Expression and secretion of the Candida wickerhamii extracellular β-glucosidase gene,bglB, in Saccharomyces cerevisiae

The yeast Candida wickerhamii exports a cell-associated β-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active β-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-μ replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the α-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity. Received: 28 March / 19 July 1996     

A gene homologous to Saccharomyces cerevisiae SNF1 appears to be essential for the viability of Candida albicans.

The SNF1 gene of Saccharomyces cerevisiae (ScSNF1) is essential for the derepression of catabolic repression. We report here the isolation and characterization of an SNF1 homolog from Candida albicans (CaSNF1) which is apparently essential for the viability of this organism. The putative amino acid sequence of CaSNF1 has 68% identity with that of ScSNF1 and can restore the S. cerevisiae snf1 delta mutant's ability to utilize sucrose. Disruption of one of the CaSNF1 alleles resulted in morphological changes and decreased growth rates but did not modify the carbon source utilization pattern. Repetitive unsuccessful attempts to generate a snf1/snf1 homozygote by disruption of the second allele, using various vectors and approaches, suggest the lethal nature of this mutation. Integration into the second allele was possible only when a full-length functional SNF1 sequence was reassembled, further supporting this hypothesis and indicating that the indispensability of Snf1p prevented the isolation of snf1/snf1 mutants. The mutant bearing two disrupted SNF1 alleles and the SNF1 functional sequence maintained its ability to utilize sucrose and produced stellate colonies with extensive hyphal growth on agar media. It was demonstrated that in a mouse model, the virulences of this mutant and the wild-type strain are similar, suggesting that hyphal growth in vitro is not an indicator for higher virulence.     

Influence of sub-inhibitory concentrations of conventional antifungals on metabolism of Candida albicans and on its adherence to polystyrene and extracellular matrix proteins.

Five antifungal agents with different mechanisms of action were compared for their ability to affect mitochondrial dehydrogenase activity and adherence capacity of Candida albicans to polystyrene and extracellular matrix proteins. Only amphotericin B inhibited mitochondrial dehydrogenase activity when the culture medium was supplemented with galactose. 5-Fluorocytosine and terbinafine did not affect this activity, whereas itraconazole and fluconazole improved it. Furthermore, in these experimental conditions, the effect of sub-inhibitory concentrations of antifungals on adherence was dependent on the tested antifungal and the adherence surface: amphotericin B inhibited adherence to polystyrene and fibrinogen, but improved adherence to extracellular matrix. For all surfaces tested, when culture medium was supplemented with galactose, fluorocytosine did not affect adherence, and itraconazole, fluconazole and terbinafine inhibited adherence. Our results also confirmed the influence of the carbohydrates: sub-minimum inhibitory concentrations (MIC) of itraconazole increased or did not modify the mitochondrial metabolism of yeasts when the culture medium was supplemented with galactose, but this antifungal always decreased mitochondrial metabolism when the culture medium was supplemented with glucose. These data indicate that antifungals used below their MIC values can have various effects. It is important to distinguish the effects of antifungals on the metabolism of C. albicans from effects on its adherence capacity. The former effects are linked to the viability of the yeast and the latter depends on the colonization of cellular as opposed to inert surfaces.     

The Candida albicans PMM1 gene encoding phosphomannomutase complements a Saccharomyces cerevisiae sec 53-6 mutation

We have constructed an ordered-array genomic DNA library of the pathogenic dimorphic fungus Candida albicans which facilitates the rapid cloning of C. albicans genes by hybridisation. Using the Saccharomyces cerevisiae SEC53 gene encoding phosphomannomutase as a hybridisation probe we have cloned the C. albicans homologue, PMM1, and determined its sequence. This gene shows high similarity, both at the nucleotide (76.2%) and amino-acid (77.7%) level, to the S. cerevisiae SEC53 gene. We have used the C. albicans PMM1 gene, in single copy, to transform temperature-sensitive S. cerevisiae sec53-6 mutant cells, which are defective in PMM activity at 37°C, to growth at 37°C. The C. albicans PMM1 gene is thus the structural and functional equivalent of the SEC53 gene.     

Characterization of the Saccharomyces cerevisiae FCY1 gene encoding cytosine deaminase and its homologue FCA1 of Candida albicans

By functional complementation of a fcy1 null mutant of Saccharomyces cerevisiae, we have cloned and characterized the FCY1 gene, encoding cytosine deaminase in Saccharomyces cerevisiae, and its homologue FCA1, encoding cytosine deaminase in Candida albicans. Disruption of FCY1 resulted in high resistance to 5-fluorocytosine (10⁻² M) and in total loss of cytosine deaminase activity. By contrast the transformation by FCY1 or FCA1 of the haploid FCY1-disrupted host strain restored sensitivity to 5-fluorocytosine and allowed growth on cytosine, as a source of pyrimidine, or ammonium. FCA1 as opposed to FCY1 contains an intron. FCA1 and FCY1 encode respectively 150- and 158- residue proteins of 60% identity. Both Fcy1p and Fca1p share common motifs with cytidine and CMP deaminases, but homology with cytosine deaminase of E. coli could not be detected. Received: 21 August / 12 September 1996     

Expression of the Candida albicans Gene ALS1 in Saccharomyces cerevisiae Induces Adherence to Endothelial and Epithelial Cells

To identify genes encoding adhesins that mediate the binding of Candida albicans to endothelial cells, a genomic library from this organism was constructed and used to transform Saccharomyces cerevisiae. These transformed organisms were screened for adherence to endothelial cells, and a highly adherent clone was identified. The adherence of this clone to endothelial cells was over 100-fold greater than that of control S. cerevisiae transformed with the empty plasmid. This clone also exhibited enhanced adherence to epithelial cells. The C. albicans gene contained within this clone was found to be ALS1. These results indicate that ALS1 may encode a candidal adhesin.     

Molecular cloning and genetic characterization of the Saccharomyces cerevisiae NGS1/MRE11 gene

The Saccharomyces cerevisiae ngs1-1 mutant was previously identified by its enhanced sensitivity to simple DNA-alkylating agents such as methyl methanesulfonate but not to UV. Molecular cloning and sequencing of NGS1 as a putative DNA-alkylation repair gene revealed that it is identical to MRE11, a gene that is involved in DNA recombinational repair. In order to investigate functional domains of the Mre11 protein, nucleotide-sequence alterations of a number of mre11 mutant alleles, including ngs1-1, mre11-1 (ts), mre11-2, mre11-3 and mre11-58, were determined. Most of these mutations map to the N-terminus of Mre11, emphasizing the importance of this highly conserved domain. The ngs1-1 and mre11-3 mutants carry nonsense mutations resulting in truncated proteins. Missense mutations were found in mre11-1 (ts), mre11-2 and mre11-58, of which mre11-2 and mre11-58 mapped to the conserved phosphoesterase domains, indicating the involvement of these motifs in the formation and/or processing of DNA double-strand breaks. Finally, mitotic-recombination assays show that the mre11Δ mutation enhances inter-chromosomal recombination but decreases the intra-chromosomal deletion frequency. In addition, MRE11 appears to play different roles during spontaneous and alkylation-induced homologous mitotic recombination. Received: 19 August / 18 September 1998     

Molecular cloning and characterization of a novel gene of Candida albicans,CDR1, conferring multiple resistance to drugs and antifungals

By functional complementation of a PDR5 null mutant of Saccharomyces cervisiae, we have cloned and sequenced the multidrug-resistance gene CDR1 of Candida albicans. Transformation by CDR1 of a PDR5-disrupted host hypersensitive to cycloheximide and chloramphenicol resulted in resistance to cycloheximide, chloramphenicol and other drugs, such as the antifungal miconazole, with collateral hypersensitivity to oligomycin, nystatin and 2,4 dinitrophenol. Our results also demonstrate the presence of several PDR5 complementing genes in C. albicans, displaying multidrug-resistance patterns different from PDR5 and CDR1. The nucleotide sequence of CDR1 revealed that, like PDR5, it encodes a putative membrane pump belonging to the ABC (ATP-binding cassette) superfamily. CDR1 encodes a 1501-residue protein of 169.9 kDa whose predicted structural organization is characterized by two homologous halves, each comprising a hydrophobic region with a set of six transmembrane stretches, preceded by a hydrophilic nucleotide binding fold.     

Antibodies against glucan, chitin, and Saccharomyces cerevisiae mannan as new biomarkers of Candida albicans infection that complement tests based on C. albicans mannan

Antibodies against Saccharomyces cerevisiae mannan (ASCA) and antibodies against synthetic disaccharide fragments of glucans (ALCA) and chitin (ACCA) are biomarkers of Crohn's disease (CD). We previously showed that Candida albicans infection generates ASCA. Here, we explored ALCA and ACCA as possible biomarkers of invasive C. albicans infection (ICI). ASCA, ALCA, ACCA, and Candida mannan antigen and antibody detection tests were performed on 69 sera obtained sequentially from 18 patients with ICIs proven by blood culture, 59 sera from CD patients, 47 sera from hospitalized subjects colonized by Candida species (CZ), and 131 sera from healthy controls (HC). ASCA, ALCA, and ACCA levels in CD and ICI patients were significantly different from those in CZ and HC subjects (P < 0.0001). In ICI patients, these levels increased as infection developed. Using ASCA, ALCA, ACCA, and Platelia Candida tests, 100% of ICIs were detected, with the kinetics of the antibody response depending on the patient during the time course of infection. A large number of sera presented with more than three positive tests. This is the first evidence that the detection of antibodies against chitin and glucans has diagnostic value in fungal infections and that these tests can complement more specific tests. Future trials are necessary to assess the value of these tests in multiparametric analysis, as well as their pathophysiological relevance.     

Molecular cloning of a second phospholipase B gene, caPLB2 from Candida albicans.

Accumulating evidence suggests that phospholipase B, secreted by pathogenic fungi such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, functions as one of the virulence factors. In the present study, we have attempted to clone phospholipase B gene from C. albicans. By RT-PCR analysis with degenerate primers based on conserved regions of phospholipase B from Saccharomyces cerevisiae, Penicillium notatum and Torulaspora delbrueckii two similar but different cDNA fragments were obtained. One corresponded to the partial sequence of caPLB1, recently cloned phospholipase B gene from C. albicans by a different approach (Leidich et al.: J Biol Chem 1998; 273: 26078-86). The other fragments contained sequences similar to the corresponding sequences of phospholipase B from other fungi. The presence of two related genes was confirmed by Southern and Northern blot analyses. The full length of the second C. albicans phospholipase B gene (caPLB2) encoded a putative protein with 608 amino acids and contained a potential signal peptide sequence and a putative catalytic region, which are found in phospholipase B from other fungi. Consistent with the findings of caPLB1, caPLB2 also lacks a cluster of hydrophobic amino acids at the COOH-terminal, which may function as a signal of glycosylphosphatidylinositol anchor.     

Cloning and Characterization of CAD1/AAF1, a Gene from Candida albicans That Induces Adherence to Endothelial Cells after Expression in Saccharomyces cerevisiae

Adherence to the endothelial cell lining of the vasculature is probably a critical step in the egress of Candida albicans from the intravascular compartment. To identify potential adhesins that mediate the attachment of this organism to endothelial cells, a genomic library from C. albicans was used to transform a nonadherent strain of Saccharomyces cerevisiae. The population of transformed yeasts was enriched for highly adherent clones by repeated passages over endothelial cells. One clone which exhibited a fivefold increase in endothelial cell adherence, compared with S. cerevisiae transformed with vector alone, was identified. This organism also flocculated. The candidal DNA fragment within this adherent/flocculent organism was found to contain a single 1.8-kb open reading frame, which was designated CAD1. It was found to be identical to AAF1. The predicted protein encoded by CAD1/AAF1 contained features suggestive of a regulatory factor. Consistent with this finding, immunoelectron microscopy revealed that CAD1/AAF1 localized to the cytoplasm and nucleus but not the cell wall or plasma membrane of the transformed yeasts. Because yeasts transformed with CAD1/AAF1 both flocculated and exhibited increased endothelial cell adherence, the relationship between adherence and flocculation was examined. S. cerevisiae expressing either of two flocculation phenotypes, Flo1 or NewFlo, adhered to endothelial cells as avidly as did yeasts expressing CAD1/AAF1. Inhibition studies revealed that the flocculation phenotype induced by CAD1/AAF1 was similar to Flo1. Thus, CAD1/AAF1 probably encodes a regulatory protein that stimulates endothelial cell adherence in S. cerevisiae by inducing a flocculation phenotype. Whether CAD1/AAF1 contributes to the adherence of C. albicans to endothelial cells remains to be determined.     

Cross-reacting human and rabbit antibodies to antigens of Histoplasma capsulatum, Candida albicans, and Saccharomyces cerevisiae.

Using Western blots of electrophoretically separated antigens, we show that human antibodies react most frequently to antigens shared by three fungi (Histoplasma capsulatum, Candida albicans, and Saccharomyces cerevisiae). Reactivity to antigens specific for individual fungi was relatively uncommon. The pattern of reactivity could not distinguish infected patients from uninfected controls. Rabbits immunized with extracts of each fungus also produced antibodies to cross-reactive or shared antigens of the other two fungi. Furthermore, preimmune sera showed similar but lower reactivity with the same fungal antigens. We believe that the preimmunization antibodies, which probably resulted from earlier fungal colonization or inapparent infections, predisposed the immune responses elicited by the vaccinations. A similar mechanism likely explains the results with human sera.     

Molecular cloning of the PEL1 gene of Saccharomyces cerevisiae that is essential for the viability of petite mutants

The PEL1 gene of Saccharomyces cerevisiae is essential for the cell viability of mitochondrial petite mutants, for the ability to utilize glycerol and ethanol on synthetic medium, and for cell growth at higher temperatures. By tetrad analysis the gene was assigned to chromosome III, centromere proximal of LEU2. The PEL1 gene has been isolated and cloned by the complementation of a pel1 mutation. The molecular analysis of the chromosomal insert carrying PEL1 revealed that this gene corresponds to the YCL4W open reading frame on the complete DNA sequence of chromosome III. The putative Pel1 protein is characterized by a low molecular weight of approximately 17 kDa, a low codon adaptation index, and a high leucine content.     

The homologue of the Saccharomyces cerevisiae RIM9 gene is required for ambient pH signalling in Candida albicans

In fungi, ambient pH sensing relies on the conserved Rim101 signalling pathway. All components of the pathway have been shown to be functionally conserved in the pathogenic yeast Candida albicans except for Rim9p which, in other fungi, has been suggested to be involved in this process. Here we report that, in C. albicans, the RIM9 homologue is required for Rim-dependent signalling. We also show that overexpressing Vps32p, an endosomal component required for transduction of the pH signal, does not bypass defects in upstream components such as Rim9p, Rim21p and Rim8p.     

Multivariate analyses of cellular carbohydrates and fatty acids of Candida albicans, Torulopsis glabrata, and Saccharomyces cerevisiae.

Quantitative data of major cellular carbohydrates distinguished Candida albicans or Torulopsis glabrata from Saccharomyces cerevisiae but not C. albicans from T. glabrata. Multivariate analyses of both carbohydrate and fatty acid variables (I. Brondz, I. Olsen, and M. Sj?str?m, J. Clin. Microbiol. 27:2815-2819, 1989), however, differentiated all three species.     

The effect of aminocandin (HMR 3270) on the in-vitro adherence of Candida albicans to polystyrene surfaces coated with extracellular matrix proteins or fibronectin

Aminocandin is a new representative of the echinocandins that could potentially affect the cellular morphology and metabolic status of Candida albicans cells within biofilms. This study investigated the influence of a sub-inhibitory concentration (MIC/2) of aminocandin on in-vitro growth of C. albicans and subsequent fungal adherence to plastic surfaces coated with fibronectin or extracellular matrix (ECM) proteins. Eleven strains of C. albicans were studied, of which six were susceptible and five were resistant to fluconazole. All 11 strains were susceptible to aminocandin in vitro, regardless of the culture medium used for the microdilution method. Aminocandin induced a significant (p <0.005) decrease in adherence when polystyrene was coated with ECM gel (ten strains) or fibronectin (seven strains). Growth in medium containing aminocandin (MIC/2) decreased the adherence of five (ECM gel) or three (fibronectin) of the six strains susceptible to fluconazole, and inhibition was observed for all five (ECM gel) or four (fibronectin) of the five fluconazole-resistant strains. Overall, the study demonstrated the anti-adherent properties of aminocandin with fluconazole-susceptible strains, and suggested that this activity was at least equivalent with fluconazole-resistant strains. Thus, the ability of aminocandin to inhibit the first step in the development of C. albicans biofilms appeared to be independent of the in-vitro resistance of C. albicans to fluconazole.