role of Pir1 in the construction of the Candida albicans cell wall
Searches in a Candida albicans database (http://genolist.pasteur.fr/CandidaDB/) identified two Individual Protein Files (IPF 15363 and 19968) whose deduced amino acid sequences showed 42 % and 45 % homology with Saccharomyces cerevisiae Pir4. The two DNA sequences are alleles of the same gene (CaPIR1) but IPF 19968 has a deletion of 117 bases. IPF 19968 encodes a putative polypeptide of 364 aa, which is highly O-glycosylated and has an N-mannosylated chain, four cysteine residues and seven repeats. Both alleles are expressed under different growth conditions and during wall construction by regenerating protoplasts. The heterozygous mutant cells are elongated, form clumps of several cells an…
In Candida parapsilosis the ATC1 Gene Encodes for an Acid Trehalase Involved in Trehalose Hydrolysis, Stress Resistance and Virulence
An ORF named CPAR2-208980 on contig 005809 was identified by screening a Candida parapsilosis genome data base. Its 67% identity with the acid trehalase sequence from C. albicans (ATC1) led us to designate it CpATC1. Homozygous mutants that lack acid trehalase activity were constructed by gene disruption at the two CpATC1 chromosomal alleles. Phenotypic characterization showed that atc1Δ null cells were unable to grow on exogenous trehalose as carbon source, and also displayed higher resistance to environmental challenges, such as saline exposure (1.2 M NaCl), heat shock (42°C) and both mild and severe oxidative stress (5 and 50 mM H2O2). Significant amounts of intracellular trehalose were …
Specific stress-induced storage of trehalose, glycerol and D-arabitol in response to oxidative and osmotic stress in Candida albicans.
Candida albicans exponential yeast cells are able to face environmental challenges by mounting a rapid and efficient "general stress response". Here we show that one of the main components of this response consists of the intracellular protective accumulation of the non-reducing disaccharide trehalose and two polyols, glycerol and D-arabitol, an accumulation that occurs in a stress-specific dependent manner. Thus, oxidative exposures promoted a marked increase in both trehalose and D-arabitol in the wild type strain, RM-100, whereas the glycerol content remained virtually unaffected with respect to basal levels. In contrast, osmotic challenges induced the significant storage of glycerol acc…
Oligonucleotide-capped nanoporous anodic alumina biosensor as diagnostic tool for rapid and accurate detection of Candida auris in clinical samples.
[EN] Candida auris has arisen as an important multidrug-resistant fungus because of several nosocomial outbreaks and elevated rates of mortality. Accurate and rapid diagnosis of C. auris is highly desired; nevertheless, current methods often present severe limitations and produce misidentification. Herein a sensitive, selective, and time-competitive biosensor based on oligonucleotide-gated nanomaterials for effective detection of C. auris is presented. In the proposed design, a nanoporous anodic alumina scaffold is filled with the fluorescent indicator rhodamine B and the pores blocked with different oligonucleotides capable of specifically recognize C. auris genomic DNA. Gate opening modul…
A study of the Candida albicans cell wall proteome
Considering the importance of proteins in the structure and function of the cell wall of Candida albicans, we analyzed the cell wall subproteome of this important human pathogen by LC coupled to MS (LC-MS) using different protein extraction procedures. The analyzed samples included material extracted by hydrogen fluoride-pyridine (HF-pyridine), and whole SDS-extracted cell walls. The use of this latter innovative procedure gave similar data as compared to the analysis of HF-pyridine extracted proteins. A total of 21 cell wall proteins predicted to contain a signal peptide were identified, together with a high content of potentially glycosylated Ser/Thr residues, and the presence of a GPI mo…
Genomic response programs of Candida albicans following protoplasting and regeneration
Transcription profiling of Candida albicans cells responding to the elimination of the wall (protoplasts) and posterior regeneration was explored. DNA microarrays were used to measure changes in the expression of 6039 genes, and the upregulated genes during regeneration at 28 degrees C were assigned to fourteen categories. A total of 407 genes were upregulated during the process, of which 144 reached a maximum after 1 h. MKC1, a gene encoding a member of the regulatory pathway involved in cell wall integrity was overexpressed. Time-dependent expression divided the genes into 40 clusters. Clusters 1-19 were highly expressed initially (time 0) and downregulated following incubation, whereas t…
Glycoprotein molecules in the walls of Schizosaccharomyces pombe wild-type cells and a morphologically altered mutant resistant to papulacandin B
SUMMARY: Schizosaccharomyces pombe cell walls contain two major glycoprotein species, I and II, with molecular masses of 2 x 106 and 5 x 105 Da respectively, as determined by gel filtration chromatography and PAGE. The ratio of sugar to protein is higher in species I than in species II. Much of the sugar in both glycoproteins (about 85% in wild-type cells) is O-linked to the peptide moiety. The morphological sph1 mutant is resistant to papulacandin B, and its cell wall contains less glycoprotein II (but not less glycoprotein I) than the parental wild-type strain, although glycoprotein II is still synthesized and released into the growth medium. Papulacandin B largely reverses the morphologi…
Phenotypic characterization and adhesive properties of vaginal Candida spp. strains provided by the CHU Farhat Hached (Sousse, Tunisia).
Abstract Background Vulvovaginal candidiasis is a common infection among women worldwide, being Candida albicans the most commonly isolated species. Therefore, controlling this opportunistic yeast is one of the key factors for reducing nosocomial infection. Aims We investigated several virulence properties of 28 vaginal strains of Candida isolated from Tunisian women suffering from vulvovaginitis. We also analyzed the virulence properties of a clinical Candida krusei strain and five Candida reference strains. Methods Candida strains were subjected to microscopic analysis and culture in Candida ID2 chromogenic medium. The adhesive properties of these strains were estimated by the microtiter …
The ATC1 gene encodes a cell wall-linked acid trehalase required for growth on trehalose in Candida albicans.
After screening a Candida albicans genome data base, the product of an open reading frame (IPF 19760/CA2574) with 41% identity to Saccharomyces cerevisiae vacuolar acid trehalase (Ath1p) was identified and named Atc1p. The deduced amino acid sequence shows that Atc1p contains an N-terminal hydrophobic signal peptide and 20 potential sites for N-glycosylation. C. albicans homozygous mutants that lack acid trehalase activity were constructed by gene disruption at the two ATC chromosomal alleles. Analysis of these null mutants shows that Atc1p is localized in the cell wall and is required for growth on trehalose as a carbon source. An Atc1p endowed with acid trehalase activity was obtained by …
Molecular organization of the cell wall of Candida albicans and its relation to pathogenicity.
Candida albicans is one of the most important opportunistic pathogenic fungi. Weakening of the defense mechanisms of the host, and the ability of the microorganism to adapt to the environment prevailing in the host tissues, turn the fungus from a rather harmless saprophyte into an aggressive pathogen. The disease, candidiasis, ranges from light superficial infections to deep processes that endanger the life of the patient. In the establishment of the pathogenic process, the cell wall of C. albicans (as in other pathogenic fungi) plays an important role. It is the outer structure that protects the fungus from the host defense mechanisms and initiates the direct contact with the host cells by…