Properties of a particulate squalene epoxidase from Candida albicans

The properties and requirements of squalene epoxidase and effects of some inhibitors were investigated in the pathogenic yeast Candida albicans. A washed 'microsomal' fraction converted radiolabelled squalene to 2,3-oxidosqualene and lanosterol. Minimum requirements for activity were molecular oxygen, NADH or NADPH, and FAD. Epoxidase activity was stimulated by up to 100% by addition of the soluble cytoplasmic fraction, which itself contained negligible epoxidase activity. This stimulation was most powerful at low concentrations of enzyme, or high concentrations of squalene. Divalent cations did not stimulate activity and EDTA was not inhibitory. An apparent Km for squalene of 50 microM was determined in the presence of soluble cytoplasm. Epoxidase activity was destroyed by Triton X-100, deoxycholate or Cu2+, and partially inhibited by thiol reagents, rotenone and antimycin A. The enzyme was not inhibited by cyanide or by several inhibitors of cytochrome P-450.     

Virulence genes in the pathogenic yeast Candida albicans

In recent years, the incidence of fungal infections has been rising all over the world. Although the amount of research in the field of pathogenic fungi has also increased, there is still a need for the identification of reliable determinants of virulence. In this review, we focus on identified Candida albicans genes whose deletant strains have been tested in experimental virulence assays. We discuss the putative relationship of these genes to virulence and also outline the use of new different systems to examine the precise effect in virulence of different genes.     

Siderophore production by the pathogenic yeast, Candida albicans

Biochemical assays were used to determine that some strains of Candida albicans were capable of simultaneous secretion of both the hydroxamate and phenolate-type siderophores when grown in a deferrated medium at 37 degrees C. All isolants of C. albicans released hydroxamate-type siderophores into the culture medium; whereas, approximately 40% of the strains simultaneously secreted phenolate-type siderophores. The presence of phenolate and hydroxamate-type siderophores in the culture medium was further confirmed by assaying the culture media with type specific siderophore-dependent bacterial auxotrophs. This is the first report showing production of both classes of siderophores by a pathogenic yeast.     

Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans

Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full-length DHODH and N-terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.     

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.     

Cloning and characterization of mitochondrial methionyl-tRNA synthetase from a pathogenic fungi Candida albicans

A genomic sequence encoding mitochondrial methionyl-tRNA synthetase (MetRS) was determined from a pathogenic fungi Candida albicans. The gene is distinct from that encoding the cytoplasmic MetRS. The encoded protein consists of 577 amino acids (aa) and contains the class I defining sequences in the N-terminal domain and the conserved anticodon-binding amino acid, Trp, in the C-terminal domain. This protein showed the highest similarity with the mitochondrial MetRSs of Saccharomyces cerevisiae and Shizosaccharomyces pombe. The mitochondrial MetRSs of these fungi were distinguished from their cytoplasmic forms. The protein lacks the zinc binding motif in the N-terminal domain and the C-terminal dimerization appendix that are present in MetRSs of several other species. Escherichia coli tRNA^Met was a substrate for the encoded protein as determined by genetic complementation and in vitro aminoacylation reaction. This cross-species aminoacylation activity suggests the conservation of interaction mode between tRNA^Met and MetRS.     

Cloning and expression of the 3-isopropylmalate dehydrogenase gene from Candida albicans

Abstract A variety of Saccharomyces cerevisiae genes e.g. HIS3, LEU2, TRP1, URA3 , are expressed in Escherichia coli and have been isolated by complementation of mutations in the corresponding E. coli genes [1]. The LEU2 gene was one of the first S. cerevisiae genes to be isolated in this way [2], and its isolation led to the development of transformation systems for S. cerevisiae [3,4]. The leuB gene in E. coli [5] and the LEU2 gene in S. cerevisiae [6] both code for 3-isopropylmalate dehydrogenase (3-IMDH; EC 1.1.1.85) which is essential for the biosynthesis of leucine in both organisms. This paper describes the cloning of a fragment of C. albicans DNA carrying the gene for 3-IMDH which will be useful in the development of transformation methods in C. albicans .     

Expression and characterization of protein geranylgeranyltransferase type I from the pathogenic yeast Candida albicans and identification of yeast selective enzyme inhibitors

Protein geranylgeranyltransferase type I (GGTase I) is a heterodimeric zinc metalloenzyme catalyzing protein geranylgeranylation at cysteine residues present in C-terminal signature sequences referred to as CaaX (X=Leu) motifs. We have studied GGTase I as a potential antifungal target and recently reported its purification and cloning from the yeast Candida albicans (Ca GGTase I), an important human pathogen. Here, we report the high yield bacterial expression of Ca GGTase I by coexpression of maltose binding protein fusion proteins of both the alpha (Ram2p) and beta (Cdc43p) subunits. The cleaved and purified recombinant Ca GGTase I was demonstrated to be functional and structurally intact as judged by the presence of one equivalent of a tightly bound zinc atom and the near stoichiometric formation, isolation and catalytic turnover of a geranylgeranyl pyrophosphate-GGTase I complex. Kinetic analysis was performed with a native substrate protein, Candida Cdc42p, which exhibited significant pH dependent substrate inhibition, a feature not observed with other Ca GGTase I substrates. Prenyl acceptor substrate specificity was studied with a series of peptides in which both the CaaX motif, and the sequence preceding it, were varied. The prenyl acceptor K(M)s were found to vary nearly 100-fold, with biotinyl-TRERKKKKKCVIL, modeled after a presumably geranylgeranylated Candida protein, Crl1p (Rho4p), being the optimal substrate. A screen for inhibitors of Ca GGTase I identified compounds showing selectivity for the Candida versus human GGTase I. The most potent and selective compound, L-689230, had an IC(50) of 20 nM and >12,500-fold selectivity for Ca GGTase I. The lack of significant anti-Candida activity for any of these inhibitors is consistent with the recent finding that GGTase I is not required for C. albicans viability [R. Kelly et al., J. Bacteriol. 182 (2000) 704-713].     

Complete inventory of ABC proteins in human pathogenic yeast, Candida albicans

The recent completion of the sequencing project of the opportunistic human pathogenic yeast, Candida albicans (http://www.ncbi.nlm.nih.gov/), led us to analyze and classify its ATP-binding cassette (ABC) proteins, which constitute one of the largest superfamilies of proteins. Some of its members are multidrug transporters responsible for the commonly encountered problem of antifungal resistance. TBLASTN searches together with domain analysis identified 81 nucleotide-binding domains, which belong to 51 different putative open reading frames. Considering that each allelic pair represents a single ABC protein of the Candida genome, the total number of putative members of this superfamily is 28. Domain organization, sequence-based analysis and self-organizing map-based clustering led to the classification of Candida ABC proteins into 6 distinct subfamilies. Each subfamily from C. albicans has an equivalent in Saccharomyces cerevisiae suggesting a close evolutionary relationship between the two yeasts. Our searches also led to the identification of a new motif to each subfamily in Candida that could be used to identify sequences from the corresponding subfamily in other organisms. It is hoped that the inventory of Candida ABC transporters thus created will provide new insights into the role of ABC proteins in antifungal resistance as well as help in the functional characterization of the superfamily of these proteins.     

Sensing the host environment: recognition of hemoglobin by the pathogenic yeast Candida albicans

Adhesion to host cells and tissues is important for several steps in the pathogenesis of disseminated Candida albicans infections. Although such adhesion is evident in vivo and for C. albicans grown in vitro in complex medium, some adhesive activities are absent when cultures are grown in defined media. However, addition of hemoglobin to defined media restores binding and adhesion to several host proteins. This activity of hemoglobin is independent of iron acquisition and is mediated by a cell surface hemoglobin receptor. In addition to regulating expression of adhesion receptors, hemoglobin rapidly induces expression of several genes. One of these, a heme oxygenase, allows the pathogen to utilize exogenous heme or hemoglobin to acquire iron and to produce the cytoprotective molecules alpha-biliverdin and carbon monoxide. The specific recognition of and responses to hemoglobin demonstrate a unique adaptation of C. albicans to be both a commensal and an opportunistic pathogen in humans.     

Purification of squalene epoxidase from rat liver microsomes

Squalene epoxidase was purified from rat liver microsomes by DEAE-cellulose, alumina C_ν gel, hydroxylapatite, CM-Sephadex C-50 and Cibacron Blue Sepharose 4B in the presence of Triton X-100. The specific activity was increased 50 fold with a yield of about 10%. On SDS-polyacrylamide gel electrophoresis, the preparation gave one major band and one minor band with apparent molecular weights of 47,000 and 27,000 daltons, respectively. The protein of 47,000 was the most probable candidate for squalene epoxidase. Squalene epoxidase activity could be reconstituted in the squalene epoxidase preparation with the addition of NADPH-cytochrome P-450 reductase, FAD, and Triton X-100.     

Emerging role of lipids of Candida albicans, a pathogenic dimorphic yeast.

It is clear that C. albicans lipids have gained tremendous importance in recent years. In addition to being a barrier for entrance of various metabolites, it also provides the site of action for the synthesis of enzyme(s) involved in cell wall morphogenesis and antifungal action. While alterations in lipid composition during a yeast to mycelia transition have been observed, in most of the studies, lipid fluctuations reported could have been due to various environmental factors involved in the induction of morphogenesis [4,5]. A clear understanding of lipid biosynthesis and metabolic blocks due to antifungal action is likely to shed further light on selective interactions of antifungals. Despite the multifacet role of lipids in various functions of this pathogenic yeast, their exact involvement is poorly understood. The situation is little better with regard to ergosterol and its metabolism. Ergosterol is, indeed, important for anti-candidal activity and appears to be involved in the morphogenesis of C. albicans. The fluctuation in phospholipid composition have led to altered properties of plasma membrane namely, membrane fluidity, transport activities and drug sensitivity, which suggest that-a critical level of individual phospholipid is important for proper functioning of the plasma membrane. What the exact role is of individual phospholipid is far from clear. Many unanswered questions relating to the role of PI and sphingomyelin in signal transduction, involvement of phospholipases in the maintenance of phospholipid composition, and role of lipid transfer proteins in assembly and asymmetry of lipids are some aspects which merit further work.     

A squalene epoxidase from Nigella sativa participates in saponin biosynthesis and mediates terbinafine resistance in yeast

Squalene epoxidase catalyzes the formation of 2,3-oxidosqualene from squalene and in plants is the last enzyme common to all biosynthetic pathways leading to an array of triterpene derivatives like phytosterols, brassinosteroid phytohormones or saponins. In this work, we present a squalene epoxidase gene (NSSQE1) from the triterpene saponin producing plant Nigella sativa. The gene product showed a high degree of homology to functional squalene epoxidases (SQEs) from Arabidopsis thaliana and was able to complement SQE deficient yeast that harboured a knockout mutation in the underlying erg1 gene. Moreover, the expression of the NSSQE1 gene in ERG1 wild type yeast revealed that NSSQE1 conferred resistance towards terbinafine, an inhibitor of fungal SQEs. The latter suggested that a terbinafine-dependent NSSQE1 selection marker system can be developed for yeast. The gene NSSQE1 was ubiquitously expressed in all plant tissues analysed, including roots where no triterpene saponins are produced. Therefore, we argue that NSSQE1 is a housekeeping gene for triterpene metabolism in Nigella sativa. Similar to triterpene saponins, NSSQE1 was up-regulated by methyl jasmonate in leaves and should also be functionally involved in saponin biosynthesis in Nigella sativa.