Surface-active properties of Candida albicans

Cell surface hydrophobicity may be an important factor contributing to the virulence of Candida yeast cells. Surface hydrophobic and surface polar groups would be required for a yeast cell to act as a surface-active agent. In this report, the surface activities of whole yeast cells were measured. Yeast cells added at 10(8)/ml reduced the surface tension (gamma s) of saline by 20% as determined by the du Nouy method. A 1% suspension of yeast cell wall fragments reduced gamma s of saline by 36%. Whole yeast cells caused a reduction in interfacial tension (gamma I) between hexadecane and saline. The reduction of gamma I was proportional to the surface hydrophobicity of the yeasts. Yeast cells grown in glucose as the sole carbon source (thus possessing a relatively more hydrophilic cell surface) reduced gamma I by 30%, whereas yeast cells grown in hexadecane (thus possessing a more hydrophobic cell surface) reduced gamma I by 41%. The reduction of gamma I was reversed upon the addition of a strong surfactant. It was also demonstrated that yeast cells blended with nonionic surfactants during growth in a glucose broth in order to change their cell surface hydrophobicity adhered to solid surfaces in direct proportion to their cell surface hydrophobicity. Thus, the surface-active properties of Candida yeast cells may significantly contribute to the accumulation of yeast cells at various biological interfaces such as liquid-solid, liquid-liquid, and liquid-air, leading to their eventual adhesion to solid or tissue surfaces.     

Purification and some properties of Candida albicans DNA polymerases.

DNA polymerases of Candida albicans were purified to near homogeneity. Three well distinguished peaks of DNA polymerase activity (Enzyme I, II and III respectively) were obtained by DEAE-Sephacel chromatography. This purification step was followed by column chromatographies on Sepharose 6B and denatured DNA-cellulose. The enzymes' molecular mass and biochemical properties, including their inhibition by aphidicolin, were studied. Molecular mass was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and was found to be 110 kDa for Enzyme I, 80 kDa for Enzyme II and 50 kDa for Enzyme III.     

Genetics of Candida albicans.

Candida albicans is among the most common fungal pathogens. Infections caused by C. albicans and other Candida species can be life threatening in individuals with impaired immune function. Genetic analysis of C. albicans pathogenesis is complicated by the diploid nature of the species and the absence of a known sexual cycle. Through a combination of parasexual techniques and molecular approaches, an effective genetic system has been developed. The close relationship of C. albicans to the more extensively studied Saccharomyces cerevisiae has been of great utility in the isolation of Candida genes and development of the C. albicans DNA transformation system. Molecular methods have been used for clarification of taxonomic relationships and more precise epidemiologic investigations. Analysis of the physical and genetic maps of C. albicans and the closely related Candida stellatoidea has provided much information on the highly fluid nature of the Candida genome. The genetic system is seeing increased application to biological questions such as drug resistance, virulence determinants, and the phenomenon of phenotypic variation. Although most molecular analysis to data has been with C. albicans, the same methodologies are proving highly effective with other Candida species.     

Genetics of Candida albicans.

Candida albicans is among the most common fungal pathogens. Infections caused by C. albicans and other Candida species can be life threatening in individuals with impaired immune function. Genetic analysis of C. albicans pathogenesis is complicated by the diploid nature of the species and the absence of a known sexual cycle. Through a combination of parasexual techniques and molecular approaches, an effective genetic system has been developed. The close relationship of C. albicans to the more extensively studied Saccharomyces cerevisiae has been of great utility in the isolation of Candida genes and development of the C. albicans DNA transformation system. Molecular methods have been used for clarification of taxonomic relationships and more precise epidemiologic investigations. Analysis of the physical and genetic maps of C. albicans and the closely related Candida stellatoidea has provided much information on the highly fluid nature of the Candida genome. The genetic system is seeing increased application to biological questions such as drug resistance, virulence determinants, and the phenomenon of phenotypic variation. Although most molecular analysis to data has been with C. albicans, the same methodologies are proving highly effective with other Candida species.     

Calcification by Candida albicans.

Candida albicans was grown in a chamically defined medium in which certain microorganisms are known to calcify. The fungus developed calcium phosphate deposits with the same X-ray diffraction maxima as biological apatite.     

Adhesins in Candida albicans.

The adherent properties of Candida albicans blastoconidia and germ tubes have long been appreciated, but little is known about the mechanisms responsible for adherence. Recently, three genes, ALA1, ALS1 and HWP1, encoding proteins with adherent properties and motifs consistent with linkage to the beta-1, 6-glucan of C. albicans cell walls have provided insight into the topology of protein adhesins. Hwp1, a developmentally regulated adhesin of germ tubes and hyphae, attaches to buccal epithelial cells by an unconventional, transglutaminase-mediated mechanism of adhesion.     

Surface-active properties of antibiotics

The critical concentrations of the mycella formation of novobiocin, mithramycin, variamycin, erythromycin, oleandomycin and lincomycin were determined with two methods by changes in the isotherms of the surface tension and in the maximum absorption of rodamine due to the antibiotic concentrations. The results obtained with the two methods were comparable.     

Virulence factors of Candida albicans.

Candidiasis is a common infection of the skin, oral cavity and esophagus, gastrointestinal tract, vagina and vascular system of humans. Although most infections occur in patients who are immunocompromised or debilitated in some other way, the organism most often responsible for disease, Candida albicans, expresses several virulence factors that contribute to pathogenesis. These factors include host recognition biomolecules (adhesins), morphogenesis (the reversible transition between unicellular yeast cells and filamentous, growth forms), secreted aspartyl proteases and phospholipases. Additionally, 'phenotypic switching' is accompanied by changes in antigen expression, colony morphology and tissue affinities in C. albicans and several other Candida spp. Switching might provide cells with a flexibility that results in the adaptation of the organism to the hostile conditions imposed not only by the host but also by the physician treating the infection.     

Cloning, purification, and properties of Candida albicans thymidylate synthase.

The thymidylate synthase (TS) gene was isolated from a genomic Candida albicans library by functional complementation of a Saccharomyces cerevisiae strain deficient in TS. The gene was localized on a 4-kilobase HindIII DNA fragment and was shown to be expressed in a Thy- strain of Escherichia coli. The nucleotide sequence of the TS gene predicted a protein of 315 amino acids with a molecular weight of 36,027. The gene was cloned into a T7 expression vector in E. coli, allowing purification of large amounts of C. albicans TS. It was also purified from a wild-type C. albicans strain. Comparison of several enzyme properties including analysis of amino-terminal amino acid sequences showed the native and cloned C. albicans TS to be the same.     

Enzyme cytochemistry of Candida albicans.

The application of a new preparation method for demonstrating the activities of hydrolytic and oxidative enzymes in Candida albicans is reported. The problem of inadequate penetration of fixatives into yeast cells has been solved by sectioning solidified pellets of the cells in the presence of glutaraldehyde, a procedure that yields a fairly well preserved ultrastructure and sufficient enzyme activities. The subcellular distribution of most specific and nonspecific phosphatases and of peroxidases is at variance with that found in mammalian cells. The activities toward beta-glycerophosphate, p-nitrophenylphosphate, adenosine triphosphate, adenosine monophosphate, thiamine pyrophosphate and glucose 6-phosphate are almost exclusively confined to the central vacuolar apparatus. Oxidative and peroxidative activities are demonstrated only in mitochondria. Specific marker enzymes for endoplasmic reticulum, plasmalemma, Golgi apparatus and peroxisomes in C. albicans are not found. The possible function of the various subcellular organelles in relation to their enzymatic content is discussed.     

Instability of Candida albicans hybrids.

Total cellular DNA content, determined by a colorimetric method, was used as an index of ploidy in Candida albicans. Mononucleate hybrids were formed by fusion of spheroplasts derived from diploid parent strains. Five hybrids, of six studied, were taken to be tetraploid on the basis of estimated DNA content. One hybrid was taken to be hexaploid or near-hexaploid. Selection for increased resistance to 5-fluorocytosine in the hybrids, which were heterozygous for resistance, resulted in isolation of variants which were of lower ploidy than the hybrids from which they originated. Variants were obtained which corresponded (in measured DNA content) to aneuploid, triploid, and diploid states. These results may form the basis of a cyclic parasexual system (2n X 2n----4n----2n) for genetic analysis of this asexual species.     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Phospholipase activity in Candida albicans.

Phospholipase A and lysophospholipase have been identified as the enzymes responsible for phospholipid hydrolysis by Candida albicans. The method used to identify and measure the activity of these enzymes is described, and the probable significance of phospholipase in the invasion of the epithelium by Candida albicans discussed.     

Septal ultrastructure in Candida albicans.

Fine structural studies on the septa of Candida albicans in vitro (when leading a saprophytic existence) and the organism in its invasive form (as a pathogen in oral candidosis) have shown that in the former the septum exhibits a unique central perforation resembling an aggregate of fine canaliculi connecting one cell to the other. In the invasive form the septum is non-perforated and appears as a solid structure. Septal ultrastructure is well characterised in many pathogenic fungi. Our observations on Candida albicans do not resemble any previous studies carried out on other deutromycetous fungi.