The effect of sucrose on the colonization of acrylic by Candida albicans in pure and mixed culture in an artificial mouth

The roles of sucrose and microbial interactions in the colonization of acrylic by Candida albicans in an artificial mouth were investigated. The acrylic was colonized by pure cultures of the organism but a visible plaque was not produced whether or not sucrose was present. Salivary bacteria had no effect in the absence of sucrose but when sucrose was present the numbers of C. albicans were significantly increased. It is suggested that this phenomenon is related to the production of extracellular polysaccharides by salivary streptococci in the presence of sucrose. In mixed culture of C. albicans and Streptococcus salivarius CJ2, growth of the latter was inhibited in the presence of sucrose. This inhibition was due to factors other than pH, although no such antagonism could be demonstrated on solid media.     

Adhesion and surface-aggregation of Candida albicans from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber

Adhesive interactions between Candida albicans and oral bacteria are generally thought to play a crucial role in the microbial colonization of denture acrylic, which may lead to denture stomatitis. This study investigated the influence of saliva on the adhesive interactions between C. albicans and Streptococcus sanguis or Actinomyces naeslundii on denture acrylic. First, bacteria were allowed to adhere to the acrylic surface from a flowing suspension, and subsequently yeasts were flowed over the acrylic surface. The organisms were assayed in the presence or absence of human whole saliva. All experiments were carried out in a parallel plate flow chamber and enumeration was done in situ with an image analysis system. In the absence of adhering bacteria, adhesion of C. albicans from buffer was more extensive than from saliva. However, in the presence of adhering bacteria, yeast adhesion from saliva was increased with respect to adhesion of yeasts from buffer, indicating that specific salivary components constitute a bridge between bacteria and yeasts. In all cases, yeast aggregates consisting of 3 to 5 yeast cells were observed adhering to the surface. A surface physico-chemical analysis of the microbial cell surfaces prior to and after bathing the microorganisms in saliva, suggests that this bridging is mediated by acid-base interactions since all strains show a major increase in electron-donating surface free energy parameters upon bathing in saliva, with no change in their zeta potentials. The surface physico-chemical analysis furthermore suggests that S. sanguis and A. naeslundii may use a different mechanism for adhesive interactions with C. albicans in saliva.     

Differential adhesion of pathogenic Candida species to epithelial and inert surfaces

Abstract Growth in medium containing 500 mM galactose is known to promote the adhesion of Candida albicans to buccal epithelial cells or to acrylic in vitro. Of 5 other Candida species tested, only C. tropicalis (one strain) showed substantially increased adhesion to buccal cells (but not to acrylic) after growth under these conditions. A second strain of C. tropicalis as well as C. stellatoidea, C. parapsilosis, C. pseudotropicalis, C. guilliermondii and Saccharomyces cerevisiae showed little or no increased adhesion to either surface. However, after growth in medium containing 50 mM glucose, C. tropicalis and C. parapsilosis were significantly more adherent to acrylic than glucose-grown yeasts of the other species, including C. albicans . These results are discussed in relation to the colonization and infection potential of the pathogenic Candida species.     

Effect of dietary carbohydrates on the in vitro epithelial adhesion of Candida albicans, Candida tropicalis, and Candida krusei

Adhesion to epithelial surfaces is considered as a critical step in the pathogenesis of oral candidosis. Therefore, the effects of the most commonly consumed dietary carbohydrates on the adhesion of Candida albicans, Candida tropicalis, and Candida krusei to monolayered HeLa cells were investigated. Adherence of C. albicans and C. tropicalis appeared significantly promoted by incubation in defined medium containing a high concentration (500 mM) of fructose, glucose, maltose, and sucrose (p < 0.001). C. albicans organisms grown in sucrose elicited maximal increase in adhesion, whereas adhesion of C. tropicalis and C. krusei was enhanced to the greatest extent when cultured in glucose. Maltose and fructose also promoted adherence of C. albicans and C. tropicalis (p < 0.001), but to a lesser extent than sucrose and glucose. On the other hand, sorbitol-grown yeasts demonstrated a marginal increase in adhesion (p > 0.01). Xylitol only significantly reduced adherence of C. albicans (p < 0.001). These results suggest that the frequent consumption of carbohydrates, such as sucrose, glucose, maltose, or fructose, might represent a risk factor for oral candidosis. The limitation of their consumption by substituting xylitol or sorbitol could be of value in the control of oral Candida colonization and infection.     

Surface hydrophobicity and adherence of Candida to acrylic surfaces

The relationship between cell surface hydrophobicities and adherence capacities to acrylic surfaces was investigated with seven laboratory strains and eighteen clinical isolates of Candida species. C. albicans was less adherent to acrylic surfaces than were other species and hardly adhered to hexadecane, whereas other strains, which had a high affinity to hexadecane, were more adherent to acrylic surfaces. A correlation was observed between the adherence capacities of Candida species to acrylic surfaces and their cell hydrophobicities. When acrylic plates were coated with human whole saliva, the contact angle of the plate became smaller than that of the nontreated plate and adherence of hydrophobic strains decreased, whereas the adherence of C. albicans was not affected.     

N-cadherin mediates endocytosis of Candida albicans by endothelial cells

Candida albicans is the most common cause of fungal bloodstream infections. To invade the deep tissues, blood-borne organisms must cross the endothelial cell lining of the vasculature. We have found previously that C. albicans hyphae, but not blastospores, invade endothelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the endothelial cell receptor that mediates the endocytosis of C. albicans. We determined that endocytosis of C. albicans was not mediated by bridging molecules in the serum and that it was partially dependent on the presence of extracellular calcium. Using an affinity purification procedure, we discovered that endothelial cell N-cadherin bound to C. albicans hyphae but not blastospores. N-cadherin also co-localized with C. albicans hyphae that were being endocytosed by endothelial cells. Chinese hamster ovary (CHO) cells expressing human N-cadherin endocytosed significantly more C. albicans hyphae than did CHO cells expressing either human VE-cadherin or no human cadherins. The expression of N-cadherin by the CHO cells resulted in enhanced endocytosis of hyphae, but not blastospores, indicating the selectivity of the N-cadherin-mediated endocytosis. Down-regulation of endothelial cell N-cadherin expression with small interfering RNA significantly inhibited the endocytosis of C. albicans hyphae. Therefore, a novel function of N-cadherin is that it serves as an endothelial cell receptor, which mediates the endocytosis of C. albicans.     

Binding of plasma proteins to Candida species in vitro

The ability of purified human albumin, fibrinogen and transferrin to bind to Candida species was measured by immunofluorescence. The proteins all bound with high avidity to germ-tubes formed by Candida albicans, but did not bind to blastospores of C. albicans or other pathogenic Candida species, not even to parent blastospores bearing germ-tubes. The extent of binding of the proteins to C. albicans germ-tubes varied between growth media and from germ-tube to germ-tube. Strains of C. albicans that did not form germ-tubes were incapable of binding any of the proteins. There was evidence that purified fibrinogen bound to germ-tubes with higher avidity than albumin and transferrin. When germ-tubes were treated with whole human plasma or serum, indirect immunofluorescence revealed that proteins were bound all over the surface of C. albicans blastospore-germ-tube units, indicating behaviour different from that seen with the purified proteins tested alone or in mixtures. C. albicans cells grown in the presence of azole antifungal agents bound purified plasma proteins in the same way as cells untreated with the drugs. The results of this study suggest that binding of host proteins to the surface of C. albicans may not be a property related directly to virulence of the fungus in vivo.     

In vitro effect of carbohydrates and enteric bacteria on adherence of Candida albicans

The adherence of Candida albicans to any cell is considered essential in the process that leads to colonization. Our objective in this study was to evaluate the effect of different carbohydrates and the presence of lactobacilli and Escherichia coli on the in vitro adherence of Candida albicans. The adherence to buccal epithelial cells was higher when growing at concentrations of galactose of 50, and 200 mM, as well as 50, 200, and 500 mM of sucrose, and 500 mM of mannose, compared with that obtained when growing in Sabouraud dextrose broth (p < 0.01). The presence of other microorganisms, such as Lactobacillus acidophilus and L. casei, caused a decrease in the in vitro adherence of C. albicans to buccal epithelial cells (p < 0.05), whereas E. coli did not modify this adherence at all.     

Membrane attack complex formation on yeast as trigger of selective release of terminal complement proteins from human polymorphonuclear leukocytes

It has recently been shown that measurable amounts of complement proteins, C6 and in particular C7, are released from human polymorphonuclear leukocytes (PMNs). The aim of the present study was to investigate the impact of opsonized Candida albicans on this release. Stimulation with opsonized C. albicans led to a rapid and sustained increase of C6 and C7 in the cell culture supernatant beginning within 5 min of placing in co-culture, whereas co-culture with unopsonized C. albicans or C. albicans mock-opsonized with inactivated human serum did not affect the release. In contrast, even after stimulation employing opsonized C. albicans, no release of the complement component C8 and only trace amounts of C9 were detected. The presence of the membrane attack complex (MAC) on C. albicans after opsonization was demonstrated by indirect immunofluorescence. Opsonization of C. albicans with human serum deficient in or depleted of a terminal complement component resulted in only minor stimulation of C6 and C7 release, although C3 deposition on the surface of C. albicans was not affected as determined by direct immunofluorescence. Detailed analyses with inactivated or deficient sera showed that detection of C6 and C7 was not due to insufficient washing of the opsonized yeast prior to co-culture and suggest that only a small proportion of these proteins was derived from the membrane bound and then cleaved off MAC. Thus, these findings imply that MAC on the fungal surface may represent an additional trigger for the release of C6 and C7 from PMNs, suggesting a new role for the terminal complement complex (TCC) on target membranes as modulator of PMN functions locally at the site of inflammation.     

Effect of ionic strength on the inactivation of micro-organisms by microwave irradiation

Microwave irradiation at 2450 MHz inactivated the cells of Escherichia coli, Staphylococcus aureus and Candida albicans suspended in a phosphate buffer. The rate of cell inactivation was proportional to that of the increase in temperature accompanied by microwave irradiation. The inactivation rates of E. coli and C. albicans were affected by addition of NaCl and KCl, but not by sucrose. The maximal inactivation effect was exerted at concentrations of 0.5-1.0 mol l-1, and the end-point temperature was the highest at the same salt concentrations. Correlation of both the electroconductivity and di-electric loss of ionic solutions with the heating by microwave irradiation was discussed.     

Effect of amigluracil and levorin on membrane permeability and protein synthesis in Candida albicans protoplasts]

It was shown that amigluracyl, a water soluble derivative of methacyl which decreased the nephrotoxic effect of polyens activated the membrane permeability in Candida albicans for a mixture of 14C-amino acids but had no significant effect on protein synthesis in this microorganism. The level of inhibition of the membrane permeability in C. albicans for the amino acids and protein synthesis in the fungus by levorin did not practically depend on the presence of amigluracyl in the incubation medium. The minimum levorin concentration inhibiting the growth of Candida albicans in the presence or absence of levorin was 0.039 gamma/ml. Therefore, amigluracyl may be used in combination with polyenic antibiotics for the treatment of mycoses.     

Cytodiagnosis of Candida organisms in cervical smears

Cervical smears and cervical scrapings cultured on Sabouraud agar from 31 women suspected of having Candida genital infections were examined in a study of the cytomorphology of this fungal infection in cervical smears. Of the 31 samples, 20 (64.5%) grew C. albicans in culture. One sample (3.2%) grew C. paratropicalis, 2 (6.4%) grew mixed C. albicans and Torulopsis glabrata and 2 (6.4%) grew T. glabrata alone. Of the 25 fungus-positive samples, 20 (80%) had fungus-positive cervical smears and 5 (20%) had fungus-negative smears. There was no instance in which the cervical smear was positive but the culture was negative. Among the cases positive for C. albicans, organisms occurred in two forms: pseudohyphae without blastospores (29.4%) and pseudohyphae with blastospores (70.6%). T. glabrata was present in the smears as budding and nonbudding yeasts. Although the sensitivity of the cervical smear in detecting fungus in culture-positive patients was only 80%, the cervical smear can still be a useful means of rapid identification of C. albicans when blastospores and pseudomycelium are present. The presence of budding or nonbudding yeast without pseudohyphae should strongly suggest a T. glabrata infection.     

Extracellular polymer of Candida albicans: isolation, analysis and role in adhesion

Extracellular polymeric material (EP) was isolated from culture supernatants of Candida albicans grown on carbon sources (50 mM-glucose, 500 mM-sucrose or 500 mM-galactose) known to promote yeast adhesion to different extents. Galactose-grown yeasts, which are the most adherent, produced more EP than sucrose-grown organisms, particularly after incubation for 5 d, while glucose-grown yeasts (the least adherent) gave the lowest yield. EP produced on all three carbon sources was of similar composition and contained carbohydrate (65 to 82%; mannose with some glucose), protein (7%), phosphorus (0.5%) and glucosamine (1.5%). Serological studies indicated that these EP preparations were immunologically identical but that galactose-grown yeasts had more antigenic determinants than sucrose-grown organisms while glucose-grown yeasts had the fewest determinants. Antigenic differences were apparent between EP preparations of some strains of C. albicans. Pretreatment of acrylic strips with EP to form a polymeric coating promoted yeast adhesion to the acrylic surface, but similar pretreatment of buccal epithelial cells with EP inhibited subsequent yeast adhesion. These results indicate that EP originates from the cell surface of C. albicans and that it contains the surface component(s), probably mannoprotein in nature, responsible for yeast adhesion.     

Role of candida albicans infection in napkin rashes

Skin scrapings, mouth swabs, and faecal specimens from children with eruptions in the napkin area and from a series of normal infants were examined for the presence of Candida albicans.This was found in 41% of all napkin eruptions but in only one of the 68 normal infants. While C. albicans is a common secondary invader of all types of napkin eruption, primary Candida infection of the skin in the napkin area is probably uncommon.No evidence was found that generalized psoriasiform or eczematous eruptions occurring in association with napkin rashes are due to an allergic response to the fungus. C. albicans is more likely to be present in a napkin rash if the organism has been found in the alimentary tract.     

A zinc finger protein from Candida albicans is involved in sucrose utilization.

A sucrose-inducible alpha-glucosidase activity that hydrolyzes sucrose in Candida albicans has been demonstrated previously. The enzyme is assayable in whole cells and was inhibited by both sucrose and maltose. A C. albicans gene (CASUC1) that affects sucrose utilization and alpha-glucosidase activity was cloned by expression in a Saccharomyces cerevisiae suc2 mutant (2102) devoid of invertase genes. CASUC1 enabled the S. cerevisiae mutant to utilize both sucrose and maltose. DNA sequence analysis revealed that CASUC1 encodes a putative zinc finger-containing protein with 28% identity to a maltose-regulatory gene (MAL63) of S. cerevisiae. The gene products of CASUC1 and MAL63 are approximately the same size (501 and 470 amino acids, respectively), and each contains a single zinc finger located at the N terminus. The zinc fingers of CASUC1 and MAL63 comprise six conserved cysteines (C6 zinc finger) and are of the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaavariable-Cys-Xaa2-Cys-+ ++Xaa6-Cys (where Xaan indicates a stretch of the indicated number of any amino acids). Both contain five amino acids in the variable region. CASUC1 also complemented the maltose utilization defect of an S. cerevisiae mutant (TCY-137) containing a defined mutation in a maltose-regulatory gene. The sucrose utilization defect of type II Candida stellatoidea, a sucrase-negative mutant of C. albicans, was corrected by CASUC1. Determinations of alpha-glucosidase activity in whole cells revealed that activity was restored in transformants cultivated on either sucrose or maltose. To our knowledge, this is the first zinc finger-encoding gene, as well as the first putative regulatory gene, to be identified in C. albicans.     

Characterisation of Candida albicans infections of haematogenous and mucosal origin in mice lacking the interferon gamma receptor protein

Mice harbouring a null deletion mutation in the IFNgamma receptor gene were used to study the role of IFNgamma responsiveness during experimental systemic candidiasis of mucosal or haematogenous origin. After intravenous (i.v.) or intranasal (i.n.) challenge with Candida albicans the progression of infection and concomitant cellular and antibody anti-C. albicans immune responses were analysed. During the week following i.v. challenge, the rate of C. albicans multiplication in kidneys, liver and spleen was faster in IFNgammaR (-/-) than IFNgammaR (+/+) mice. As a result, IFNgammaR (-/-) mice perished earlier than IFNgammaR (+/+) mice when challenged with equal numbers of live yeast cells. However, the overall susceptibility of the two mouse strains, in terms of survival against different C. albicans challenge doses over a 60-day period, was similar. No differences were found in the cellular anti-C. albicans response generated by i.v. challenge in both mouse strains. In contrast the kinetics and strength of the serum anti-C. albicans antibody responses were markedly different. Significantly stronger, predominantly IgG2a antibody responses accompanied the eventual control of C. albicans infection in IFNgammaR (-/-) mice. Following intranasal challenge, there was no difference in the rate of C. albicans clearance from the lungs of IFNgammaR (-/-) and IFNgammaR (+/+) mice. However, 48 h after challenge, large, conspicuous abscesses appeared in the lungs, liver, kidneys and spleen of IFNgammaR (-/-) mice. These abscesses were characterised by the presence of C. albicans and abundant neutrophilic infiltrates, but very few macrophages. No such abscesses developed in i.n. challenged IFNgammaR (+/+) mice. In both mouse strains, i.n. challenge induced strong systemic anti-C. albicans cellular responses, but relatively low titre systemic antibody responses. Mucosal anti-C. albicans antibody responses were detected in IFNgammaR (+/+), but not IFNgammaR (-/-) mice. Splenic adherent macrophages obtained from IFNgammaR (-/-) mice exhibited a significantly lower candidacidal activity than those of IFNgammaR (+/+) mice, and as expected, were not responsive to IFNgamma. In summary, these data suggest that IFNgamma has a role in limiting C. albicans multiplication during the early stages of infection, as well as in preventing the development of C. albicans-associated abscesses. Activation of macrophages by IFNgamma might be pivotal in mediating this role.     

Effect of Streptococcus salivarius K12 on the in vitro growth of Candida albicans and its protective effect in an oral candidiasis model

Oral candidiasis is often accompanied by severe inflammation, resulting in a decline in the quality of life of immunosuppressed individuals and elderly people. To develop a new oral therapeutic option for candidiasis, a nonpathogenic commensal oral probiotic microorganism, Streptococcus salivarius K12, was evaluated for its ability to modulate Candida albicans growth in vitro, and its therapeutic activity in an experimental oral candidiasis model was tested. In vitro inhibition of mycelial growth of C. albicans was determined by plate assay and fluorescence microscopy. Addition of S. salivarius K12 to modified RPMI 1640 culture medium inhibited the adherence of C. albicans to the plastic petri dish in a dose-dependent manner. Preculture of S. salivarius K12 potentiated its inhibitory activity for adherence of C. albicans. Interestingly, S. salivarius K12 was not directly fungicidal but appeared to inhibit Candida adhesion to the substratum by preferentially binding to hyphae rather than yeast. To determine the potentially anti-infective attributes of S. salivarius K12 in oral candidiasis, the probiotic was administered to mice with orally induced candidiasis. Oral treatment with S. salivarius K12 significantly protected the mice from severe candidiasis. These findings suggest that S. salivarius K12 may inhibit the process of invasion of C. albicans into mucous surfaces or its adhesion to denture acrylic resins by mechanisms not associated with the antimicrobial activity of the bacteriocin. S. salivarius K12 may be useful as a probiotic as a protective tool for oral care, especially with regard to candidiasis.     

Isogenic strain construction and gene targeting in Candida dubliniensis

Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. A comparison of C. albicans and C. dubliniensis at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In contrast to C. albicans, no auxotrophic C. dubliniensis strains are available for genetic manipulations. Therefore, we constructed homozygous ura3 mutants from a C. dubliniensis wild-type isolate by targeted gene deletion. The two URA3 alleles were sequentially inactivated using the MPA(R)-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. The URA3 gene from C. albicans (CaURA3) was then used as a selection marker for targeted integration of a fusion between the C. dubliniensis MDR1 (CdMDR1) promoter and a C. albicans-adapted GFP reporter gene. Uridine-prototrophic transformants were obtained with high frequency, and all transformants of two independent ura3-negative parent strains had correctly integrated the reporter gene fusion into the CdMDR1 locus, demonstrating that the CaURA3 gene can be used for efficient and specific targeting of recombinant DNA into the C. dubliniensis genome. Transformants carrying the reporter gene fusion did not exhibit detectable fluorescence during growth in yeast extract-peptone-dextrose medium in vitro, suggesting that CdMDR1 is not significantly expressed under these conditions. Fluconazole had no effect on MDR1 expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-dependent fashion, demonstrating that the CdMDR1 gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs.