Influence of growth conditions on cell surface hydrophobicity of ...

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Jun 25, 1986 - minimum essential medium with Earle salts (no. 11-110-22;. Flow Laboratories .... King, R. D., J. C. Lee, and A. L. Morris. 1980. Adherenceof ... Rotrosen, D., R. A. Calderone, and J. E. Edwards, Jr. 1986. Adherence of Candida ...

INFECTION AND IMMUNITY, OCt. 1986, p. 269-271

Vol. 54, No. 1

0019-9567/86/100269-03$02.00/0 Copyright © 1986, American Society for Microbiology

Influence of Growth Conditions on Cell Surface Hydrophobicity of Candida albicans and Candida glabrata KEVIN C. HAZEN,l2*



Department of Microbiology' and Acadiana Medical Research Fouindation,2 University of Southwestern Louisiana, Lafayette, Louisiana 70504-1007 Received 28 April 1986/Accepted 25 June 1986

The effect of cultural conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata was tested. C. albicans cells grown at room temperature were more hydrophobic than cells grown at 37°C. No consistent pattern was observed with C. glabrata. Relative hydrophobicity was found to vary with the growth phase and growth medium for both species. The implications for pathogenesis studies are discussed.

The initial steps of microbial pathogenesis require that an etiologic agent adhere to an appropriate host surface. Candida species, which cause mild to life-threatening disease in immunocompromised individuals, are no exception. For more than a decade, the adherence properties of Candida species to various surfaces, especially vaginal and buccal epithelial cells, have been the subject of numerous investigations. One goal of this research is to elucidate the binding molecules on the Candida cell surface in order to develop an effective vaccine against candidiasis. Based on experiments using vaginal and buccal epithelial cells as well as fibrin-platelet matrices as adherence targets, several investigators have suggested that one adhesin on the Candida albicans cell surface is a glycoprotein, possibly a mannoprotein (6, 8-10, 13, 15, 17). Little attention, however, has been directed at general cell surface properties, such as hydrophobicity, which could affect adherence. Recently, it was reported that Candida species display variable degrees of cell surface hydrophobicity (CSH) (5). Adherence to plastic was found to be correlated with CSH (5). Unfortunately, that study involved only three strains of C. albicans. Further, only stationary-phase cells grown at 26°C in a single medium were tested. Here, we report that culture conditions demonstrably affect the relative CSH of C. albicans and C. glabrata. A total of 19 isolates of C. albicans and 4 isolates of C. glabrata were tested. Of these, 16 C. albicans isolates and all C. glabrata isolates were obtained as fresh specimens from patients of nearby hospitals less than 2 months before the experiments. The C. albicans isolates designated B-311 (a gift from J. E. Cutler, Montana State University, Bozeman, Mont.) and UL-100 had been maintained in culture collections for over 3 years. Isolate Y-15 has an undetermined history. All organisms were maintained on Sabouraud dextrose (Emmons modified) slopes (Difco Laboratories, Detroit, Mich.) at 4°C and subcultured once a month. Confirmations of identification were determined by the germ tube test and a commercial sugar assimilation kit (API 20C; Analytab Products, Plainview, N.Y.) (14). To prepare cells for experiments, cells were subcultured three times at 24-h intervals in test medium. A modification of the qualitative water-hydrocarbon biphasic assay of Rosenberg et al. (12; for a review, see reference 11) was used to determine the relative CSH. Cells were washed with ice-cold, sterile distilled water and suspended in phosphate-urea-MgSO4 buffer *

(PUM buffer [12]) to an A600 of 0.400 (+0.010). This suspension served as the control. Portions (1.2 ml) of the suspension were placed in acid-washed glass tubes (13 by 75 mm), and each was overlaid with 0.3 ml of hydrocarbon. The phases were mixed by vortexing for 3 min. Once the phases separated, the A600 of the aqueous bottom layer (designated the treated sample) was immediately determined. The relative CSH was obtained by the following equation: % change in A6w = [(A6m of controls - A600 of treated cells)/(A600 of controls)] x 100. Preliminary experiments indicated that more than one cell wash did not significantly affect results. Also, in agreement with Klotz et al. (5), similar trends were seen with all eight hydrocarbons tested (octane, 2,2,4-trimethylpentane, cyclohexane, hexane, hexadecane, dodecenes, heptene, and xylene). Therefore, tests were restricted to octane, 2,2,4trimethylpentane, and cyclohexane. The CSH of C. albicans LGH1095 cells was found to be strongly influenced by the growth temperature. When grown in a broth medium composed of 2% (wt/vol) glucose (Mallinckrodt, Inc., St. Louis, Mo.), 0.3% (wt/vol) yeast extract (BBL Microbiology Systems, Cockeysville, Md.), and 1% (wt/vol) Bacto-Peptone (Difco) (GYEP), cells of isolate LGH1095 at room temperature (ca. 25°C) were more hydrophobic than those at 37°C (Fig. 1). Furthermore, the growth phase also influenced CSH. Exponential-phase cell CSH was significantly lower (P < 0.05 by a two-tailed Student t test) (Fig. 1, 6 to 12 h) than that of cells at the stationary phase at 25°C. Similar results were obtained for nine other isolates for which growth curves were run (these isolates are designated LGH490, LGH581, UL-19, UL-BH, Y-15, UMC9385, UMC1816, UMC1726, and UMC2746). One isolate designated UL-100 was consistently highly hydrophobic at both growth temperatures and all growth phases (Fig. 1). On the other hand, C. albicans B-311 displayed moderate hydrophobicity (percent change in A600, 30) at the stationary phase and was substantially hydrophilic (percent change in A6N, 0) at the exponential phase when gr*wn at room temperature and at any growth phase when grown at 37°C. Based on results with C. albicans, isolates, UL-100 and 1B-311 represented the most extreme cases we tested. Soll and Bedell (16) reported that C. albicans growth in the medium of Lee et al. (7) is characterized by altered morphology. Early exponential-phase CFU are typically composed of contiguous multicellular units. As growth continues, the number of spheres (buds plus mother cell) per contiguous unit decreases. To determine whether the number of spheres

Corresponding author. 269





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Time (hours) FIG. 1. Effect of growth temperature and growth phase on CSH of C. albicans LGH1095 and UL-100. Cells were grown in GYEP. At various times during growth, their relative CSH was determined with 2,2,4-trimethylpentane. The limit of variation (standard error of the mean) for all points did not exceed 5%. The differences in percent change in AW for LGH1095 cells grown at 25 versus 37°C at corresponding times- are statistically significant (P < 0.025, twotailed Student t test). The results are representative of six experiments. Exponential phase at both temperatures occurs between 6 and 12 h. per contiguous unit influences the measured hydrophobicity, we grew cells as before but at each time point the number of spheres per contiguous unit was determined. No clear tendency was seen. For instance, when the ratio equalled 1.8 for cells grown at both temperatures in one experiment, the relative hydrophobicity of cells grown at room temperature was 57%, but for cells grown at 37°C the value was 9%.

These results indicate that the differences in hydrophobicity are not due to cellular morphology but instead to cell surface properties. The effect of growth medium on CSH was also tested. The following four broth media were used: GYEP; Emmons modified Sabouraud dextrose broth (SDB); yeast-nitrogen base (Difco) supplemented with 0.5% mannose (Sigma Chemical Co., St. Louis, Mo.) (YNBM); and Auto-Pow minimum essential medium with Earle salts (no. 11-110-22; Flow Laboratories, Inc., McLean, Va.) supplemented with biotin (250 ,g/liter), glucose (9 g/liter), glycine (1 g/liter), and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (96 g/liter) and adjusted to pH 6.8 with sodium hydroxide (AP-MEM). Overall, only slight differences were obtained with SDB, YNBM, and GYEP, but cells of isolates Y-15 and LGH1095 exhibited greater CSH when grown in AP-MEM than in other fhedia (Fig. 2). In AP-MEM, a low percentage of cells had germ tubes at 24 h. Hence, the change in CSH in AP-MEM may be related to cell surface compositional changes that occur during form transition. C. albicans UL-100 was highly hydrophobic at both temperatures when grown in GYEP, SDB, and YNBM but had decreased CSH in AP-MEM at 37°C (Fig. 2). When CSHs of 18 isolates of C. albicans were compared, a definite pattern emerged. All isolates were less hydrophobic when grown at 37°C than when grown at room temper-

ature (Table 1, designated numbers 1 to 18). Only one isolate, UL-100, was substantially hydrophobic when grown at 37°C. Cells grown at room temperature varied in CSH. However, 14 of 18 strains had a CSH of greater than 60% when tested with 2,2,4-trimethylpentane. In contrast, the four isolates of C. glabrata had no common pattern (Table 1, designated numbers 19 to 22). Two isolates, UMC960 and UMC2716 (Table 1, numbers 19 and 22), behaved similarly. Their CSH patterns relative to growth temperature were opposite those measured for C. albicans (i.e., hydrophobic at 37°C and moderately hydrophobic at 25°C). The CSH pattern of isolate UMC1585 was like that of most isolates of C. albicans. Isolate UMC1615 had a CSH pattern similar to that of C. albicans UL-100. These results clearly demonstrate that environmental factors and growth phase can variably affect the relative CSH of Candida species. The significance of these results are uncertain given that the epidemiology of Candida infections is not well established (18). However, altered hydrophobicity may influence the establishment of infection. We found that C. albicans grown at room temperature displays faster kinetics of binding to fibrin-platelet matrices (8; unpublished results). Furthermore, the effect of the growth temperature and growth phase on CSH may explain earlier observations that adherence of C. albicans to vaginal epithelial cells is greater for cells grown at room temperature than for cells grown at 37°C and greater for stationary-phase cells than for exponential-phase cells (4, 6). Using mnonoclonal antibodies, Brawner and Cutler (1-3) demonstrated that C. albicans surface antigenic determinants vary with cultural conditions and morphology. Taken together with our results, these observations indicate that the cell surface of Candida species is dynamic. For this reason, investigators studying pathogenic and physiologic mechanisms of Candida species must consider their procedures for preparing cells for assays. We found, for instance,


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a A



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A BCD A B C D medium FIG. 2. Effect of culture medium on relative CSH of several isolates of C. albicans. All isolates were tested after 24 h of growth in the indicated medium as described in the text. The media that were tested include GYEP (A), SDB (B), YNBM (C), and AP-MEM (D). Symbols: 0, LGH1095; A, UL-100; *, Y-15. Each point represents the mean of at least three experiments each with three replicates. The limit of variation of each point did not exceed 5% (i.e., standard error of the mean).


VOL. 54, 1986


TABLE 1. Effect of growth temperature on relative CSH of various isolates of C. albicans and C. glabrata Change from control. A600 (range, %)


Isolates reacting with hydrocarbona: Octane RT 37°C 1-3, 9, 12-18, 20, 21 17, 19, 21 22


37°C High (80-100) 1-4, 9, 12-18, 20, 21 17, 19, 21-22 1, 9, 12-18, 20, 21 17, 19, 21 Medium (20-80) 5-8, 10, 11, 19, 22 4-8, 10, 11, 19, 22 2-8, 10, 11, 19, 22 22 Low (0-20) 1-16, 18, 20 1-16, 18, 20 1-16, 18, 20 a The category chosen is based on the mean of three experiments. Limit of variation was 5% (standard error of the mean). C. albicans isolates: 1, UMC1743; 2, UMC1118; 3, UMC1112; 4, LGH86; 5, UMC1529; 6, LGH285; 7, LGH870; 8, UMC1726; 9, Y-BH; 10, UMC2746; 11, UMC1816; 12, UMC9385; 13, LGH581; 14, RT



Y-15; 15, LGH490; 16, MSU19; 17, UL-100; 18, LGH1095. C. glabrata isolates: 19, LGH960; 20, UMC1585; 21, UMC1615; 22, UMC2746. Cells were grown at room temperature (RT) (23 to 250C) in GYEP.

that the ability of C. albicans cells to develop germ tubes and their susceptibility to environmental perturbations are influenced by the growth temperature and growth phase (K. C. Hazen and B. W. Hazen, submitted for publication). Hence, comparisons of data obtained in vitro with cells prepared under different conditions with pathogenic events must be made cautiously. This work was supported in part by a grant from the Louisiana affiliate of the American Heart Association. LITERATURE CITED 1. Brawner, D. L., and J. E. Cutler. 1984. Variability in expression of a cell surface determinant on Candida albicans as evidenced by an agglutinating monoclonal antibody. Infect. Immun. 43:966-972. 2. Brawner, D. L., and J. E. Cutler. 1986. Ultrastructural and biochemical studies of two dynamically expressed cell surface determinants on Candida albicans. Infect. Immun. 51:327-336. 3. Brawner, D. L., and J. E. Cutler. 1986. Variability in expression of cell surface antigens of Candida albicans during morphogenesis. Infect. Immun. 51:337-343. 4. King, R. D., J. C. Lee, and A. L. Morris. 1980. Adherence of Candida albicans and other Candida species to mucosal epithelial cells. Infect. Immun. 27:667-674. 5. Klotz, S. A., D. J. Drutz, and J. E. Zajic. 1985. Factors governing adherence of Candida species to plastic surfaces. Infect. Immun. 50:97-101. 6. Lee, J. C., and R. D. King. 1983. Characterization of Candida albicans adherence to human vaginal epithelial cells in vitro. Infect. Immun. 41:1024-1030. 7. Lee, K. L., H. R. Buckley, and C. C. Campbell. 1975. An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida albicans. J. Gen. Microbiol. 108:173-180.

8. Maisch, P. A., and R. A. Calderone. 1980. Adherence of Candida albicans to a fibrin-platelet matrix formed in vitro. Infect. Immun. 27:650-656. 9. Makrides, H. C., and T. W. MacFarlane. 1983. An investigation of the factors involved in increased adherence of C. albicans to epithelial cells mediated by E. coli. Microbios 38:177-185. 10. McCourtie, J., and L. J. Douglas. 1984. Relationship between cell surface composition, adherence, and virulence of Candida albicans. Infect. Immun. 45:6-12. 11. Rosenberg, M. 1984. Bacterial adherence to hydrocarbons: a useful technique for studying cell surface hydrophobicity. FEMS Microbiol. Lett. 22:289-295. 12. Rosenberg, M., D. Gutnick, and E. Rosenberg. 1980. Adherence of bacteria to hydrocarbons: a simple method from measuring cell-surface hydrophobicity. FEMS Microbiol. Lett. 9:29-33. 13. Rotrosen, D., R. A. Calderone, and J. E. Edwards, Jr. 1986. Adherence of Candida species to host tissues and plastic surfaces. Rev. Infect. Dis. 8:73-85. 14. Silva-Hutner, M., and B. H. Cooper. 1974. Medically important yeasts, p. 491-507. In E. H. Lennette, E. H. Spaulding, and J. R. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 15. Sobel, J. D., P. G. Meyers, D. Kaye, and M. E. Levison. 1981. Adherence of Candida albicans to human vaginal and buccal epithelial cells. J. Infect. Dis. 143:76-82. 16. Soll, D. R., and G. W. Bedell. 1978. Bud formation and the inducibility of pseudo-mycelium outgrowth during release from stationary phase in Candida albicans. J. Gen. Microbiol. 108:173-180. 17. Tronchin, G., D. Poulain, and A. Vernes. 1984. Cytochemical and ultrastructural studies of Candida albicans. III. Evidence for modifications of the cell wall coat during adherence to human buccal epithelial cells. Arch. Microbiol. 139:221-224. 18. Wade, J. C., and S. C. Schimpff. 1985. Epidemiology and prevention of Candida infections, p. 111-134. In G. P. Bodey and V. Fainstein (ed.), Candidiasis. Raven Press, New York.

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