The adhesion of the yeast Candida albicans to epithelial cells of human origin in vitro

?,ci,t wof Bioi Voi 26. pp. 81 to 820. 19X1 MX)3-9969/Rl,l008lS-06502.00/0 Pnnted I” Gredt Britain. All rights reserved Copyright 0 1981 Pergamon Prec* Ltd

THE ADHESION OF THE YEAST CANDIDA ALBICANS TO EPITHELIAL CELLS OF HUMAN ORIGIN IN VITRO

L. P. SAMARANAYAKE and T. W. MACFARLANE

Microbiology Unit, Department of Oral Medicine and Pathology, Glasgow Dental Hospital and School. 378 Sauchiehall Street. Glasgow. G2 332, Scotland, U.K.

Summary-Monolayers of HeLa cells were incubated with Cundida suspensions and the number of adherent yeasts per unit area of epithelial cells counted microscopically. Adhesion was proportional to the incubation time and the yeast concentration in the suspension. Pre- incubation of Candidu with sucrose facilitated their adhesion, there being a linear enhancement with increasing sucrose concentrations. The sucrose-facilitated adhesion disappeared when the yeasts were killed prior to incubation in a sucrose-containing medium, suggesting that an extracellular metabolic product could be responsible. On comparison of the adhesion of yeasts to viable and non-viable HeLa cells, greater adhesion to non-viable cells was observed. This experimental system can be used to study parameters influencing candidal adhesion to epithe- ha1 surfaces.

INTRODUCTION

Successful colonization and infection of host tissues by microorganisms depend upon an ability to adhere to host surfaces (Gibbons and van Houte, 1975; Jones, 1977). Although adhesion of bacteria to cell surfaces has been extensively investigated, there are few studies dealing with the adhesion of yeasts to epithelial surfaces. Liljemark and Gibbons (1973) used an optical method to count Cundidu albicuns attached to murine tongue and cheek cells and a similar method was described by Kimura and Pearsall (1978) for human buccal epithelial cells and by King. Lee and Morris (1980) for vaginal epithelial cells. How- ever. the use of such cells may introduce irregularities into the system as desquamated epithelial cells are likely to be contaminated with bacteria and salivary components to varying degrees (Varian and Cooke, 1980).

In order to overcome these problems, we have designed an in oitro technique to evaluate quantitat- ively the adhesion of C. ulbicuns to monolayers of human epithelial cell preparations.

MATERIALS AND METHODS

Prepurution of HeLu monolugws

HeLa cells were maintained in tissue culture flasks as monolayers in Glasgow’s modification of Eagle’s minimum essential medium (Gibco Bio-cult, Glasgow, U.K.) supplemented with 10 per cent (v/v) fetal calf serum (Gibco Bio-Cult), 0.24 mM sodium bicarbo- nate, 2 mM L-glutamine and 100 pg of penicillin and streptomycin/ml (Grist, Ross and Bell. 1974). To obtain HeLa cell suspensions. the nutrient medium was discarded and the cells were washed once with phosphate-buffered saline (PBS) 0.1 M. pH 7.2 and treated with a 0.25 per cent trypsin solution. The resulting cells were suspended in Eagle’s minimum essential medium, supplemented as above, to obtain an approximate cell concentration of lo5 cells/ml.

Three millilitres of cell suspension were introduced aseptically into each well (35 mm dia.) of a multi-well tissue culture tray (Sterilin, Teddington, Middlesex, England) containing sterile cover slips (22 x 22 mm) and incubated at 37°C in an atmosphere of 95 per cent air, 5 per cent CO* for 3 days to obtain confluent monolayers. Immediately before carrying out the adhesion assay, the nutrient medium was discarded and the monolayer was washed once with PBS.

To study the adhesion of organisms to dead epithe- ha1 cells, the monolayers were devitalized by incubating with 2.5 per cent formaldehyde in balanced salt solution for 30 min, and washed twice in PBS prior to the assay.

AItrrnurirr ccl/ lines

Although most of the present experiments were per- formed with HeLa cells, we also used other cell lines, including human embryonic kidney epithelial cells (Flow 4000, clone 2) and human intestinal epithelial cells (Intestine 407 ATCC CCM; Flow Laboratories, Irvine, Scotland).

Sugur media

A molar solution of sucrose was sterilized by Tyn- dallization over a period of 3 days. An appropriate volume of this stock solution was added aseptically to 3 per cent mycological peptone (Oxoid Ltd, Basing- stoke. England) to obtain concentrations ranging from 50 to 500 mm01 I- ‘.

Prepurution of C. albicans suspensions

Two strains of C. ulbicuns were used; MRL 3153 obtained from the Mycological Reference Laboratory, London, England and GDH 1957, which was a recent oral isolate from a case of chronic atrophic candidosis in Glasgow Dental Hospital. Both species were ident- ified and tested by so-called ‘germ-tube’ production and by sugar assimilation and fermentation techniques (Silva-Hutner and Cooper, 1974). Stock cul-

815

Xi6 I_. P. Samaranayake and T. W. MacFarlane

tures were maintained in Sabouraud’s dextrose agar slopes at 4 ‘C. and transferred monthly.

The yeast inoculum for the assay was prepared by sub-culturing the stock culture on a Sabouraud slope at 37 C for 18 h. Three to four standard loopfuls of this culture were inoculated into mycological peptone with or without added sugar and mcubated at 37 f for 18 h. The culture was centrifuged at 15OOg for 1Omin and the deposit washed once with PBS. Final yeast suspensions ranging from lo3 yeasts.‘ml to 10’ yeasts/ml were prepared by adding appropriate volumes of this deposit to PBS. The number of yeast ceils per ml was monitored in all the experiments by Inicroscopic counting in an improved Neubauer hae- mocytometer chamber (Hawksley, England). To study the effect of cell viability on adhesion, the yeasts were killed by suspending in mycological peptone supplemented with 0.5 per cent formalin for 18 h at 4 C and washed twice in PBS prior to the assay. to remove anv traces of formalin. Viable yeasts grown in myco- lo&al peptone atone were routinely included as controls in these experiments and in experiments involv- mg sucrose.

An experiment was also carried out using non- viable C, trlhi~7n.s MRL 3153 to investigate whether the adhesion related to sucrose was due to physical factors or to metabolic activity of Cm7riidu. The non- viable yeasts were incubated for 18 h in mycological peptone with and without 0.25 M sucrose. LJsing ap- propriately incubated viable yeasts as controls, the adhesion assay was carried out as described below.

To each well of the multi-wall tray containing the previously washed monolayer was added. aseptically. 2.5 ml of the final yeast suspension and incubated at 37 C for 30min on an orbital shaker (Lukham Ltd. England) at a speed of 60rev./min. The coverslips were subsequently removed from wells and placed in a coverslip carrier and washed for a total of 45 s by gentle manL7al agitation in 3 jars of sterile PBS. This technique dislodged all the loosely adherent yeasts and debris. The coverslips with the attached yeasts were then tixed in formalin in balanced salt solution for 30 min. and air-dried. The cells were Gram-stained and mounted on glass slides with Harleco synthetic resin medium. Each experiment was repeated a minimum of 6 times to obtain data suitable for statistical analysis.

The principle of stratitied random sampling (Wei- bel, 1969) was employed to obtain numerical data for each experiment. A Leitz Ortholux microscope (Leit7 Wetzlar. Germany) fitted with a sampling stage which permitted the specimen to be examined at 0.4mm intervals was used at x 400 magnification. A graticule of equal squares, mounted in the focus of the ocular. allowed standardized counts of yeast cells to be made on each monolayer. Fifty fields (2.6 mm2) of monolayer were counted for each coverslip according to the principle of stratified random sampling and the results expressed as yeast ceils per unit square mm of

monolayer. The majority of the attached yeasts were in the

blastospore stage, some with budding daughter cells

and only a very few with pseudohyphae. The follow- ing criteria were used to standardize the counts: (a) a budding yeast was considered as a unit cell if the daughter cell was smaller than the mother: (b) a hyphum was counted as a single cell: (c) areas with patchy and discontinuous monolayers were avoided during counting.

The results obtained by the stratified random sampling technique were not normally distributed. Hence non-parametric statistical analyses were applied to determine significance levels. As the study employed related samples yielding different scores which could be ranked in absolute magnitude, the Wilcoxon mat- ched-pairs signed-ranks test (Siegel, 1956) was used to evaluate the significance of the relative adherence studies.

REX LTS

The adhesion of C. Alhic~rns MKL 3153 to HeLa cells when incubated for 30 min at 37 C is shown in Fig. 1. Linear regression analysis showed a significant positive correlation (p < 0.001, r = 0.95) between the number of attached yeasts per unit area of the monolayer and the yeast concentration in the incubating suspension. However. significantly high levels of adhesion were noted only with yeast concentrations exceeding 105 yeasts per ml. As the yeast concentrations between lo6 and 10’ yeasts per ml resulted in an optimum distribution of the organisms for counting, the final yeast suspensions were adjusted to these standard concentrations in the series of experiments involving sucrose (Fig. 2).

90

IO

Yeasts per ml

Fig. 1. The effect of yeast concentration on the adherence of C. ct/hic,trns MRL 3153 to HeLa monolayers at ?7 C for

30 min. Correlation coefficient r = 0.95. p < 0.001.

Adhesion of yeasts to epithelial cells

Mycological peptone 0

+ sucrose

., Mycological peptone (control 1

I

1.0 8 1 1

2.5 5.0 7.5

Yeasts per ml (x IO6 1

c IO

Fig. 2. The adhesion of C. ulbicans MRL 3153 incubated in mycological peptone with and without sucrose supple- ments (250mM) to HeLa monolayers at 37’C for 30min.

The effect of duration of incubation on adhesion

readily (p < 0.025) to de-vitalized HeLa cells than to viable control cells (Table 2).

Regression analysis revealed a significant positive correlation between the number of attached yeasts per unit area of the monolayer and the time of incubation (r = 0.974, p < 0.001) (Fig. 3). An optimum distribution of the organisms for counting was obtained by incubating yeast inocula with the monolayers for 30 min. although a longer period of incubation resulted in increased yeast adhesion.

The rfiect of sucrose pre-incubation of Candida on adhesion

The qflkct of yeast cell ciability on adhesion

A significant (p < 0.025) reduction in adhesion to HeLa and Flow 4000 cells of both strains of C. albicans was observed when they were rendered non- viable with formalin (Table 1).

When C. albicans MRL 3153 was pre-incubated in sucrose media a significant positive correlation was observed (p < 0.001, r = 0.98) between the number of adherent yeasts and the sucrose concentration (Figs 2 and 4). There was a significant difference in adherence between viable and non-viable yeasts cultured in sucrose containing media (p < 0.025). However, no significant difference in adhesion between non-viable yeasts cultured in media with or without sucrose was observed (Table 3).

DISCUSSION

The effect of HeLa cell uiahility on adhesion That successful colonization and infection of the C. dhicans MRL 3153 whether viable or non-viable host by a microorganism depends upon its initial ca-

or grown in sucrose containing media adhered more pacity to adhere to host tissues has been well docu-

I I 1 1 1

0 IO 20 30 40 50 60 Time (min)

Fig. 3. The effect of incubation period on the adhesion of C. ulbicans MRL 3153 to HeLa monolayers at 37,-C. Mean of 3 experiments. Yeast concentration = 7 x lo6 yea&ml.

Correlation coefficient r = 0.974, p < 0.001.

Table I. Adhesion of 2 strains of viable and non-viable C. albicans to HeLa and Flow 4000 monolayers

Yeast

C. alhicans MRL 3153

C. albicuns GDH 1957

HeLa cells Flow 4000 cells

Yeasts per mm2 P* Yeasts per mm2 P* No. of expts of monolayer Wilcoxon’s of monolayer Wilcoxon’s

Viability? per cell line (*SE) test (&SE) test

Viable 138(f15) llO(_t46) 6 < 0.025 < 0.025

Non-viable 34(kll) 45(+23)

Viable 214(+29) 169(*19) 6 < 0.025 < 0.025

Non-viable 52(*10) 49(k13)

* Significance of difference between viable and non-viable yeasts. t Yeasts rendered non-viable by incubating in formalin for 18 h at 4 ‘C.

818 L. P. Samaranayakc and 7‘. W. MacFarlane

Table 2. Adhesion of C. alhicuns MRL 3153 to viable and non-viable (formalin-treated) HeLa monolayers

Yeasts incubated int

Yeasts mm’ of monolayer (+ SE) ____ I’

No. of expts Viable HeLa Non-viable HeLa Wilcoxon’s test

Mycological peptone (MP) (viable) 6 128 (jai) 189(?43) < 0.025

MP + formalin (non-viable) 6 41 (*XI 82(121) < 0.025

MP + 250mM sucrose 6 149 (+26) 255 ( + 37) < 0.025

* Significance of difference between adhesion to viable and nonviable HeLa cells t Period of incubation: 18 h at 37°C.

z g 400

+I

"E E 300-

/

I %

f 200- 1 *

0 / !

>

/

1

loo- P

0 I ’ I 1 1

0 100 200 300 400

Sucrose concentration (m mol IV)

Fig. 4. The effect of pre-incubating c‘. ulhicun.\ MRL 3153 in mycological peptone supplemented with varying sucrose concentrations on adhesion to HeLa monolayers at 37 C

for 30 min. Correlation coefficient r = 0.98, p < 0.001,

mented for certain oral bacteria, and various quantitative assays to determine bacterial adhesion have been described (Gibbons and van Houte, 1971; Brsta- vik, Kraus and Henshaw, 1974; Olssen and Krasse. 1976; Liljemark and Schauer, 1977). Only a few reports deal with the adhesion of the fungal pathogen C. albicans to epithelial cells. Liljemark and Gibbons (1973) studied the in-km adhesion of candida to murine buccal epithelial cells; Kimura and Pearsall (1978. 1980) and King et a/. (1980) employed in-citro optical methods to quantify the number of yeasts attached to desquamated human epithelial cells of buccal and

vaginal origin, respectively. The present method uses cpithelial monolayers of human origin in tissue culture and appears to have a number of advantages over previous methods. Buccal epithelial cells are in- evitably contaminated with commensal flora which are often difficult to dislodge even after repeated washings (Sklavounou and Germaine, 1980; Varian and Cooke, 1980). Moreover, these cells are continu- ously bathed in saliva and perhaps contaminated with serum and food debris to varying degrees prior to collection. Indeed, King er u/. (1980) pointed out that the actual numbers of adherent yeasts varied con- siderably with vaginal epithelial cells from different donors. They also noticed a considerable day-to-day fluctuation in adherence values when the cells were tested on a daily basis.

The eukaryotic cell lines we employed exhibit more uniform characteristics and should be amenable to a wider range of experiments. The effect of various oral bacteria, saliva and serum could be studied under more controlled conditions using the system, due to the uncontaminated nature of the epithelial monolayers (Samaranayake and MacFarlane, 1981). The system might be used to evaluate the ability of chemi- cals to interfere with the adhesion process.

The stratified random sampling technique used here is more bias-free (Weibel, 1969) than random- sampling techniques others have used for microbio- logical procedures (8rstavik et ul., 1974; Kimura and Pearsall. 1978; Varian and Cooke, 1980). The quantitative determination of minimum field size is an im- portant aspect of sampling techniques. To arrive at our figure of 50 fields per coverslip, we used the method of accumulative mean plots suggested by Chalkley (1943), and found that the reproducibility of

Table 3. The effect of sucrose on the adhesion of viable and non-viable formalin-treated C. uhicms (MRL 3153) to HeLa monolayers

Viability* of yeast cells Pre-treatment-I No. of expts

Yeasts/mm2 of P monolayer (+ SE) Wilcoxon’s test

Viable MP + sucrose Non-viable MP + sucrose

Non-viable MP Non-viable MP + Sucrose

91 (f9) 6 38(&X)

< 0.025

45(+7) 6 3X(*8)

NS

* Yeasts rendered non-viable by incubating in formalin for IX h at 4 C. MP. Mycological peptone, NS. Not significant.

t Either viable or non-viable yeasts were incubated in mycological peptone or mycological peptone supplemented with 250 mM sucrose for 18 h at 37’ C.

Adhesion of yeasts to epithelial cells 819

the duplicate counts per coverslip varied only l-7 per cent from the mean. The present method is a simple, reproducible and versatile method of studying yeast adhesion.

Although HeLa cells are not closely related to buccal epithelial cells, candidal adhesion to both cell types showed striking similarities. For example, in both systems, adhesion was detectable above lo4 yeasts cells/ml of incubation mixture (Fig. 1) and for- mol-killed non-viable yeasts adhered significantly less than the viable yeasts (Kimura and Pearsall, 1978). Others have used HeLa cells and other cultured cells to study bacterial adherence in vitro (Swanson, 1973: Fitzgerald et al., 1977; Hartley, Robbins and Rich- mond, 1978).

Our findings indicate that sucrose, even at very low concentrations, facilitates the in-vitro adhesion of yeasts to epithelial cells. This suggests that the intake of dietary sucrose by subjects could favour the colonization of the mouth by C. a&cans. We have shown that C. alhicans cultured in glucose and sucrose-supplemented peptone media adhere to acrylic surfaces in significantly greater numbers than organisms grown in peptone alone (Samaranayake. McCourtie and Macfarlane, 1980b); these findings have been con- firmed by Douglas and McCourtie (1980) and are furthermore supported by a number of in-lilo obser- vations. Russell and Jones (1973) observed that rats fed on sucrose-rich diets carried C. u1hicun.s for longer than the control rats. Olsen and Birkeland (1976) found that sucrose rinses initiate and aggravate chronic atrophic human candidosis: Shuttleworth and Gibbs (1960) suggested that a high carbohydrate intake was an aetiological factor in oral candidosis of 4 elderly denture wearers. Similarly, Ritchie et ul. (1969) reported an aggravation of symptoms in patients with chronic atrophic candidosis when their carbohydrate intake was raised above the normal levels. Excess dietary sucrose stimulates yeast growth in mixed saliva (Knight and Fletcher. 1971) and this would also contribute to increased oral carriage of candida.

As sucrose enhanced the adhesion only in viable cultures of C. ulhicans, it is possible that an extracellular metabolic product of the organism is responsible for the adhesion. Scanning electron microscopy of sucrose-treated candida reveals rough, uneven cellular surfaces when compared with smooth cell walls of control organisms (Samaranayake and MacFarlane, 1980a) and transmission electron microscopy has revealed a ruthenium-red positive, hbro-floccular material on the surfaces of both glucose- and sucrose-

grown yeast cells (Douglas and McCourtie, 1980). The adherence of a number of oral commensal bacteria cultured in sucrose-containing media is increased owing to the production of extracellular polysacchar- ides (Gibbons and van Houte. 1975).

As a majority of the superficial cells of the oral mucosa are non-viable (Albright. Selinger and Reed, 1980) we simulated in-ric?o conditions by carrying out the adhesion assay with non-viable HeLa cells. Sig- nificantly higher numbers of yeasts attached to non- viable cells than to the viable controls. However. the enhanced adhesion of viable candida induced by sucrose persisted irrespective of the viability of the HeLa cells. As the mechanism by which C. ulhicuns

adhere to mucosal surfaces is, as yet, unknown, the significance of the above findings in the pathogenesis of human candidal infections remains to be deter- mined.

Acknowledyements-This study was supported by a Grant from the Scottish Home and Health Department.

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The adhesion of the yeast Candida albicans to epithelial cells of human origin in vitro