Levels of plasma membrane H+-ATPase do not change during growth and morphogenesis of Candida albicans

Abstract A transient rise in the PM-ATPase activity was observed at the time of commitment of Candida albicans cells to either bud or hyphal formation. However, the changes in PM-ATPase activity did not correlate with the level of enzyme protein detected by ELISA. It was found to be fairly constant during differentiation, implying that there was no de novo synthesis of the protein. Post-translational modification(s) of enzyme protein is suggested to account for variation in PM-ATPase activity during morphogenesis.     

Microbial lipases as virulence factors

Up to now, lipases of microbial origin are known to be very useful in a palette of industrial applications. But it becomes more and more obvious that extracellular lipases also play a role during microbial infections. This review will focus on the virulent traits of these secreted enzymes from bacteria and fungi. Special emphasis will be laid on Candida albicans research. This human pathogenic fungus possesses a lipase gene family, which is expressed and differentially regulated under a variety of in vitro conditions. First results show that this isoenzyme family is also expressed during an experimental infection.

In addition, putative functions of extracellular lipases of pathogenic micro-organisms will be discussed.     

Clinical isolates of yeast produce a gliotoxin-like substance

Candida infections are major causes of morbidity in compromised human hosts, but our understanding of the virulence of Candida remains incomplete. The possibility that toxic fungal metabolites belonging to the chemical class epipolythiodioxopiperzine (ETP), which are reported to possess immunomodulating and antiphagocytic properties may be produced by Candida species was investigated. Reversed phase HPLC analysis of flash evaporated chloroform extracts of 7 day cultures of clinical Candida isolates grown in Minimal Essential Medium (MEM) with 5% fetal calf serum revealed the presence of a compound which eluted at the same time as the ETP, gliotoxin. Of 50 strains of yeast tested, 32 produced this gliotoxin-like material. This material was tested for other properties of ETP type toxins including the presence of mercaptans (Ellman reaction), ultraviolet absorbance spectrum and antibacterial activity against Micrococcus lutea. These tests revealed gliotoxin-like material from yeast cultures to be similar to commercially supplied gliotoxin. This represents the first report of the presence of ETP-like compounds in yeast and raises the possibility that ETP's may contribute to the virulence of the organism.     

Influence of Temperature and Oxygen Concentration on the Fermentation Behaviour of Candida Stellata in Mixed Fermentation with Saccharomyces Cerevisiae

Summary Candida stellata is a wine-yeast species that has been proposed for use as a starter in the wine industry in combination with selected Saccharomyces cerevisiae cultures. To improve our knowledge on the metabolic interactions between these two yeast species, we have investigated the influence of temperature and oxygen concentration on their fermentation behaviour in mixed fermentation. Trials were carried out with free and immobilized C. stellata cells followed by an inoculum of a commercial strain of S. cerevisiae, to determine the biomass evolution and the oenological profiles of the resulting wines. We show that the oxygen concentration does not influence the fermentation behaviour of the C. stellata cells, while a low temperature (16 °C) is a critical condition for the metabolic activity of C. stellata in free-cell trials. The immobilization procedures produce a significant improvement in the metabolic activity of C. stellata, with a consequent enhancement of the glycerol content also at the 16 °C fermentation temperature. High cell concentrations and the immobilization system appear to positively influence the fermentation behaviour of C. stellata. These results are of interest for the practical application of this process in winemaking.     

Hybridization of Candida albicans through fusion of protoplasts

Protoplasts of complementing auxotrophs of Candida albicans can fuse in the presence of polyethylene glycol and generate prototrophic cells. The yields of prototrophs from fusion mixtures depend greatly on the particular combinations of auxotrophies involved but not on other features of the strain backgrounds of protoplasts. The initial cellular products of fusions isolated on selective media are heterokaryons which replicate slowly but also segregate single parental nuclei into blastospores in high frequency. Karyogamy within heterokaryons produces hybrid nuclei which, on segregation, give rise to rapidly growing, uninucleate substrains. Analyses of the substrains show that hybrid nuclei either stabilize as diploid or undergo random loss of chromosomes to stabilize at various levels of aneuploidy prior to segregation. Chromosome losses and radiation induced mitotic crossing-over can effect recombination for parental auxotrophic markers in hybrids; patterns of recombination for ade^r and arg markers provide the first documented example of chromosomal linkage in C. albicans. Thus, protoplast fusions offer opportunities otherwise unavailable for applying the incisive tools of genetic recombination to analysis of this important, asexual yeast.     

Crystalline formate dehydrogenase from Candida methanolica

Abstract Formate dehydrogenase (EC 1.2.1.2) was purified about 38-fold with an overall yield of 76% from a methanol-utilizing yeast, Candida methanolica (ATCC26175), in 4 steps and, by adding polyethylene glycol, the enzyme was crystallised for the first time. The final preparation appeared to be homogeneous by the criteria of polyacrylamide electrophoresis and analytical centrifugation. Compared with the yeast formate dehydrogenases so far reported, the purified enzyme exhibited higher specific activity (7.52 U/mg).     

A dihydrofolate reductase gene from Candida albicans: molecular cloning

The dihydrofolate reductase gene from Candida albicans has been cloned and partially characterized. A genomic bank from C. albicans strain 10127/5 was constructed in Escherichia coli and screened for trimethoprim resistance. A plasmid pMF1, carrying the resistance marker was isolated and characterized by restriction mapping and Southern blotting. Cells harbouring pMF1 were as sensitive as the parental cells to a wide spectrum of antibacterial agents, except for trimethoprim; the dihydrofolate reductase activity from these cells was trimethoprim resistant.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.     

Conservation and dispersion of sequence and function in fungal TRK potassium transporters: focus on Candida albicans

TRK proteins – essential potassium (K⁺) transporters in fungi and bacteria, as well as in plants – are generally absent from animal cells, which makes them potential targets for selective drug action. Indeed, in the human pathogen Candida albicans , the single TRK isoform (CaTrk1p) has recently been demonstrated to be required for activity of histidine-rich salivary antimicrobial peptides (histatins). Background for a detailed molecular investigation of TRK-protein design and function is provided here in sequence analysis and quantitative functional comparison of CaTrk1p with its better-known homologues from Saccharomyces cerevisiae . Among C. albicans strains (ATCC 10261, SC5314, WO-1), the DNA sequence is essentially devoid of single nucleotide polymorphisms in regions coding for evolutionarily conserved segments of the protein, meaning the four intramembranal [membrane–pore–membrane (MPM)] segments thought to be involved directly with the conduction of K⁺ ions. Among 48 fungal (ascomycete) TRK homologues now described by complete sequences, clades (but not the detailed order within clades) appear conserved for all four MPM segments, independently assessed. The primary function of TRK proteins, 'active' transport of K⁺ ions, is quantitatively conserved between C. albicans and S. cerevisiae . However, the secondary function, chloride efflux channeling, is present but poorly conserved between the two species, being highly variant with respect to activation velocity, amplitude, flickering (channel-like) behavior, pH dependence, and inhibitor sensitivity.     

Complete mitochondrial genome sequences of three Nakaseomyces species reveal invasion by palindromic GC clusters and considerable size expansion

Here we report the sequence of three mitochondrial genomes from yeasts of the Nakaseomyces clade that includes the pathogenic yeast Candida glabrata , namely, that of Kluyveromyces delphensis, Candida castellii and Kluyveromyces bacillisporus . The gene content is equivalent to that of C. glabrata , but reveals the existence of new group I introns in COX1 and CYTB and new potential intronic endonucleases. Gene order is highly rearranged in these genomes, which contain numerous palindromic GC clusters. The more GC nucleotides these elements contain, the longer and more AT-rich are the intergenes containing them, leading to a direct relationship between the number of Gs and Cs within the elements and the size of the genomes. Thus, there is a fivefold difference in size between the smallest and the largest mitochondrial genome, with the largest being the most AT-rich overall. Sequences are available under EMBL accession numbers FM995164 , FM995165 , and FM995166 .