Morphogenesis in Candida albicans

This review will survey environmental controls on the morphology of Candida albicans, describe the cellular and ultrastructural events associated with morphological transitions in this fungus, and attempt to relate biochemical phenomena that have been reported to be associated with dimorphic change to C. albicans cell biology. The synthesis of the cell wall of C. albicans and its control remain largely undiscovered, but it is clear that the cell wall is the principal component involved in shape determination. Possible models for C. albicans dimorphism will be critically reviewed.     

Traversal of Candida albicans across human blood-brain barrier in vitro

Candida albicans is an opportunistic pathogen, which primarily affects neonates and immunocompromised individuals. The pathogen can invade the central nervous system, resulting in meningitis. At present, the pathogenesis of C. albicans meningitis is unclear. We used an in vitro model of the human blood-brain barrier to investigate the interaction(s) of C. albicans with human brain microvascular endothelial cells (BMEC). Binding of C. albicans to human BMEC was time and inoculum dependent. Invasion of C. albicans into human BMEC was demonstrated by using an enzyme-linked immunosorbent assay based on fluorescent staining of C. albicans with calcoflour. In contrast, avirulent Candida mutant strains and nonpathogenic yeast Saccharomyces cerevisiae were not able to bind and invade human BMEC. Morphological studies revealed that on association with human BMEC, C. albicans formed germ tubes and was able to bud intracellularly. Transmission electron microscopy showed various stages of C. albicans interactions with human BMEC, e.g., pseudopod-like structures on human BMEC membrane and intracellular vacuole-like structures retaining C. albicans. Of interest, C. albicans was able to bud and develop pseudohyphae inside human BMEC without apparent morphological changes of the host cells. In addition, C. albicans penetrates through human BMEC monolayers without a detectable change in transendothelial electrical resistance and inulin permeability. This is the first demonstration that C. albicans is able to adhere, invade, and transcytose across human BMEC without affecting monolayer integrity. A complete understanding of the interaction(s) of C. albicans with human BMEC should contribute to the understanding of the pathogenic mechanism(s) of C. albicans meningitis.     

Fingerprinting Candida albicans

A new method of typing Candida albicans based on immunoblotting is described. Isolates were disrupted by a mixture of enzymic pretreatment with alpha-mannosidase followed by sonication. They were then stained using a modified ELISA system by a rabbit hyperimmune serum raised against a single isolate, C. albicans NCTC 3153. The 190 isolates examined from the London Hospital produced 16 different types. Type 1 accounted for 43% of the isolates and was the commonest type outside the intensive care unit. Type 2 caused an outbreak of systemic candidosis on the intensive care unit. The technique was much more sensitive than the serotyping and morphotyping methods and lacked the phenotypic variability of the biotyping procedure previously used to define the outbreak. The gel-to-gel variation precludes its use in large scale epidemiological work. Its value lies in identification of outbreaks so that they can be controlled by the introduction of measures to prevent cross-infection.     

Direct modulation of human neutrophil behaviour by Candida albicans

We examined the direct effect of unopsonized yeast particles of Candida albicans on two aspects of neutrophil behaviour, namely adherence and 3H-deoxyglucose uptake. The data show that brief exposure of C. albicans to human neutrophils resulted in decreased ability of the neutrophils to adhere to Dacron fibre and take up deoxyglucose. This inhibitory effect was further shown to be dependent on yeast concentration and on the integrity of the yeast cell wall. Additional experiments indicate that this effect was direct rather than indirect through soluble mediators in the supernatant. Interference experiments with glucan and mannan, the two major polysaccharide components of the yeast cell wall, suggest that the ligand in direct interaction between C. albicans and neutrophil contains glucan. Finally, it was shown that nonpathogenic species of Candida such as C. krusei, C. parapsilosis and C. guilliermondii did not display neutrophil-modulatory properties while the occasionally pathogenic C. tropicalis did. These results indicate that C. albicans has the ability to circumvent neutrophil defence mechanisms, and may in part explain the propensity of this fungus to cause infection.     

Immunoglobulin levels and antibody to Candida albicans in human cervicovaginal secretions

Human cervicovaginal secretions were examined for immunoglobulin content and for antibody to Candida albicans. The predominant immunoglobulin class of the secretions was IgA, accounting for approximately 65% of the total immunoglobulin. Most of the IgA was eluted from Sephadex G-200 in the excluded peak and was associated with secretory component; it therefore had the characteristics of `secretory IgA''. With increasing age, the IgA content fell and the IgG content rose in cervicovaginal secretions. No IgE could be detected in the secretions. Antibody to C. albicans was found to be predominantly of the IgA class.     

Candida albicans and selenium

Although low selenium levels have been recorded in infants, no specific human disorder has been linked to low selenium status. The incidence of thrush, the common enteric fungal infection caused by Candida albicans, has increased markedly with antibiotic therapy and research has provided evidence that its colonization leads to competition for Coenzyme Q10 (CoQ10) in the host. Furthermore it is now known that ubiquinones are essential in heart muscle for oxidative phosphorylation in the mitochondrial respiratory chain and considered that glutathione peroxidase (GSHPx) in the mitochondria protects ubiquinone from oxidation.     

Adherence of Candida albicans to human buccal epithelial cells.

The adherence of Candida albicans to human buccal epithelial cells after 2 h at 37 degrees C was significantly greater in human saliva than in phosphate-buffered saline. in saliva, viable fungi adhered much better than did nonviable fungi, and this adherence was greater at 37 than at 25 degrees C. Viable yeasts, preincubated in saliva for 90 min at 37 degrees C before being washed and mixed with epithelial cells in phosphate-buffered saline, adhered better than nonviable yeasts or yeasts preincubated in phosphate-buffered saline. Enhanced adherence in saliva appeared to be associated with germination of the yeast cells. Conditions permitting germination (growth in tissue culture medium 199 at 37 degrees C but not at 25 degrees C) also supported enhanced adherence. After germination had occurred, the fungi could be killed with Formalin without interfering with their rapid and efficient adherence to epithelial cells. These data indicate that the enhanced adherence of C. albicans observed after incubation in saliva is related to changes in the fungi, rather than to a requirement for prolonged interaction between fungi and epithelial cells.     

Genetic relationship between human and animal isolates of Candida albicans

Analyzing Candida albicans isolates from different human and animal individuals by Ca3 fingerprinting, we obtained no evidence for host-specific genotypes and for the existence of species-specific lineages, even though a certain degree of separation between human and animal isolates was found. Therefore, animals could potentially serve as reservoirs for human Candida infection.     

Characterization of Candida albicans adherence to human vaginal epithelial cells in vitro

Certain environmental, physical, and biochemical aspects of Candida albicans adherence to human vaginal epithelial cells were characterized by using an in vitro radiometric adherence assay. Blastospores harvested from cultures grown at 25 degrees C adhered to vaginal epithelial cells in significantly greater numbers than did blastospores isolated from cultures grown at 37 degrees C. C. albicans viability was not essential for adherence, but severe methods used to kill the blastospores did reduce their attachment. The addition of sodium chloride, divalent cations, sugars, mannan, or mannoprotein to the assay had no effect on attachment. Pretreatment of the blastospores with detergents, salts, urea, glycosidases, lipase, or pepsin did not affect adherence, but treatment with reducing agents or five proteolytic enzymes did render C. albicans nonadherent. Cell wall fragments prepared from C. albicans, but not from Candida krusei, adhered to vaginal epithelial cells. Loss of adherence after the cell walls were treated with alpha-mannosidase or papain suggests that cell wall mannoprotein is an essential component of the C. albicans adhesin.     

UV-induced instability in Candida albicans hybrids

Summary Auxotrophic variants were obtained following UV-irradiation of Candida albicans hybrids which were heterozygous (+/+/-/-/) for various genetic markers (met, ade, his, lys). Some variants contained less DNA (per cell) than did the hybrids from which they originated; such variants were considered to arise in a process which resulted in generalized reduction in ploidy. These results provide the basis for a cyclic parasexual system (2n × 2n 4n 2n) for genetic analysis in this amictic diploid species.     

Natural auxotrophic heterozygosity in Candida albicans

Candida albicans is imperfect and diploid. This unusual genetic state permits the occurrence of strains heterozygous for recessive deleterious alleles. These alleles can be brought to homozygosity and therefore expression by UV irradiation-induced mitotic crossing over. Heterozygosity for recessive auxotrophic alleles is found in 10 to 15% of the strains. A wide variety of auxotrophic alleles have been found in such natural heterozygotes but one type, termed Suf +/- was found at an unusually high frequency. Some 5 to 10% of strains are of the Suf +/- type. Suf- homozygous auxotrophs are defective in sulfite reductase. Such auxotrophs require a source of reduced sulfur such as methionine or cysteine, alternatively inorganic thiosulfate will serve as a supplement. Complementation analysis of a variety of Suf- auxotrophs established that 11 out of 12 strains were in a single complementation group.     

Genomic analysis in Candida albicans]

With the recent advances in DNA sequencing technology, a succession of entire genome sequences have been published. A number of genome projects are underway in pathogenic fungi. From these, we present the history and current status of the genomic analysis of Candida albicans. The sequencing project for this organism has been undertaken at Stanford University, and is now nearing the end.     

High-frequency switching in Candida albicans.

Most strains of Candida albicans are capable of switching frequently and reversibly between a number of phenotypes distinguishable by colony morphology. A number of different switching systems have been defined according to the limited set of phenotypes in each switching repertoire, and each strain appears to possess a single system. Switching can affect many aspects of cellular physiology and morphology and appears to be a second level of phenotypic variability superimposed upon the bud-hypha transition. The most dramatic switching system so far identified is the "white-opaque transition." This system dramatizes the extraordinary effects switching can have on the budding cell phenotype, including the synthesis of opaque-specific antigens, the expression of white-specific and opaque-specific genes, and the genesis of unique cell wall structures. Switching has been demonstrated to occur at sites of infection and between episodes of recurrent vaginitis, and it may function to generate variability in commensal and infecting populations for adaptive reasons. Although the molecular mechanisms involved in the switch event are not understood, recent approaches to its elucidation are discussed and an epigenetic mechanism is proposed.     

Quorum-sensing system in Candida albicans]

The bacterial behavior system controlled by the cell density is described as quorum-sensing. The system is triggered via autoinducers. Various kinds of autoinducers have been identified from different bacteria. Quorum-sensing signals via autoinducers are involved in regulation of important virulences such as exotoxin, protease, and pigment production. Therefore, this system in pathogenic bacteria has a critical role in the regulation of bacterial pathogenicity. In the pathogenic fungus Candida albicans, an extracellular quorum-sensing molecule that regulates hyphal formation by this organism has been identified in recent years. Candida albicans has been shown to form biofilm on many medical devices, therefore quorum-sensing in this organism has been especially focused on from the aspect of biofilm formation.     

Changes in external trehalase activity during human serum-induced dimorphic transition in Candida albicans

Yeast-like cells (blastoconidia) of Candida albicans growing exponentially on a glucose-containing medium (YPD) exhibited low external trehalase activity and stored a negligible amount of intracellular trehalose. The addition of human serum at 37 degrees C to exponential cultures promoted a high degree of germ-tube formation with no significant changes in trehalase activity or trehalose content. In contrast, stationary cells accumulated a large amount of trehalose, while external trehalase remained at a low and practically constant level. However, resting cultures were unable to enter the dimorphic program, except when they were supplemented with fresh YPD and serum together. Only under these conditions was trehalase activated and trehalose hydrolyzed. Specific inhibition of external trehalase by validoxylamine A caused a certain delay in, and a lower level of, germ-tube formation, but did not totally block the dimorphic conversion. These results suggest that external trehalase is not involved in the serum-induced morphological transition in C. albicans.     

An improved transformation protocol for the human fungal pathogen Candida albicans

Commonly used protocols for the transformation of the dimorphic human fungal pathogen Candida albicans rely on established methods for the yeast Saccharomyces cerevisiae. With respect to transformation efficiency, however, there is a great difference between these two organisms when using the lithium acetate procedure. Here we present a modified version of this protocol for use with C. albicans. Among the different parameters tested, two turned out to be particularly relevant and, when combined, resulted in an up to 10-fold increase in transformation efficiency (400-500 integrative transformants) compared with previous protocols: first, adjusting the heat shock applied to the cells to 44 degrees C for C. albicans instead of 42 degrees C for S. cerevisiae and, second, treating C. albicans cells with lithium acetate in an overnight incubation instead of for 30 min as used for S. cerevisiae. With these modifications, the lithium acetate procedure becomes a very efficient and reliable tool for C. albicans transformation.     

Variations in affinity to Candida albicans in vitro among human buccal epithelial cells

Using an in-vitro adherence assay it was observed that the number of Candida albicans cells that attached to individual buccal mucosal cells varied greatly. Three mucosal-cell characteristics--state of aggregation, size and viability--that might influence yeast adhesion in vitro were studied. The number of attached yeast cells per mucosal cell varied from 0 to 32. The majority of buccal cells (88%) had none or very few yeasts attached, whereas a minority of cells (12%) bound more than one half of all the attached yeasts. In donors whose buccal cells had large numbers of attached yeasts this percentage increased and the number of cells with no attached yeast cells fell. Cells of an intermediate size (36-70 micron) had a greater affinity for yeasts than did cells of other sizes. Buccal cell viability appeared not to be necessary for adhesion of yeasts. No significant differences were observed in the number of yeast cells attached to single buccal cells compared with attachment to buccal cells within sheets. It would appear, therefore, that there are distinct subpopulations of epithelial cells with high and low affinity for attachment by C. albicans in vitro. Mucosal cell size or viability might influence this affinity.