Candida albicans to Human Epithelial Cells

Transcription

1 INFECTION AND IMMUNITY, JUlY 1989, p /89/ $02.00/0 Copyright C) 1989, American Society for Microbiology Vol. 57, No. 7 Participation of Yeast Cell Surface Hydrophobicity in Adherence of Candida albicans to Human Epithelial Cells KEVIN C. HAZENt Department of Microbiology, University of Southwestern Louisiana, Lafayette, Louisiana Received 12 January 1989/Accepted 19 March 1989 Recent studies have revealed that hydrophobic cells of the opportunistic pathogenic fungus Candida albicans are more virulent than hydrophilic cells. One critical step in the pathogenic process is adherence to host tissues. Adherence of C. albicans to epithelial tissues is mediated primarily by specific adhesin-receptor interactions, but whether cell surface hydrophobicity (CSH) of the yeast cells may also contribute has not been definitively demonstrated. Nineteen isolates of C. albicans were grown in Sabouraud dextrose broth at either 23 or 37 C and tested for CSH by a polystyrene microsphere assay and for the ability to adhere to HeLa cells, a human cervical epitheloid carcinoma cell line. For 13 isolates, growth at 23 C resulted in significantly higher levels of CSH than did growth at 37 C. Three isolates were hydrophobic and two were hydrophilic regardless of growth temperature. One isolate was more hydrophobic after growth at 37 C. Of the isolates that were more hydrophobic after growth at 23 C, 86.5% (11 of 13) were also more adherent to HeLa cells. Growth temperature did not appear to determine adherence ability, as all isolates that did not differ in CSH after growth at either temperature also did not differ in ability to adhere. No correlation (r = 0.44) was obtained between CSH and adherence when the isolates grown at 23 C were evaluated as a group. Higher correlation (r = 0.65) was obtained when the isolates were grown at 37 C. Interestingly, a significantly positive correlation between CSH and adherence was obtained when individual isolates were analyzed. To accomplish this analysis, the isolates were allowed to vary in CSH over time in tissue culture medium without serum, and the corresponding adherence values determined. Only isolates that varied in CSH by greater than 10% were used. Correlation statistical analysis in which the coefficient of determination (r2) was calculated indicated that poor correlation between CSH and adherence for the isolates evaluated as a group was likely due to the fact that CSH had little effect on adherence once a moderately high level of CSH was attained. These results indicate that CSH is involved in adherence but is not the predominant mechanism and that the effect of CSH on adherence is isolate dependent. As with other microbial diseases, establishment of candidiasis requires the initial attachment of the etiologic agent to host tissues (2, 16). Numerous studies demonstrate that adherence of Candida albicans, the most frequent agent of candidiasis, to host tissues is primarily mediated by a mannoprotein adhesin on the yeast cell surface (reviewed in references 16 and 30). Participation of yeast cell surface hydrophobicity (CSH) in facilitating adherence of C. albicans to human tissues has not been extensively studied, yet C. albicans CSH may represent the virulence factor that allows yeast cells, grown at room temperature, to kill mice faster than cells grown at 37 C do (1). CSH has been shown to facilitate attachment of bacterial cells to host tissues (27). Interestingly, certain conditions that influence adherence of C. albicans to epithelial and endothelial tissues have also been shown in separate studies to affect CSH similarly. For instance, room temperature-grown yeast cells are more adherent to vaginal and buccal epithelial cells and are more hydrophobic than are cells grown at 37 C (12, 13, 14, 24, 35). Stationary-phase cells are more adherent and more hydrophobic than are exponential-phase cells (9, 14, 21). Germ tubes elaborated by C. albicans are more adherent than are yeast cells (20, 31, 34, 36). Germ tubes are also highly and invariably hydrophobic regardless of whether the mother cell displays CSH (11, 12). Proteolytic enzyme treatment of C. albicans results in lowered adherence and CSH (24; unpublished results). t Present address: Division of Biological Sciences, University of Montana, Missoula, MT Despite these suggestive observations, the contribution of CSH of C. albicans to adherence to epithelial cells remains unclear. Several studies indicate that CSH is not involved in adherence to vaginal or buccal epithelial cells, but these findings are countered by other results indicating participation of CSH in adherence to these epithelial cells as well as intestinal epithelial cells (17-19, 23, 25, 28). Disagreement among studies in which the investigators directly tested the role of CSH in adherence appears to be due to several factors. In some cases, only one isolate of C. albicans was tested (4, 18, 19, 23). This poses the problem that the single isolate is not representative of the species. When multiple isolates were used, other problems detracted from the results; for instance, yeast cells may have autoaggregated during the assay, only one yeast cell growth medium was used, or the assay used to determine CSH levels did not discriminate between cell populations that differ in CSH (17, 18, 25, 28). The latter problem is particularly associated with hydrophobic interaction chromatography, in which yeast cells may be mechanically trapped within the hydrophobic resin, thus leading to false high s (28; unpublished observations). Another problem that must be considered in studies of the role of CSH in adherence is that expression of CSH by C. albicans is a dynamic process (9). Hydrophobic and hydrophilic cells can switch CSH status within 30 min (9). Dynamic changes in CSH during the performance of an adherence assay could lead to spurious results. Recently, Klotz and Penn (23) reported that adherence of C. albicans to human intestinal epithelial cells is mediated by multiple mechanisms, namely, electrostatic, hydrophobic, 1894

2 VOL. 57, 1989 HYDROPHOBICITY-MEDIATED ADHERENCE OF C. ALBICANS 1895 and specific adhesin-receptor interactions. It is reasonable to expect that the distribution, quantity, and composition of the particular molecules responsible for each mechanism could differ among yeast isolates. For this reason, studies in which the evaluation of the relationship between CSH and adherence is based on values obtained with multiple isolates, all grown under similar conditions, could cause misleading results. In this study, the role of CSH in adherence of C. albicans to human epithelial cells was investigated. Multiple C. albicans isolates and different growth media, growth temperatures, and epithelial cells were used. The relationship of CSH to adherence among isolates and for each isolate varying the CSH were evaluated by correlation statistical methods. The results demonstrated that CSH contributes to adherence of C. albicans to human epithelial cells but is not the primary attachment mechanism. MATERIALS AND METHODS Organisms and growth conditions. A total of 19 isolates of C. albicans were used in this study. One isolate, LGH1095, has been used as the archetype of the species in several of our earlier studies concerning CSH of C. albicans (9, 11-13) was originally obtained from a skin lesion of a patient appearing at the Tulane University Mycology Unit (6). Four isolates, CK2, CK4, CK39, and CK117, were kindly provided by Carol A. Kauffman (Veterans Administration Medical Center, Ann Arbor, Mich.) and were tested within 4 months of receipt. C. albicans isolate B311 was purchased from the American Type Culture Collection, Rockville, Md. (accession no ). s 132-8C, 132- C3, EIO-010, and 031-1E are natural hydrophilic and hydrophobic variants of C. albicans isolate LGH1095. These variants were isolated by a repetitive limiting dilution method as described elsewhere (10). UL200 was cultured from an oral lesion of a patient suffering acquired immunodeficiency syndrome. All other isolates were originally obtained from either oral lesions or routine gynecological specimens of patients at nearby hospitals during the past 1 to 3 years. Stock cultures of all isolates were maintained on Sabouraud dextrose agar slants (Difco Laboratories, Detroit, Mich.) and routinely subcultured onto fresh slants every 1 to 2 months. To prepare cells for experiments, the isolates were grown in Sabouraud dextrose broth (SDB) at either 23 or 37 C with constant aeration by gyrorotary shaking (130 rpm) for 24 h (stationary phase) and subcultured two to four times (13). s 132-8C and 132-C3 were transferred every 48 h when grown at 37 C. In some experiments, glucose (2%)- yeast extract (0.3%)-Bacto-Peptone (1%; Difco) (GYEP) or yeast nitrogen base containing 1% glucose (YNBG) was substituted for SDB. When cells were grown in GYEP, the culture conditions were similar to those for growth in SDB. YNBG, however, required that the cells be grown for 48 h before harvesting for CSH and adherence tests. Under these various culture conditions, all of the C. albicans isolates grew in the yeast form. Human epithelial cells. HeLa cells, derived from a human cervical carcinoma (CCL2; American Type Culture Collection) were maintained as monolayer cultures in 25-cm2 tissue culture flasks (Bellco Glass, Inc., Vineland, N.J.) under 5% CO2 at 37 C. The cells were grown in Dulbecco's modification of Eagle minimal essential medium (DMEM; Flow Laboratories, Inc., McLean, Va.) plus glutamine (0.584 g/liter), sodium bicarbonate (3.7 g/liter), penicillin (100 U/ ml), streptomycin (100,ug/ml), and 5% defined equine serum (Hyclone, Logan, Utah) (herein this medium is referred to as DMEM with serum). The final ph of the medium was 7.2 before filter sterilization. Human intestinal epithelial (HIE) cells (CCL6; American Type Culture Collection) were also maintained as monolayer cultures under 5% CO2 at 37 C. These cells were grown in Eagle MEM with Earle salts plus glutamine, bicarbonate (2 g/liter), penicillin, and streptomycin. Fetal bovine serum (10%; Cell Culture Laboratories, Cleveland, Ohio) was also added to the medium. Human buccal epithelial (HBE) cells were obtained by gently scraping with a sterile swab the inside cheek of a male volunteer. The cells were released from the swab by mild agitation in sterile phosphate-buffered saline (PBS; 0.05 M sodium phosphate, M NaCl [final ph, 7.4]). The cells were washed five times with cold PBS and adjusted to 5 x 105 cells per ml of PBS. Monolayer adherence assays. The monolayer adherence assay of Samaranayake and MacFarlane (32, 33) was used with some modifications. To prepare monolayers for adherence tests, HeLa and HIE cells were detached from tissue culture flasks by trypsinization and suspended in fresh growth medium, and the cell concentration was determined with a hemacytometer. The cell concentration was adjusted to 1 x 105 (HeLa) or 2 x 105 (HIE) cells per ml, and 2-ml portions were placed into tissue culture plates (35 mm2; Falcon; Becton Dickinson Labware, Oxnard, Calif.). The plates were incubated under 5% CO2 at 37 C until confluent monolayers were formed. The cells were replenished with fresh growth medium every 2 to 3 days. Preliminary experiments demonstrated that more than 90% (HeLa) and 85% (HIE) of the cells after detachment with trypsin were viable, as determined by trypan blue exclusion. Yeast cells were prepared for the adherence experiments by first washing stationary-phase cells three times with cold saline (0.145 M NaCl in distilled water). The cell concentration was then determined by direct counts with a hemacytometer. The concentrated washed cell stock (typically more than 2 x 108 cells per ml) was kept at 0 to 4 C until needed (no longer than 1 h). Epithelial cell monolayers were washed three times with warm (37 C) DMEM or Eagle MEM without serum. At least 7 ml of wash medium was used per plate. Yeast cells were then diluted in cold saline to achieve a cell concentration of 2 x 103/ml. At this point, the last wash medium was removed from the epithelial cell monolayers. The yeast cells were then diluted 1:10 into prewarmed (37 C) DMEM or Eagle MEM without serum. The diluted suspension was gently mixed, and 1.0 ml was added to each monolayer (i.e., ca. 2 x 102 yeast cells per plate). The plates were incubated without agitation at 37 C under 5% CO2 for the desired time. The actual inoculum size for the monolayers in each experiment was determined by mixing 0.1 ml of the penultimate yeast cell suspension (ca. 2 x 103 cells per ml) into 2.5 ml of molten brain heart infusion agar (Difco) and pouring the mixture into 35-mm2 tissue culture plates. Once solidified, the pour plates were incubated at 37 C for 24 h (or 48 h for slower growers), and the colonies were enumerated. The inoculum size was based on the average colony number from quintuplicate samples. After incubation, the inoculated monolayers were washed three times with warm tissue culture medium without serum and overlaid with molten brain heart infusion agar. The plates were then incubated at 37 C for 24 or 48 h. The mean number of colonies from quintuplicate samples was determined, and the percent adherence for the isolate was calcu-

3 1896 HAZEN lated on the basis of the inoculum control representing 100% adherence. Preliminary experiments indicated that more than 95% of the cells inoculated onto the monolayers were recoverable in the wash fluid and attached to the monolayers. Also, when yeast cells were added to monolayers and the monolayers were immediately washed, less than 1% of the yeast cell inoculum attached to the epithelial cells, indicating that simple mechanical manipulation was not responsible for apparent yeast cell adherence. Other preliminary studies demonstrated that more than 95% of all attached yeasts gave rise to colonies. HBE cell adherence assay. The buccal cell adherence assay of Centeno et al. (3) was used with some modifications. Equal volumes (0.5 ml) of HBE cells and C. albicans isolate LGH1095 yeast cells (5 x 105 and 1 x 107 cells per ml, respectively, in PBS) were mixed in acid-washed borosilicate glass scintillation vials and incubated at 37 C for 1 h with constant agitation with a wrist-action shaker (1200 of arc, 19 oscillations per min). Nonadherent yeast cells were separated from the HBE cells by filtration (12-p.m-pore-size filter; Nuclepore Corp., Pleasanton, Calif.). Each filter was washed three times with a total of 50 ml of prewarmed (37 C) PBS and air dried at room temperature for 24 h. The filters were then stained with a saturated saffranin solution for 30 s and washed twice with distilled water. By this procedure, yeast cells appear orange against pink epithelial cells. In each assay, five areas of the filter, representing the center and four peripheral quadrants, were counted. The number of yeast cells attached to at least 200 epithelial cells in each sample was determined, and the average from triplicate samples was considered the relative adherence ability of the yeast cell population. Each experiment was repeated at least twice. Assessment of CSH. Yeast CSH was determined with a polystyrene microsphere assay, which has been shown to be effective in assessing CSH of yeast and hyphal cell populations and individual cells (9, 11, 12). The procedure for yeast cells is described in detail elsewhere (9, 12). The glassware in all steps must be either acid washed or washed in 2% Contrad 70 (Curtin Matheson Scientific, Houston, Tex.) and rinsed seven times with deionized water. Yeast cells are washed and suspended in ice-cold sodium phosphate buffer (0.05 M, ph 7.2) to achieve a concentration of 2 x 106 cells per ml. Blue polystyrene latex microspheres having a low surface charge density and either ± or ± 0.010,um in diameter (Serva Fine Biochemicals, Westbury, N.Y.) are diluted from a 10% solids stock to achieve an approximate concentration of 9 x 108 microspheres per ml of cold phosphate buffer. Equal volumes (100 p.l) of cell and microsphere suspensions are placed in test tubes (12 by 75 mm), rapidly equilibrated to room temperature, and rapidly mixed for 30 s on a flat-top vortexer (Maxi-Mix; Thermolyne, Dubuque, Iowa). The percent hydrophobicity of the cell population is determined on the basis of the number of cells from at least 100 cells with three or more attached microspheres. This criterion is based on earlier studies which demonstrated that it corresponds to CSH levels obtained with a modified aqueous-hydrocarbon biphasic assay (12). Subsequent studies using hydrophobic bondbreaking agents such as methylumbelliferone have further demonstrated that microsphere attachment to yeast cells is mediated by hydrophobicity interactions (8; unpublished observations). To determine CSH during time course experiments, a stock yeast cell suspension was prepared in sterile saline as described above for the monolayer adherence assays. The INFECT. IMMUN. same cell suspension was used for the adherence assay and for assessing CSH when the incubation period in each assay was 30 min or less. If the total incubation period in each assay was longer, a fresh, washed yeast cell stock suspension was prepared from the same yeast cell subculture for each assay. Preliminary experiments showed that no change in CSH of yeast cells occurred between 24 and 27 h of growth in SDB and GYEP. Yeast cells were diluted to 5 x 106/ml of cold saline and then diluted 1:10 into the appropriate prewarmed tissue culture medium without serum (total volume was 3.0 ml). After mixing, the suspensions were incubated at 37 C under 5% C02 for the desired time period in order to mimic the conditions used for the adherence assays. After incubation, the suspensions were centrifuged for 30 to 45 s at 1,000 x g and washed twice with cold (4 C) sterile distilled water. The cell pellet was then suspended to 0.5 to 1.0 ml of cold phosphate buffer (i.e., approximately 1.5 x 106 to 3.0 x 106 cells per ml). Equal volumes (100 p.l) of this cell suspension and microspheres were mixed, and CSH was evaluated as described above. By this method, the relative amount of germination could also be evaluated. Statistical analysis. Yeast cells grown at 23 and 37 C were compared for CSH and for adherence ability by a two-tailed Student t test (38). To determine the relationship between CSH and adherence, a commercial computer program (Cricket Graph; Cricket Software, Malvern, Pa.) was used to determine the coefficient of determination (r2) for a simple regression line generated from a given set of CSH and adherence values. The product-moment correlation coefficient (r) was calculated from the coefficient of determination. The interpretative importance of these values is discussed by Ullman (37). Probability values based on the calculated r were determined from the tables provided by Wardlaw (38). RESULTS Effect of incubation time on CSH and adherence to HeLa cell monolayers. Previous studies from our laboratory demonstrated that upon incubation in synthetic defined media, hydrophilic cells become hydrophobic within 1 h (9). Under the conditions used for the HeLa cell monolayer adherence assay, a similar result was obtained with isolates LGH1095 and UMC9385 (Fig. 1) and most of the other isolates used in this study (not shown). Hydrophilic cells, however, generally did not manifest increased levels of CSH until 30 min into incubation at 37 C. With prolonged incubation, adherence by hydrophilic and hydrophobic cells to HeLa cell monolayers increased (Fig. 1). At the initial time points when the CSH levels between 23 C- and 37 C-grown cells of an isolate were significantly different, the corresponding level of adherence was also significantly different. This trend was less apparent at the later time points, when the difference in CSH between 23 Cand 37 C-grown cells was less pronounced. These data indicate that incubation of yeast cells with monolayers for 15 min is the appropriate period to use for comparing hydrophobic and hydrophilic cells for adherence capabilities without the potentially confusing effect of dynamic changes in CSH. Unless otherwise indicated, this incubation period was used in subsequent experiments. Effect of growth temperature on CSH and adherence. Fourteen isolates of C. albicans that displayed significantly different CSH levels when grown at 23 and 37 C were assessed for adherence to HeLa cell monolayers. Three of the isolates (132-8C, 132-C3, and 031-1E) were derived from the parent isolate LGH1095. Of the 14 isolates, 13 were more

4 VOL. 57, 1989 HYDROPHOBICITY-MEDIATED ADHERENCE OF C. ALBICANS 1897 a) C.) a) C.) L. C) B Time (min) FIG. 1. Effect of incubation time on adherence (solid symbols) and expression of CSH (open symbols) of initially hydrophobic, 23 C-grown (squares) and initially hydrophilic, 37 C-grown (circles) cells of C. albicans isolates LGH1095 (A) and UMC9385 (B). Yeast cells were incubated in DMEM without serum and exposed to HeLa cell monolayers for the adherence assay. Changes in CSH in DMEM without serum were determined as described in Materials and Methods. Each point represent the mean from at least three experiments, each having either quadruplicate or quintuplicate samples. The standard deviation for each point does not exceed 8%. hydrophobic when grown at 23 C than when grown at 37 C (Table 1). Of these 13 isolates of yeast cells obtained by growth at 23 C, 11 (84.6%) were significantly more adherent than 37 C-grown cells to the HeLa cell monolayers (Table 1). Yeast cells grown at 23 and 37 C of the other two isolates (CK117 and 031-lE) appeared to adhere at similar levels (Table 1). Similar to the results obtained in the time course experiments (Fig. 1), when CSH levels of a particular isolate grown at both temperatures were relatively high (>40%), the corresponding adherence levels tended to be similar (e.g., isolates 031-1E, CK39, and UL100 in Table 1). One isolate (CK39) was less hydrophobic after growth at 23 C than when grown at 37 C. Interestingly, the CSH levels were high (>80%), and the adherence levels were not significantly different. Yeast cells from five C. albicans isolates previously grown at 23 and 37 C were equally hydrophobic by 15 min in DMEM at 37 C (Table 2). The 23 C- and 37 C-grown yeast cells for each isolate also adhered at similar levels to HeLa cell monolayers (Table 2). Whether the cells were hydrophobic or relatively hydrophilic did not appear to influence the result. These data indicate that growth temperature is not responsible for the differences seen in adherence levels for isolates that vary in CSH when grown at the two temperatures. Inter- and intraisolate comparisons between CSH and adherence to HeLa cells. CSH and adherence levels for each isolate at 15 min into incubation were analyzed by correlation statistical methods to determine whether these param- TABLE 1. Effect of CSH on adherence of C. albicans to HeLa cells for isolates that had different levels of CSH when grown at 23 and 370C' % Adherence LGH (0.6)b 8.9 (1.5) 28.0 (4.8) 9.6 (4.7) UMC (80)b 1.0 (0.7) 14.9 (5.2)b 0.8 (0.2) LGH (2.4)b 1.3 (1.0) 17.2 (0.3)b 6.1 (1.6) UMC (8.8)b 3.6 (2.4) 23.2 (1.5)b 8.2 (2.2) UMC (9.5)' 8.3 (1.7) 16.7 (6.8)c 3.7 (1.1) 132-C3d 48.7 (6.5)b 10.1 (3.6) 22.7 (2.2)b 13.5 (1.5) UL (3.9)b 0.5 (0.5) 18.2 (8.2)c 8.6 (1.7) 132-8Cd 97.6 (1.9)e 34.1 (17) 21.6 (3.1)b 7.7 (1.8) CK (0.1)e 79.4 (7.3) 49.0 (10.1)' 28.6 (1.0) LGH (0.3)b 0.6 (0.1) 24.8 (6.3)' 4.2 (1.6) UL (1.4)e 55.0 (12.0) 19.4 (1.8)b 8.1 (1.2) CK (0.2)b 3.6 (1.8) 33.2 (3.2) 31.3 (11.7) 031-1Ed 99.4 (0.4)b 89.4 (0.8) 27.5 (10.0) 19.0 (5.4) CK (3.3)e 100 (0) 38.9 (16.8) 31.0 (5.6) " Values correspond to the levels obtained at 15 min into the adherence assay period and represent the means (standard deviations of the populations) from at least three experiments, each run with quintuplicate samples. b p < 0.01 (two-tailed Student t test). P < 0.05 (two-tailed Student t test). "Derived from C. albicans isolate LGH1095. P < 0.02 (two-tailed Student t test). eters are related. If the combined data for 23 C- and 37 Cgrown cells are used, a positive correlation is obtained (r = 0.67, P < 0.001). On the basis of the data for individual temperatures, a positive correlation was obtained for cells grown at 37 C (r = 0.65, P < 0.05) but not for cells grown at 23 C (r = 0.44, P > 0.05). The latter result may be due to the fact that a high percentage (84.7%) of isolates were highly hydrophobic (s of greater than 75%), as the correlation-of-determination value was low (r2 = 0.19). For 11 of 12 isolates (91.7%) that diplayed varying levels of CSH (at least a 10% range) during the 60-min adherence assay, a significant correlation between CSH and adherence was obtained (Table 3). Growth temperature did not appear to affect the CSH-adherence relationship for individual isolates, since both 23 C- and 37 C-grown cells displayed significant correlations (Table 3). In this analysis, a greater than 10% range was used as the criterion to assess the relationship between CSH and adherence because the mean maximum and minimum s for isolates displaying CSH variability of 10% or less during the 60-min incubation period were not significantly different (as determined by the Student t test). TABLE 2. Effect of CSH on adherence of C. albicans to HeLa cells for isolates that had similar levels of CSH after growth for 15 min in SDB at 23 and 37 C" % Adherence Hydrophobic B (2.9) 90.5 (2.5) 44.2 (11.4) 18.2 (6.6) E10-O10b 93.0 (6.4) 96.8 (1.4) 26.9 (10.8) 25.3 (3.7) CK (0.1) 98.5 (1.7) 26.7 (5.0) 23.7 (1.6) Hydrophilic (20.6) 1.3 (1.8) 24.1 (4.1) 18.4 (3.9) UMC (0.2) 3.1 (1.5) 4.8 (1.2) 7.4 (3.0) " Values represent means (standard deviations of the populations) of at least three experiments, each run with quintuplicate samples. Values for all pairs were not significant by a two-tailed Student t test. bderived from C. albicans isolate LGH1095.

5 1898 HAZEN TABLE 3. Correlation statistical analysis of CSH and adherence values of each isolate of C. albicans that displayed at least a 10% change in CSH during the 60-min HeLa cell adherence assay Growth Range" temp CSH I value ( C) value Adherence < C < UMC < UMC < < UMC < UL < LGH < UMC < LGH < UMC < C NSb 0.66 a Based on the respective means obtained at 15, 30, 45, and 60 min for each isolate from at least three experiments, each having quintuplicate samples. b NS, Not significant (P > 0.1). Effect of growth medium on CSH and adherence. Three isolates of C. albicans were grown to stationary phase in GYEP and YNBG broths, and their CSH and adherence levels were determined. For these experiments, 15-min values were used and HeLa cell monolayers were the adherence targets. Two isolates (LGH1095 and LGH870) displayed significantly different levels in CSH and adherence after growth at 23 and 37 C regardless of the growth medium (Table 4). The CSH levels of the yeast cell populations of the third isolate did not differ regardless of growth medium, and the corresponding adherence levels were also not significantly different. These results indicate that growth medium was not responsible for the positive correlation between CSH and adherence obtained with cells grown in SDB. CSH and adherence of C. albicans to HIE and HBE cells. The influence of CSH on adherence of four C. albicans isolates to HIE cells was determined. All four isolates displayed high levels of hydrophobicity when grown at 23 C in SDB and low levels when grown at 37 C (Table 5). The hydrophobic cells for each isolate adhered more than the hydrophilic cells did. C. albicans isolate LGH1095 was also tested for the effect of CSH on adherence to HBE cells. After growth in SDB, the CSH levels of 23 C- and 37 C-grown yeast cells were significantly different (97.1 ± 0.9 versus 1.5 ± 0.8%, respectively; P < 0.001). Their adherence levels were also significantly different (491 ± 38 versus 29 ± 7 yeast cells, respec- TABLE 5. CSH and adherence to HIE cells of C. albic(ans yeast cells grown at 23 and 37 C" % Adherence LGH (0.8)" 4.0 (3.7) 34.1 (2.6)' 16.1 (4.7) LGH (0)" 14.0 (2.1) 41.0 (4.7)" 25.2 (1.6) (1.1)" 0.5 (0.4) 41.8 (2.5) 8.0 (3.5) UL (0.3) 1.8 (0.5) 37.5 (6.9)' 14.6 (5.0) " All isolates were grown in SDB as described in Materials and Methods. Values were obtained after incubation for 15 min in Eagle MEM and represent the arithmetic mean (standard deviation) from at least two experiments, each having quadruplicate samples. "P < 0.01 (two-tailed Student t test). P < 0.02 (two-tailed StLudent t test). "P < 0.05 (two-tailed Student t test). tively, per 200 HBE cells; P < 0.001). Coadherence was, however, especially apparent for the 23 C-grown cells, possibly because of the high concentration of hydrophobic cells used in the assay. DISCUSSION INFECT. IMMUN. Evaluation of the role of yeast CSH in adherence of C. albicans to human epithelial cells is complicated by the participation of several mechanisms (e.g., ionic and van der Waals) that can strengthen or weaken the attraction between cell surfaces (22, 23). The predominant and strongest mechanism for adherence appears to involve adhesin-receptor interactions in which the C. albicans adhesin is a mannoprotein (5, 16, 22, 24). Hydrophobic interactions are believed to contribute to adherence by maintaining the fidelity of the adhesin-receptor bonds (2, 29, 30). Hydrophobic forces are, however, long-range, reversible forces, and adhesinreceptor bonds are short range (15, 27). Thus, hydrophobic interactions can significantly increase the number of successful contacts between two surfaces. One difficulty in assessing how CSH affects adherence is that expression of surface hydrophobicity by C. albicans can be dynamic (9). This phenomenon was particularly evident in the study described here, as initially hydrophilic cells of various isolates became more than 80% hydrophobic within 1 h in incubation in the synthetic media used in monolayer adherence assays (9; Fig. 1). Because of this problem, CSH and adherence of the C. albicans isolates used in this study were compared on the basis of the values obtained at 15 min of incubation. Intra- and interisolate comparisons of CSH and adherence were conducted to limit the potential equivocation that could Medium TABLE 4. Effect of yeast cell growth medium on adherence and CSH of cells grown at 23 and 370C" %/ Adherence GYEP LGH (0.5)* 5.1 (0.9) 37.3 (4.7)' 20.4 (3.4) LGH (0)' 49.7 (26.7) 43.1 (0.3)" 35.1 (1.9) CK2 100 (0). NS" 99.2 (0.2) 32.4 (4.5). NS 31.5 (5.7) YNBG LGH (0.4)" 20.7 (8.9) 39.7 (11.5)" 14.5 (4.6) LGH (0.8)" 28.5 (6.4) 54.7 (2.0)" 18.6 (2.1) CK (1.0), NS 100 (0) 38.1 (10.6), NS 32.6 (9.1) a Values represent the means (standard deviations of the populations) from three experiments, each run with at least quadruiplicate samples. b p < 0.01 (two-tailed Student t test). P < 0.02 (two-tailed Student t test). d NS, Not significant (two-tailed Student t "P < 0.05 (two-tailed Student t test). test).

6 VOL. 57, 1989 HYDROPHOBICITY-MEDIATED ADHERENCE OF C. ALBICANS 1899 occur because of (i) the participation of multiple mechanisms contributing to adherence and (ii) dynamic changes in CSH. The results revealed that, in most cases, for individual isolates differing in CSH after growth in SDB at 23 and 37 C, the more hydrophobic cells adhered to a greater extent than did hydrophilic cells to HeLa monolayers (Table 1). This finding was not attributable to growth temperature, as yeast cells of several isolates had similar CSH and adherence values after growth at the two temperatures (Table 2). Growth temperature did appear to influence the degree of correlation between CSH and adherence when yeast cells grown at 23 and 37 C of all isolates were compared as a single population. Although a good correlation was obtained (r = 0.67), the correlation for just 23 C-grown cells was poor (r = 0.44) but was good for 37 C-grown cells (r = 0.65). The difference in correlation between CSH and adherence for cells grown at the two temperatures appears to be due to the high degree of variability in adherence obtained with 23 Cgrown isolates, as many of these were already highly hydrophobic. This observation suggests that CSH and adherence behave as independent variables once a high level of yeast CSH is attained. This interpretation is supported by the low coefficient-of-determination value (r2 = 0.19) that is used when the degree of correlation between an independent variable (e.g., CSH) and a dependent variable (adherence) is desired (37). (The product-moment correlation coefficient [r-] is used when both variables are independent, which is. in fact, the situation in the experiments described here.) A similar argument could explain the poor relationship between CSH and adherence when the comparisons are restricted to isolates that express similar levels of hydrophobicity or to a single isolate that is hydrophilic under multiple growth conditions (18, 28). Better correlation between adherence and CSH was obtained when the analyses were limited to individual isolates (Table 3). Interestingly, a linear relationship between adherence and CSH was noted in many cases (as determined by the high r value). In these analyses, varying s and the corresponding adherence values were obtained by allowing the yeast cells of an isolate to incubate for different time periods in test medium as opposed to growing cells in various media, as has been done by others (17). By using the same medium to obtain different levels of CSH, the influence of medium effects on adhesin expression could be minimized. In a further attempt to compensate for the influence of multiple mechanisms in adherence and allow the effect of CSH alone to be evaluated, several natural variants of C. (albicans isolate LGH1095 were tested. The criterion for choosing a particular variant was that the variant must display small differences in CSH when grown at 23 and 37 C. The supposition for this analysis is that these variants would display the least difference in surface composition in terms of the molecules not involved in CSH but possibly involved in adherence (adhesins) while having a difference in the surface molecules that determine CSH. This supposition was not tested here. Correlation analyses revealed that adherence was correlated to CSH but not strongly (r = 0.805). Growth temperature influenced the degree of correlation. The 23 C-grown cells were less likely to show a relationship between CSH and adherence (r = for 23 C-grown cells; r = 0.81 for 37 C-grown cells). Here, again, once a relatively high level of CSH is obtained (>40%), the influence of CSH on adherence is not linearly related. These results suggest that CSH contributes to adherence but is not the primary mechanism. This suggestion is supported by recent experiments in which tunicamycin-treated cells are highly hydrophobic but are poorly adherent (7; unpublished observations). Tunicamycin apparently inhibits mannosylation of the protein moiety of the adhesion during intracellular processing (26). Coadhesion has been suggested to contribute to the high level of adhesion seen with hydrophobic cells (18, 23). This mechanism of attachment appears to be particularly operative in buccal epithelial cell studies in which a high concentration of yeast cells is used (21, 25). Although coadhesion may explain the high levels of adherence of hydrophobic yeast cells to HBE cells, this mechanism did not appear to be involved in the monolayer assays in which colony counts were used. If hydrophobic cells coadhered, then fewer colonies would be expected from these cells than from hydrophilic cells. This was not observed. Preliminary microscopic examinations indicated that coadherence did not occur under the conditions of the assay, possibly because a low concentration of yeast cells (2 x 102 cells per ml) was used. Taken together, these results demonstrate that CSH is involved in adherence of most isolates of C. albic-ans to human epithelial cells. Hydrophobic interactions are not the only mechanisms mediating adherence that must be considered, although they can represent the predominant force during initial contact between two surfaces (15, 27). Adhesin-receptor interactions, which involve dipole-dipole and hydrogen bonds, and electrostatic forces are also important and may predominate (16, 23). Which adherence mechanism is operative is dependent on the target host tissue, the surface composition of the yeast cell, and the surface molecules that are exposed. This study demonstrates that the macromolecules available for adherence can change within 30 min. This observation suggests that hydrophilic, endogenous C. albiccans can become hydrophobic rapidly under appropriate stimulation and thus possibly more likely to cause disease. With the demonstrated importance of CSH in adherence, the range of pathogenic events affected by CSH during development of candidiasis becomes greater. Previous investigations have demonstrated that CSH affects yeastto-hypha conversion and avoidance of polymorphonucleated neutrophil killing (1, 9, 11, 12). CSH is also involved in attachment of C. albicans to plastics, which is believed to explain how plastic medical devices can serve as the nidus for disseminated candidiasis (22, 30). These individual pathogenic events are not mutually exclusive, as germinated cells have been shown to adhere better than yeast cells to epithelial cells (20, 36). During the experiments described here, initially hydrophobic cells were observed to produce germ tubes sooner (typically by no earlier than 45 min) and in greater abundance than did the corresponding initially hydrophilic cells (data not shown). This observation agrees with our previous results concerning dynamic expression of CSH (9). Interestingly, germinated cells are hydrophobic regardless of the hydrophobicity status of the mother cell (11, 12). Whether the same hydrophobic molecules of germ tubes are expressed by yeast cells and are involved in adherence has not yet been determined. ACKNOWLEDGMENTS This work was supported in part by Public Health Service grant A from the National Institute of Allergy and Infectious Diseases and by a grant from the American Diabetes Association- Louisiana Affiliate.

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