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.     

Adherence of Candida albicans and other Candida species to mucosal epithelial cells.

To study the possible involvement of candidal adherence in mucosal colonization, we examined the in vitro adherence capabilities of seven Candida species. Adherence was evaluated by direct microscopic examination and by a quantitative radiometric adherence test. The results indicate that C. albicans adheres to vaginal and buccal epithelial cells to a significantly greater degree (P less than 0.01) than the other species tested. C. tropicalis and C. stellatoidea demonstrated moderate adherence capabilities, while C. parapsilosis adhered only to a slight degree. Other species failed to interact with isolated mucosal cells. These findings suggest that there is a relationship between the adherence capabilities of the Candida species and their abilities to colonize mucosal surfaces, since those species which adhere are those which most frequently colonize mucosal surfaces. C. albicans was found to be adherent under a variety of environmental conditions. Stationary-phase blastospores of C. albicans were found to be more adherent than logarithmic-phase yeasts, and larger blastospore cell-to-epithelial cell ratios resulted in greater adherence values. The actual number of adherent yeasts varied considerably when epithelial cells were obtained from different donors.     

Fimbria-mediated adherence of Candida albicans to glycosphingolipid receptors on human buccal epithelial cells.

Candida albicans is an opportunist fungal pathogen that has the ability to adhere to host cell surface receptors via a number of adhesins. Yu et al. (L. Yu, K. K. Lee, K. Ens, P. C. Doig, M. R. Carpenter, W. Staddon, R. S. Hodges, W. Paranchych, and R. T. Irvin, Infect. Immun. 62:2834-2842, 1994) described the purification and initial characterization of a fimbrial adhesin from C. albicans. In this paper, we show that C. albicans fimbriae also bind to asialo-GM1 [gangliotetraosylceramide: beta Gal(1-3)beta GalNAc(1-4) beta Gal(1-4)beta Glc(1-1)Cer] immobilized on microtiter plates in a saturable and concentration-dependent manner. C. albicans fimbrial binding to exfoliated human buccal epithelial cells (BECs) was inhibited by asialo-GM1 in in vitro binding assays. The fimbriae interact with the glycosphingolipid receptors via the carbohydrate portion of the receptors, since fimbriae were observed to bind to synthetic beta GalNAc(1-4)beta Gal-protein conjugates and the disaccharide was able to inhibit binding of fimbriae to BECs in in vitro binding assays. We conclude from these results that the C. albicans yeast form expresses a fimbrial adhesin that binds to glycosphingolipids displayed on the surface of human BECs.     

Relationship between germination of Candida albicans and increased adherence to human buccal epithelial cells.

A strong correlation was shown between germination and increased adherence of Candida albicans to human buccal epithelial cells, indicating that germination or other changes in the fungi accompanying germination were responsible for enhanced adherence. Partial inhibition of germination by cysteine resulted in a comparably lower adherence. Preferential adherence of germinated fungi occurred in competition assays with nongerminated and germinated fungi. The enhanced adherence to human mucosal cells of germinated C albicans could represent one mechanism contributing to the pathogenicity of the organism.     

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.     

Adherence of Candida albicans to oral epithelial cells differentiated by Papanicolaou staining

OBJECTIVE: To examine the relative adherence of Candida albicans to oral epithelial cells differentiated by Papanicolaou staining. METHODS: Oral epithelial cells were collected from 10 healthy adults (five male, five female) and counted. Equal volumes of oral epithelial cells and candida were mixed and incubated. The epithelial cells from this mix were collected by filtration through 10 microns polycarbonate membrane filters. Cells retained on the membrane filters were stained with crystal violet followed by Papanicolaou stain. The number of yeast attached to each of 100 red, orange, and green staining oral epithelial cells was determined by direct microscopic examination. RESULTS: C albicans had a higher level of adherence (p < 0.001) to red staining oral epithelial cells (mean (SD) number of candida attached to 100 oral epithelial cells 562 (159)) than to cells staining either orange (105 (47)) or green (161 (66)). CONCLUSIONS: Oral epithelial cell variability for candidal adherence is confirmed. The technique provides an opportunity to examine the relation between oral epithelial cell type and oral candidosis in specific groups, such as tobacco smokers, where increased epithelial cell keratinisation and candidal colonisation has been reported.     

Adherence of Haemophilus influenzae to buccal epithelial cells.

The role of adherence of Haemophilus influenzae to epithelial surfaces in the pathogenesis of infection is unknown. Fluorescent-antibody and radiolabeled adherence methods were adapted to study H. influenzae adherence to human buccal epithelial cells. By the fluorescent-antibody method, 19 of 21 (90%) nontypable H. influenzae strains were found to be adherent compared with 2 of 42 (5%) type b strains (P less than 0.0001). Using a radiolabeled adherence method, we found that 9 of 12 (75%) nontypable H. influenzae strains were adherent to buccal epithelial cells whereas only 3 of 32 (9%) type b strains were adherent (P = 0.001). Results of H. influenzae adherence examined by both methods correlated significantly (P = 0.01). H. influenzae adherence to adult pharyngeal, nasal, and buccal epithelial cells was comparable. Type b H. influenzae did not adhere to the buccal epithelial cells of well children, children with H. influenzae type b disease, or children with upper respiratory infections. In contrast, nontypable H. influenzae did adhere to the buccal epithelial cells of well children and children with upper respiratory infections. These observed in vitro differences in adherence between nontypable and type b H. influenzae strains may explain differences in colonization, pathogenesis, and types of infection due to nontypable and type b H. influenzae.     

Adherence of cell surface mutants of Candida albicans to buccal epithelial cells and analyses of the cell surface proteins of the mutants.

Strains of Candida albicans, selected on the basis of their reduced agglutination with a polyclonal anti-Candida antiserum, were tested for their adherence to human buccal epithelial cells (BEC). Of four strains, one (A9V2) had reduced binding to BEC in vitro. Adherence of wild type (wt) yeast cells (A9), as measured by the percentage of BEC with adhering Candida cells, was 73.4% +/- 3.8% compared with 49.3% +/- 3.1% for A9V2 (P less than 0.01). From yeast cells of A9 and A9V2, whole-cell extracts and dithiothreitol-, Zymolyase-, or beta-mercaptoethanol-solubilized cell extracts were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting). From dithiothreitol-solubilized cell extracts, proteins with molecular masses of 55 to 60, 80 to 84, 115, and 165 kDa were observed from wt (A9) cells but were highly reduced in amount or absent from A9V2 cells. Western blot profiles of Zymolyase-solubilized extracts from both A9 and A9V2 were similar in appearance, while 55-, 80- to 84-, 115-, and 165-kDa proteins were observed only in A9 cells extracted with beta-mercaptoethanol. Strain A9V4, also selected by reduced agglutination but which adhered as well as strain A9, lacked the 80- to 84-kDa and 115-kDa proteins but otherwise was similar to strain A9. These results indicate that the 55- to 60- and 165-kDa proteins may be related to an adhesin function in C. albicans. The differences observed in the protein profiles of the wt adhering strain and its derived nonadhering mutant are similar to those described for another matched pair of C. albicans strains.     

Role of specific determinants in mannan of Candida albicans serotype A in adherence to human buccal epithelial cells.

Candida albicans serotype A (C. albicans A) possesses a specific antigen, designated antigen 6, which resides in mannans on the cell surface. To determine the role of the mannan moiety of the C. albicans cell wall in adherence to buccal epithelial cells, we used antigen 6-deficient mutants which had been isolated by screening with an agglutinating monoclonal antibody against antigen 6 (MAb-6). 1H nuclear magnetic resonance spectral analysis of the purified mannans from the mutants showed a loss of the signals related to that beta-linkage of the side chains. Moreover, acetolyzed fragments of the mutant mannans showed a decreased amount of mannohexaose and mannopentaose. The mutant yeast cells exhibited significantly reduced ability to adhere both to exfoliated buccal epithelial cells and to a human buccal cell line. A number of strains of C. albicans A, C. tropicalis, and C. glabrata, all of which bear antigen 6, showed significantly higher adherence to the cell line than did those of C. albicans serotype B, which lack antigen 6. The whole mannan from the C. albicans A parent inhibited the adherence of C. albicans A to epithelial cells dose dependently, whereas mannan from a mutant strains did not. Moreover, C. albicans A treated with MAb-6 or polyclonal factor 6 serum showed reduced adherence. A close correlation was found between adhesive ability and agglutinability with MAb-6 in the C. albicans A parent, the antigenic mutants, and their spontaneous revertants. These results suggest that so far as mannan adhesion is concerned, serotype A-specific determinants are largely involved in the mechanisms of adherence of C. albicans A to human buccal epithelial cells.     

Adherence of Enterobacteriaceae to human buccal cells.

A preliminary examination has been made of the adherencae isolated from sputa. The radioadherence method was found to correlate well with the conventional light-microscope adherence technique. Saturation of buccal-cell binding sites with bacteria occurred when less than 10% of the buccal-cell surface was occupied. The adherence of Enterobacter aerogenes to buccal cells was impaired by the prior adherence of bacilli of either the same strain, or of a strain of Klebsiella pneumoniae.     

Adherence of Haemophilus influenzae to human buccal and pharyngeal epithelial cells: relationship to pilation

An improved understanding of the role of pili in adherence of Haemophilus influenzae type b to human epithelial cells (EC) would enhance knowledge of the pathogenesis of H. influenzae b infections. In this study a highly sensitive in-vitro assay allowed the quantitative assessment of H. influenzae b adherence to EC. The degree of adherence was influenced by incubation time, temperature, bacteria/EC ratio, EC type and the growth phase of the bacteria. Most serially subcultured (SC) capsular type-b strains originally isolated from cerebrospinal fluid, blood, nasopharynx or throat gave similar low degrees of adherence, as did representative single strains of capsular types a, c, d, e and f. SC non-capsulated H. influenzae strains adhered in significantly greater numbers than most SC capsulated strains (p less than 0.001). One SC type-b strain isolated from a throat, with stable piliation, adhered in very high numbers despite capsulation. Piliated subpopulations selected from type-b capsulated strains adhered in greater numbers than did their parent strains. These data suggest that capsulation of H. influenzae is a deterrent to adherence of the bacteria to EC. However, the presence of pili may allow type-b organisms to overcome the effects of capsulation.     

Evidence for mannose-mediated adherence of Candida albicans to human buccal cells in vitro.

Various lectins and sugars were used to study the possible role of saccharide-containing moieties on the surface of Candida albicans and human buccal cells in the adherence of this yeast to mucosal surfaces. The lectins possessed affinities for several different sugar moieties and were used to pretreat C. albicans or buccal cells before mixing and incubating in the adherence assay. It was found that concanavalin A, a lectin that recognizes mannose and glucose, inhibited adherence of the pretreated yeasts to buccal cells and also inhibited adherence of pretreated buccal cells to nonpretreated yeast cells. Adherence was restored by preincubating the concanavalin A with a mannose derivative, but preincubation of concanavalin A with other sugars did not produce this effect. Lectins that do not recognize mannose had no effect on adherence. The presence of alpha-D-methyl mannopyranoside in the incubation medium during the assay inhibited adherence, whereas other sugars did not. Germinated yeasts adhered to buccal cells more effectively than nongerminated cells and were more susceptible to adherence inhibition by concanavalin A than were nongerminated yeasts. Thus, mannose-containing moieties on the surface of C. albicans and buccal cells could mediate the adherence of this yeast to human epithelium.     

Modulation of Candida albicans attachment to human epithelial cells by bacteria and carbohydrates.

The effects of carbohydrates (mannose and dextrose). Escherichia coli 07KL. and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells.     

Adherence of clinical isolates of Saccharomyces cerevisiae to buccal epithelial cells.

A number of isolates of Saccharomyces cerevisiae have been associated with disease in immunocompromised individuals. Such isolates display a variety of characteristics that enable colonization and persistence in the host. The aim of the work presented here was to establish whether clinical isolates of S. cerevisiae were capable of adhering to epithelial tissue. Adherence to host tissue has been shown to be crucial to the virulence of the pathogenic yeast Candida albicans, and identification of this ability in S. cerevisiae might indicate a role for adherence in tissue colonization by this emerging pathogen. Clinical S. cerevisiae isolates were found to be capable of adhering to exfoliated buccal epithelial cells (BECs) but to a lesser degree than C. albicans. In contrast to the situation evident with C. albicans, the adherence of S. cerevisiae isolates to BECs was not influenced by the carbon source in which the yeast was grown. Treatment of S. cerevisiae with trypsin or proteinase K resulted in a significant reduction in adherence ability while adherence was unaffected by treatment of cells with mannosidase, thus indicating a possible role for proteins rather than mannoproteins in the adherence of S. cerevisiae to BECs.     

Inhibition by sugars of Candida albicans adherence to human buccal mucosal cells and corneocytes in vitro.

The adherence and inhibition of adherence of Candida albicans to epithelial cells was studied for human cells obtained from skin (corneocytes) and buccal mucosa. The yeast adhered to both kinds of cells, although in somewhat greater numbers to buccal mucosal cells. Adherence to the cells of different individuals was variable, but the ratios of values for the two kinds of cells from a single subject were quite constant. Inhibition of adherence was produced by several sugars, including the aminosugars mannosamine, glucosamine, and galactosamine. The pattern of inhibition produced by the sugars was similar for the two types of cells. Pretreatment of the yeast with mannosamine, followed by dilution to a subinhibitory concentration, produced some inhibition of yeast-buccal mucosal cell attachment, indicating some direct interaction between the sugar and the fungal cell. These data suggest that the mechanisms whereby C. albicans attaches to corneocytes and to buccal mucosal cells are probably similar.     

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.     

Deletion of ALS5, ALS6 or ALS7 increases adhesion of Candida albicans to human vascular endothelial and buccal epithelial cells.

Candida albicans yeast forms deleted for ALS5, ALS6 or ALS7 are more adherent than a relevant control strain to human vascular endothelial cell monolayers and buccal epithelial cells. In the buccal and vaginal reconstituted human epithelium (RHE) disease models, however, mutant and control strains caused a similar degree of tissue destruction. Deletion of ALS5 or ALS6 significantly slowed growth of the mutant strain; this phenotype was not affected by addition of excess uridine to the culture medium. These studies demonstrate similar phenotypic characteristics for the als5Delta/als5Delta, als6Delta/als6Delta and als7Delta/als7Delta strains that are not observed in any of the other C. albicans alsDelta/alsDelta isolates.     

Eikenella corrodens adherence to human buccal epithelial cells.

The mechanism of Eikenella corrodens adherence to human buccal epithelial cells in vitro was studied. Initial experiments to determine the optimal conditions for adherence of E. corrodens to buccal epithelial cells showed that adherence was dependent on time, temperature, bacterial concentration, and pH. Different strains of E. corrodens varied in their ability to adhere, and strain 1073 showed the greatest ability in adherence. Strain 1073 was selected for studies of adherence mechanisms. Trypsin treatment or heating (100 degrees C, 10 min) of the bacterial cells abolished their capacity to adhere to buccal epithelial cells. Treatment of buccal epithelial cells with trypsin also abolished adherence of E. corrodens 1073, whereas neuraminidase treatment of buccal epithelial cells enhanced the adherence. The adherence was inhibited by ethylenediaminetetraacetic acid and restored by adding Ca2+. The adherence was remarkably inhibited by sugars containing D-galactose and n-acetyl-D-galactosamine. Treatment of neuraminidase-treated epithelial cells with sodium metaperiodate or alpha- and beta-galactosidase did not decrease the adherence. These data suggest that adherence of E. corrodens 1073 to human buccal epithelial cells may require the interaction of lectin-like proteins on the bacterial surface with galactose-like receptors on the surface of epithelial cells.     

Adherence of Neisseria meningitidis to human epithelial cells.

Carrier strains of Neisseria meningitidis are recovered almost solely from the posterior pharynx and they are often nongroupable or rough. Invasive strains can be serogrouped (encapsulated). We studied adherence of both carrier- and patient-derived serogroupable and nongroupable meningococci to buccal epithelial and posterior pharyngeal cells. Fresh meningococcal isolates attached significantly better to pharyngeal than to buccal cells (P = 0.01). Strains that could be serogrouped adhered less than nongroupable strains (P less than 0.05). Meningococci passed in vitro became hemagglutinin negative and nonpiliated. Hemagglutinin-negative meningococci always adhered less to both epithelial cell types than the hemagglutinin-positive variants of the same strain. These results indicate that meningococcal pili probably mediate attachment to oropharyngeal cells, but encapsulation may reduce adherence. Localization of meningococci in the posterior pharynx is in part explained by the receptivity of the epithelial cells in this area for meningococci.     

Adherence of skin bacteria to human epithelial cells.

Aerobic and anaerobic bacteria isolated from human axillae were tested for their capacity to adhere to buccal epithelial cells, immortalized human epithelial (HEp-2) cells, and undifferentiated and differentiated human epithelial cells. In general, both aerobic and anaerobic diphtheroids adhered better to differentiated human epithelial cells than to HEp-2 and undifferentiated human epithelial cells (P less than 0.05). Mannose, galactose, fucose, N-acetyl-D-glucosamine, and fibronectin were also assayed for their capacity to inhibit the adherence of diphtheroids to human epithelial cells. A great deal of variability was observed in the capacity of the latter compounds to inhibit the attachment of aerobic diphtheroids to undifferentiated and differentiated epithelial cells. Overall, mannose appeared to be best at inhibiting the adherence of the aerobic diphtheroids to undifferentiated human epithelial cells. Galactose, fucose, N-acetyl-D-glucosamine, and fibronectin showed a greater capacity to inhibit attachment of aerobic diphtheroids to differentiated than to undifferentiated human epithelial cells. The inhibition of adherence to differentiated human epithelial cells varied with the microorganism and the compound tested; however, the highest and most consistent inhibition of adherence (76.1 to 88.6%) was observed with a 5% solution of N-acetyl-D-glucosamine. The in vitro adherence and adherence inhibition assays presented here demonstrate that a number of adhesins and receptors are involved in the adherence of skin bacteria to human epithelial cells and receptors on human epithelial cells are apparently altered during differentiation.