Vol. 56, No. 11

Role of Yeast Cell Growth Temperature on Candida albicansVirulence in Mice

PENNY P. ANTLEY AND KEVIN C. HAZEN*Department of Microbiology, University of Southwestern Louisiana, Lafayette, Louisiana 70504-1007

Received 25 April 1988/Accepted 5 August 1988

Previous studies have suggested that yeast cell growth temperature may influence the relative virulence of theopportunistic dimorphic fungus Candida albicans. To test this possibility, mice were challenged with C. albicansyeast cells which were grown at either room temperature or 37°C, and their survival was monitored daily. Micewhich received room temperature-grown cells died faster. The interaction of glycogen-elicited polymorphonu-cleated neutrophils (PMNs) with C. albicans yeast cells grown at the two temperatures was examined, becausePMNs have been shown to have a critical role in preventing development of candidiasis in normal individuals.In the absence of serum (i.e., nonopsonic conditions), more PMNs conjugated and engulfed C. albicans cellsgrown at room temperature than those grown at 37°C. However, PMNs were less able to kill cells grown atroom temperature than cells grown at 37°C. Cells grown at room temperature also produced abundant germtubes after engulfment and were thus more likely to escape killing by phagocytes. These results suggest thatcells grown at room temperature are more virulent because they are less likely to be killed by phagocytes andare more likely to disseminate. The possibility that expression of cell surface hydrophobicity is involved in theseevents is discussed.

Several physiological characteristics of the dimorphicfungus Candida albicans appear to enhance the ability of theorganism to cause disease. These virulence factors includegerm tube formation, exoenzyme production, release oftoxins, and antigenic variation (2-4, 17, 27, 29). The magni-tude of expression of these characteristics by C. albicans isisolate dependent (4, 27, 29). We have recently demon-strated that growth temperature can influence expression ofsome of these virulence characteristics (13). For instance,the ability to form germ tubes is greater for cells grown atroom temperature (RT) than for cells grown at 37°C (13).Also, RT-grown cells appear to be less sensitive to toxicsubstances, such as detergents, and to the germinationinhibitor designated morphogenic autoregulatory substance(11, 13). Other investigators have shown that growth tem-perature may influence the ability of C. albicans to adhere toepithelial tissue (21), which is an important initial step inestablishing infection (33, 37). Cells grown at RT adhered toepithelial tissue better than cells grown at 37°C did. Inter-estingly, cells that form germ tubes adhere better than yeastcells (19, 36). Taken together, these observations suggestthat cells grown at RT may be more virulent than cells grownat 37°C.An additional physiological characteristic of C. albicans

which may influence virulence is expression of cell surfacehydrophobicity (CSH). Previous studies have shown thatCSH expression by C. albicans yeast cells is dependent ongrowth temperature: cells grown at RT are more hydropho-bic than cells grown at 370C (12, 16). CSH has been shown toenhance adherence of pathogenic bacteria to host surfaces,but whether this is true for C. albicans adherence is not clear(18, 20, 24, 28, 30, 32). However, expression of CSH doesappear to be required for germination by C. albicans tooccur and could thus indirectly affect pathogenesis (14a, 15).These results again suggest that yeast cell growth tempera-ture may influence the relative virulence of C. albicans.Here we report the results of our initial studies concerning

* Corresponding author.

the possibility that yeast cell growth temperature increasesC. albicans virulence and consider the relationship of CSHto phagocytosis. We determined that cells grown at RT weremore virulent than cells grown at 37°C. Although cells grownat RT were conjugated to and engulfed by polymorphonu-cleated neutrophils (PMNs) more than cells grown at 37°Cwere, they were killed less often. Following engulfment byPMNs, cells grown at RT elaborated germ tubes sooner thancells grown at 37°C did, which may have allowed them toescape from the PMNs.

MATERIALS AND METHODSOrganism and growth conditions. C. albicans LGH1095

(originally obtained from a blood culture of a septicemicpatient) was used throughout this study. Identification,maintenance, and physiological characteristics of this isolatehave been described elsewhere (13). Yeast cells were pre-pared for experiments as described previously (13). Cellswere subcultured 3 times every 24 h in Sabouraud dextrosebroth (Difco Laboratories, Detroit, Mich.). Each subculturewas grown to the stationary phase with constant aeration(120 rpm; Incubator-Shaker; New Brunswick Scientific Co.,Inc., Edison, N.J.) at either RT (ca. 22 to 25°C) or 37°C.Cells were harvested by centrifugation (400 x g) and washedtwice with either Hanks balanced salts solution (HBSS)buffered with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 6 g/liter) at pH 7.2 (HBSSH) or withRPMI 1640 medium (pH 7.2). The CFU and spherical unitconcentrations were determined by direct counts with ahemacytometer (13). A CFU was defined here as any appar-ently contiguous unit of cells or microcolony of blastoco-nidia. We have shown previously that greater than 99% ofthe CFU are viable (13). Mother cells and buds were eachdesignated as a sphere (13). Stock cell suspensions were keptat 0 to 4°C on ice until they were needed (no longer than 2 h).Heat-killed yeast cells were prepared by exposing cell sus-pensions to 100°C for 15 min, followed by two washes withice-cold HBSSH or RPMI 1640 medium.

Hydrophobicity assays. The CSH of C. albicans was de-

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termined by a polystyrene microsphere assay. This assayhas been shown to yield results similar to those of anaqueous hydrocarbon biphasic assay (12, 16). In the micro-sphere assay, equal volumes (100 ,u) of C. albicans yeastcells (1 x 106 to 2 x 106 CFU/ml) and blue-dyed, polystyrenelatex beads (diameter, 0.845 + 0.001 mm; 9.02 x 10'microspheres per ml; Serva Fine Biochemicals, Westbury,N.Y.) were placed in an acid-washed glass tube (12 by 75mm) and equilibrated to RT. The mixture was then vortexedvigorously for 30 s, and the percentage of hydrophobic CFUwas immediately determined. A CFU was considered hydro-phobic if it had three or more attached microspheres. Whengrown at RT under the conditions stated above, C. albicansLGH1095 typically had a hydrophobicity value of >95%.When grown at 37°C, the value was <10%. The growthtemperature-dependent variation in CSH is typical of thisspecies and has been described before (12, 13, 16).The microsphere hydrophobicity assay can, with minor

modifications, also be applied to mammalian cells (14). Inexperiments in which PMNs were used, the CSH value ofthe PMN population was always >85%. Using contact anglemeasurements, other investigators have shown that PMNsare highly hydrophobic (for a review, see reference 1).

Animals. Male and female outbred CD1 mice ranging inage from 6 to 10 weeks were obtained from our departmentalcolony and were used for survival and phagocytosis exper-iments. In individual experiments, animals of only one sexwere used as the source of PMNs. Mice were provided withfood and water ad libitum and housed under conditions thatwere in compliance with the guidelines of the AmericanAssociation for the Accreditation of Laboratory AnimalCare. When required, fresh mouse serum was obtained bypuncture of the retroorbital plexus of ether-anesthetizedanimals followed by removal of clots.

Survival experiments. Mice were injected intravenouslywith 0.2 ml of HBSS containing either 4 x 105 or 2 x 106CFU of C. albicans grown at RT or 37°C. The survival ofeach group (12 to 15 mice per group) was monitored every 24h. The experiments were terminated when the deaths of theremaining mice would not affect the statistical significance ofthe data. Two groups of mice were statistically compared bythe Mann-Whitney U test, in which the null hypothesis wasthat the difference in the days of median death of the twogroups was no greater than that expected from randomsampling fluctuations (41).PMN populations. CD1 mice were injected intraperitone-

ally with 2.5 ml of 0.5% glycogen (Sigma Chemical Co., St.Louis, Mo.) in physiological saline. Three to four hourslater, the peritoneum of each mouse was lavaged twice with5 ml of HBSS without calcium or magnesium (GIBCOLaboratories, Grand Island, N.Y.). The peritoneal exudatecell suspensions were centrifuged at 285 x g for 5 min.Erythrocytes were removed by lysis with Tris-bufferedammonium chloride, as described by Mishell and Shiigi (25),except that fetal bovine serum was not included. The peri-toneal exudate cells were then pelleted and washed twicewith the appropriate test medium (HBSSH or RPMI 1640).Cell viability was determined by trypan blue exclusion, andthe cell concentration was determined by direct counts witha hemacytometer. Typically, the percentage of PMNs in theperitoneal exudate was >98%, as determined by Wright-Giemsa staining (Anderson Laboratories, Fort Worth, Tex.).Exudates were not used if the concentration of PMNs wasless than this value or if cell viability was less than 95%.

Single-cell conjugation assay. Conjugation experimentswere done by a modification of the procedure of Grimm and

Bonavida (10). PMNs and C. albicans yeasts were washed,and hydrophobicity levels were determined. PMNs in HBSSwithout Mg2+ and Ca2' and without HEPES were adjustedto 5 x 106 cells per ml of yeast cells to 2.5 x 106 CFU/ml.Equal volumes of each cell suspension were mixed and keptat 30°C for 5 min. The cell suspension was then centrifugedat RT for 5 min at 100 x g, and a portion was transferred toa hemacytometer. Conjugates were counted immediately byexamination at x400 magnification. A conjugate was consid-ered as any PMN with one or more attached yeast cells. Thepercentage of total conjugation was determined by countingthe number of conjugates per 100 PMNs.Monolayer assay for conjugation and engulfment. PMN

monolayers were prepared by placing 0.5 ml of either 2 x 106or 1 x 106 PMN cell suspensions on sterile glass cover slips(22 by 22 mm; Corning Glass Works, Corning, N.Y.) andincubating the cover slips for 1 h at 37°C in a humidified 5%CO2 atmosphere (26). Following incubation, monolayerswere rinsed with warm medium to remove nonadherentcells. Monolayers were then covered with 0.5 ml of a yeastcell suspension (2 x 106 CFU/ml) grown at RT or 37°C andincubated at 37°C in 5% CO2 for various time periods. Testmedia in these experiments included HBSSH and RPMI1640 each with or without 5% (vol/vol) fresh mouse serum.The monolayers were gently washed with cold medium andallowed to air dry. Prior to microscopic examination, themonolayers were stained with Wright-Giemsa stain. Thepercentage of conjugation and engulfment (the phagocyticactivity index [1]) was determined by observing 200 PMNsper monolayer at x 1,000 magnification. A PMN was consid-ered to have conjugated with a yeast cell if the yeast cell wasabutted to the PMN, but no observable membrane encircledthe yeast cell. If the PMN membrane surrounded the yeastcell, then the PMN was considered as having engulfed theyeast cell.

Radiometric engulfment assay. Engulfment of yeast cellsby glycogen-elicited PMNs was also evaluated by the[3H]uridine uptake method of Bridges et al. (5). Equalvolumes (100 ,ul) of PMNs (5 x 106 cells per ml) and C.albicans (1 x 106 CFU/ml) were placed in tissue culture-treated (polystyrene), round-bottom centrifuge tubes (16 by150 mm; Corning) and tumbled at 8 rotations per min for 30min or 1 h at 37°C. Control samples containing just PMNs,yeast cells, or PMNs with heat-killed yeast cells were alsoincluded. After incubation, 100 ,ul of each sample was mixedwith 100 ,ul of test medium and 20 pul of medium containing0.5 mCi of [5,6-3H]uridine (specific activity, 47.1 Ci/mmol;Dupont, NEN Research Products, Boston, Mass.). Thesemixtures were incubated at 37°C for 1 h. Cells were collectedonto glass fiber filters (GFC; Whatman, Inc., Clifton, N.J.)and washed with at least 10 ml of distilled water; this wasfollowed by washing with at least 10 ml of 2 mM uridine(Sigma) in water. The filters were dried overnight. Theamount of radioactivity associated with each sample wasthen determined by scintillation spectrometry (model LS2000 scintillation spectrometer; Beckman Instruments, Inc.,Fullerton, Calif.) with Aquasol 2 (Dupont, NEN ResearchProducts) as the scintillation cocktail. Each experiment hadtriplicate samples, and background radioactivity uptake (up-take by PMNs alone) was subtracted from each test sample.The percentage of engulfment was calculated by comparingthe amount of [3H]uridine uptake by viable yeast cells aloneversus that by yeast cells mixed with PMNs by using thefollowing formula: percent engulfment = {1 - [(cpm testsample - cpm PMN control)/(cpm yeast control - cpm Dyeasts)]} x 100, where the test sample is the yeast cell and

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PMN mixture, the PMN control is PMNs alone, yeastcontrol is viable yeast cells alone, and D yeasts is theheat-killed yeast cell control.

Phagocytic killing assay. PMN candidacidal activity wasdetermined by the microtiter killing assay described byCutler and Thompson (6). Each well of a 96-well, flat-bottommicrotiter plate was precoated with 1% (wt/vol) bovineserum albumin in physiological saline for 1 h at RT and thenwashed with saline. To each well, 200 [L of C. albicans yeastcell suspension (104 CFU/ml of either HBSSH or RPMI 1640medium) was added. The plate was centrifuged (360 x g, 5min, RT), and the supernatants were removed by flicking theplate. Glycogen-elicited PMNs were suspended in HBSSHor RPMI 1640 medium with or without 5% fresh mouseserum to a concentration of 2.5 x 105 cells per ml, and 200 p.lof the appropriate suspension was added to each well. Theplate was incubated at 37°C for 1 h under 5% C02, afterwhich the plate was centrifuged at 360 x g for 10 min. PMNswere lysed by three consecutive washes with distilled water.In order to ensure that yeast cells were retained in the wellsduring the PMN lysis step, the washes for each well werepooled and then plated onto Sabouraud dextrose agar. Theplates were incubated for 48 h at 37°C, to observe yeastcolony formation. Molten corn meal agar was added to eachwell (200 pI per well), and the microtiter plate was incubatedat 37°C under 5% CO2. After 1 h, the number of germ tubesper five high-power fields was determined for each sample.Similarly, at 4 to 5 h, the number of microcolonies per fivehigh-power fields was determined. Germination and subse-quent microcolony formation were used as an indication ofviability (6). Percent killing was determined by comparingthe sample wells with control wells which received onlyyeast cells.

RESULTSEffect of yeast cell growth temperature on virulence. Out-

bred CD1 mice were inoculated intravenously with C. albi-cans yeast cells grown at either RT or 37°C on day 0 andmonitored for percent survival every 24 h. The mediansurvival times for male mice infected with cells grown at RTand 37°C were 10 and 14 days, respectively. In female mice,these times were 9 and 12 days, respectively. The mediansurvival times for male and female mice challenged with C.albicans were significantly less (P < 0.05; Mann-Whitney Utest) when the inoculated cells were grown at RT than whenthey were grown at 37°C (Fig. 1). Similar results, althoughwith some variation in the rapidity of death, were obtainedwhen these experiments were repeated and when bothconcentrations of C. albicans were tested (data not shown).These results were not due to differences in the number ofspherical units of C. albicans administered to the micebecause the ratio of spherical units per CFU were similar inthe cell populations grown at RT and 37°C (ca. 1.8:2.1).PMN-yeast cell conjugation. The influence of yeast cell

growth temperature on nonopsonically mediated conjugationwith PMNs was evaluated by using a modification of asingle-cell binding assay developed previously for naturalkiller cell and target cell studies (10). Glycogen-elicitedPMNs were mixed with C. albicans yeast cells, and thepercentage of PMNs that formed conjugates with the yeastcells was determined following incubation for 5 min at 30°C.PMNs mixed with C. albicans formed more conjugates whenthe yeast cells were grown at RT than when they were grownat 37°C (9.7 + 0.7 versus 2.2 + 0.9%, respectively; results ofthree experiments each conducted with triplicate samples).For this assay, low levels of conjugation are typical (10).

100

80

-a

I-

4m~

60

40

20

C-

4 8 12 16 20 24

Time (Days)FIG. 1. Effect of yeast cell growth temperature on virulence in

male (A) and female (B) mice. Mice were injected intravenouslywith a total of 0.2 ml of HBSSH containing 4 x 105 CFU of C.albicans grown at RT (Oi) or 37°C (*). Percent survival wasmonitored every 24 h. Each group consisted of 12 to 15 mice. Ineach experiment, when the entire population of one of the groupshad died, the percent survival of the other group was not monitoredfurther. This was done because subsequent deaths in the remaininggroup would not alter the statistical significance of the data (seetext).

Increased conjugation with C. albicans grown at RT withPMNs was also obtained in the monolayer assay when theincubation medium (HBSSH or RPMI 1640) did not containserum (Table 1). In this assay, conjugation was followedduring a 1-h incubation period. Although PMNs conjugatedcells grown at RT more at 15 and 30 min, higher levels ofconjugation were obtained with yeast cells grown at 37°C by60 min (Table 1). This was probably due to the fact that theinitially conjugated cells grown at RT were engulfed, thuslowering the apparent number of conjugated cells. Whenfresh mouse serum obtained from CD1 mice was incorpo-rated into the test medium, no difference in conjugation wasobserved (data not shown).PMN engulfment of C. albicans. The effect of yeast cell

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TABLE 1. Effect of yeast cell growth temperature on conjugationbetween PMNs and C. albicans

Test Expt Yeast cell % PMNs forming conjugates ata:medium no.

growthmedium no. temp ('C) 15 min 30 min 60 min

RPMI 1640 1 RTb 13.7 ± 0.6c 24.2 ± 0.9g 4.1 ± 1.1c37 2.6 ± 1.5c 17.4 ± 3.7e 47.2 ± 1.5c

2 RT 27.9 ± 1.6c 28.3 ± 2.5d 0 ± 0'37 5.8 ± 1.6c 16.6 ± 1.1d 27.5 ± 0.8c

HBSSH 1 RT 8.2 ± 2 0d 1.6 ± 0.3c 0 ± OC37 2.7 + 0.9d 5.0 0.5c 2.1 + 0.8c

2 RT 19.5 ± 2.5d 25.1 ± 1.4c 6.8 ± 1.7e37 11.1 1.2d 7.5 1.Oc 11.0 ± 2.3e

a Conjugation of C. albicans to PMNs was determined by the monolayerassay, as described in the text. PMNs were exposed to C. albicans fordifferent time periods, after which the monolayers were gently washed toremove unattached. C. albicans. The monolayers were than air dried prior tostaining. Values are arithmetic means ± standard deviations of at least threesamples; 200 PMNs were examined per replicate. Probabilities were calcu-lated with a two-tailed Student t test (41).

b RT was ca. 22 to 25°C.C p < 0.01.d p < 0.05.' Not significant.

growth temperature on the ability of glycogen-elicited PMNsto engulf C. albicans under nonopsonizing conditions (i.e.,in the absence of serum) was assessed by the monolayerassay described by Morrison and Cutler (26). The percentageof PMNs that engulfed C. albicans was greater for cellsgrown at RT throughout the 1-h incubation period than it wasfor cells grown at 37°C, regardless of the test medium (Table2). These results were confirmed with the [3H]uridine uptakeassay of Bridges et al. (5) (Table 3). Similar to the resultsobtained with conjugation, when fresh mouse serum was

TABLE 2. Effect of yeast cell growth temperature onPMN engulfment of C. albicansa

Test Expt Yeast cell Phagocytic activity index atb:Test Exptgrowthmedium no. temp (°C) 15 minc 30 mine 60 min

RPMI 1640 1 RTd 14.2 ± 2.7 38.7 ± 4.3 50.4 + 2.7c37 1.1 ± 0.2 15.5 ± 0.8 36.5 ± 0.3c

2 RT 8.5 ± 0.7 54.6 ± 4.9 57.0 ± 0.8c37 0 0 2.8 ± 0.8 21.7 t 0.9c

HBSSH 1 RT 27.8 ± 2.4 40.3 ± 1.2 49.1 ± 4.8e37 13.0 ± 0.8 30.0 + 1.2 44.9 ± 2.1l

2 RT 23.5 ± 2.3 35.0 ± 2.2 70.5 ± 6.1c37 5.3 ± 0.4 12.8 ± 0.7 23.2 ± 0.8c

a As determined by the monolayer assay of Morrison and Cutler (26).b The phagocytic activity index is defined here as the percentage of PMNs

with engulfed C. albicans and was determined with the monolayer assay asdescribed in the text. PMNs were exposed to C. albicans 15, 30, or 60 min.The PMN monolayers were then washed to remove unattached yeast cells andair dried prior to staining. Values are arithmetic means + standard deviationsof at least three samples; 200 PMNs were examined per replicate. Probabili-ties were calculated with a two-tailed Student t test (41).

c P < 0.01 for all values.d RT was ca. 22 to 25°C.eNot significant.

TABLE 3. Effect of yeast cell growth temperature onPMN engulfment of C. albicansa

% [3H]uridine uptake by engulfedExpt no. Yeast cell yeast cells atb:growth temp('C)--

30 minc 60 mind

1 RTe 63.8 ± 3.8 61.5 ± 12.937 6.5 3.7 6.5± 6.3

2 RT 46.6 ± 3.4 73.4 ± 6.337 3.7 ± 1.2 25.8 ± 9.8

3 RT 85.2 ± 4.5 89.3 ± 6.237 46.3 ± 5.0 57.3 ± 13.1

aAs determined by the radiometric method of Bridges et al. (5).b [3HJuridine uptake by engulfed yeast cells was determined as described in

the text. PMNs were exposed to C. albicans for either 30 or 60 min, afterwhich the [3H]uridine uptake by engulfed yeast cells was determined. Valuesare arithmetic means ± standard deviations from at least three samples.Probabilities were calculated with a two-tailed Student t test (41).

C P < 0.01 for all values.d P < 0.05 for all values.eRT was ca. 22 to 25°C.

present in the test medium, no difference in engulfment byPMNs of C. albicans grown at RT or 370C was observed.PMN killing of yeast cells. Results of the monolayer

engulfment assay also suggested that RT-grown cells couldescape killing by PMNs by elaboration of germ tubes, as hasbeen described by others (8, 9). This was evident by the highdegree of germination that occurred by 60 min into incuba-tion for cells grown at RT but the lack of germination by cellsgrown at 37°C (Fig. 2).

Other experiments were performed to determine whetherPMNs killed cells grown at RT less than they did cells grownat 37°C. Several phagocyte killing assays were tried initially.These included viability staining, radiometric methods, anddimethylthiazolyl-diphenyltetrazolium bromide cleavage (5,22, 23). Unfortunately, these assays were insufficiently sen-sitive or caused a loss of hydrophobic cells because ofattachment to plastic tubes or caps such that highly variableresults were obtained. When the microtiter plate assay ofCutler and Thompson (6) was used, however, these prob-lems were avoided.When HBSS without serum was used in the assay, cells

grown at RT were killed less than cells grown at 37°C (Table4). However, the addition of serum to the medium resulted insimilar levels of killing between the cells grown at thedifferent temperatures (Table 4). When RPMI 1640 mediumwas substituted for HESS, essentially identical results wereobtained (data not shown).

DISCUSSIONPrevious studies concerning the influence of growth tem-

perature on various physiological attributes of C. albicanssuggested that cells grown at RT may be more virulent thancells grown at 37°C (13). The survival experiments reportedhere appear to support this idea. Thus, regardless of whetherthe etiologic agent of candidiasis in a patient is eitherexogenously introduced (for example, via contaminatedfoods) or derived from the endogenous microbiota of thehost (40), the ability to behave like a RT-grown cell mayhave important ramifications for the relative success of theorganism to incite infection. We have recently demonstratedthat cells grown at 37°C are capable of expressing surfacehydrophobicity during initial growth of yeast cells and priorto germination (14a).

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FIG. 2. Appearance of C. albicans yeast cells grown at RT (A) or

37°C (B) after exposure to PMNs for 60 min in RPMI 1640 medium.The cells grown at RT showed prominent germ tubes extending outof the PMN (panel A, thin arrows), while cells grown at 37°C were

primarily in the yeast form or only had germ tube initials (panel B,thick arrow).

PMNs have been strongly implicated to have a criticalfunction in preventing the development of candidiasis innoncompromised hosts (9, 31, 35). Under nonoposonizingconditions, PMNs conjugated and engulfed yeast cells grownat RT more than they did cells grown at 37°C. Given that theglycogen-elicited PMNs were highly hydrophobic and that,

as shown by others (34, 38, 39), nonopsonic phagocytosis ofbacteria is mediated by hydrophobic interactions, we sug-gest that the differences obtained in the conjugation andengulfment results between cells grown at RT and 37°C isdue to CSH. Cells grown at RT are more hydrophobic thancells grown at 37°C (12, 13, 16). This suggests that hydro-phobic interactions are also involved in nonopsonic phago-cytosis of C. albicans. Further support for this suggestioncomes from the conjugation and engulfment data in which a

difference was obtained between cells tested in HBSSH andRPMI 1640 medium (Tables 1 and 2). In general, RPMI 1640medium caused lower levels of conjugation and engulfmentof RT-grown cells. This agrees with our earlier observationthat RPMI 1640 medium does not support hydrophobicinteractions as well as HBSSH does (14).PMNs appeared to be less capable of killing cells grown at

RT than they were of killing cells grown at 37°C (Table 4).The reasons for this difference are not clear. However,several explanations based on the data presented here andelsewhere are possible. Elaboration of germ tubes by en-

gulfed yeast cells has been shown to provide a mechanism bywhich C. albicans can escape a phagocyte (7, 8). In our

experiments, RT-grown cells produced abundant germ tubesby 60 min into the phagocytic assay, and the germ tubesexuded from the PMNs (Fig. 2). In contrast, cells grown at37°C germinated poorly. Another reason for the difference inkilling may be related to the relatively greater sensitivity ofcells grown at 37°C to potential growth inhibitors (13). It isunknown whether cells grown at RT are similarly lesssensitive than cells grown at 37°C to the oxidative andnonoxidative killing mechanisms of PMNs. These explana-tions are not mutually exclusive, because a yeast cell mustbe able to withstand PMN killing mechanisms during theprocess of germination.CSH may also be involved in avoidance of PMN killing.

Initially, hydrophobic yeast cells have been shown to pro-duce germ tubes sooner than hydrophilic yeast cells (14a).This appears to be due to the requirement that hydrophiliccells become hydrophobic prior to germination (14a).Taken together, our results suggest that enhanced viru-

lence of RT-grown yeast cells in mice previously unsensi-tized to C. albicans can be explained in part by their

TABLE 4. Effect of yeast cell growth temperature on killing of C. albicans by glycogen-elicited PMNsa

% Killing":

Expt no. growth temp lC) HBSSH HBSSH + MS"

lhd 4h 1h' 4he

1 RTf 56.8 ± 8.1 57.6 ± 2.3"d 46.6 ± 5.2 55.7 ± 10.137 87.7 ± 9.3 97.6

± 2.8" 44.7±

1.4 65.9 ± 17.6

2 RT 10.1 ± 2.6 8.6 ± 1.3' 15.1 + 5.0 20.0 ± 8.937 34.7 ± 7.3 25.5 ± 1.79 37.9 + 8.1 43.4 ± 15.3

3 RT 35.4 ± 0.7 43.2 ± 6.5" 37.8 ± 0.9 76.1 ± 3.657.3 ± 8.9 65.8 ± 7.7d 35.5 ± 6.5 87.0 ± 5.1

"As determined by the method of Cutler and Thompson (6).b PMNs were exposed to C. albicans for 1 h at a ratio of 25:1 (PMN:C. albicans). PMNs were then lysed, and the percentage of C. albicans yeast cells which

produced germ tubes by 1 h or microcolonies by 4 to 5 h was determined. This percentage was subtracted from 100% to yield the percent killing. Values arearithmetic means ± standard deviations of at least two experiments, each of which had three samples. Probabilities were calculated with a two-tailed Student Itest.

C MS, Fresh, normal mouse serum."P < 0.05 for all values.Not significant.

f RT was ca. 22 to 25°C.g p < 0.01.

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GROWTH TEMPERATURE AND C. ALBICANS VIRULENCE

interaction with phagocytes. We speculate that hydrophobic(and more virulent) yeast cells are engulfed rapidly by PMNsbut are only poorly killed. This provides sufficient time forthese yeast cells to elaborate structures such as germ tubesand extracellular materials that make them less susceptibleto killing during subsequent encounters with phagocytes andmore capable of successful colonization and dissemination(8, 19, 35, 36). We believe that these initial events are relatedto CSH. When opsonins were available, however, the influ-ence of CSH on phagocytic interactions was less pro-

nounced. This observation does not detract from the poten-tial importance of CSH in pathogenesis, as there are areas ofthe human body that are common sites for candidiasis butthat are not unlikely to be exposed to high levels of comple-ment (e.g., the vagina and the gastrointestinal tract).Our interpretations concerning the possible role of CSH in

candidiasis are based on experiments in which hydrophobicand hydrophilic cells were obtained by growth at RT and37°C, respectively. It is possible, of course, that other cellwall differences exist between these cells, and these differ-ences could explain the results reported here. However,other investigators have shown that at least the initial eventsof phagocytosis under nonopsonic conditions are mediatedby hydrophobic interactions (1). Nonetheless, given that a

multiplicity of immune mechanisms is needed to combat C.albicans during the development of disease, further studiesare needed to establish definitively the importance of CSH inavoiding immune system elimination.

ACKNOWLEDGMENTS

We thank Beth W. Hazen (Acadiana Medical Research Founda-tion, Lafayette, La.) for excellent technical assistance with portionsof this study.

This study was supported in part by a grant from the AmericanDiabetes Association, Louisiana Affiliated (to K.C.H.).

LITERATURE CITED1. Absolom, D. R. 1986. Basic methods for the study of phagocy-

tosis. Methods Enzymol. 132:95-180.2. Barrett-Bee, K., Y. Hayes, R. G. Wilson, and J. F. Ryley. 1985.A comparison of phospholipase activity, cellular adherence andpathogenicity of yeasts. J. Gen. Microbiol. 131:1217-1221.

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