Farnesol Concentrations Required To Block Germ Tube Formation in Candida albicans in the Presence and Absence of Serum

Concentrations of (E,E)-farnesol needed to inhibit germ tubeformation were determined for Candida albicans strains A72 andSC5314 by using six different conditions known to trigger germination.For defined media, 1 to 2 µM farnesol was sufficient.However, with serum at 2 to 20%, up to 250 µM farnesolwas required. Farnesol blocked germ tube formation but did notblock elongation of existing germ tubes.

INTRODUCTION

Top

Introduction

Fungal yeast-mycelium dimorphism is of interest because of theeconomic and medical importance of dimorphic fungi and becausethese organisms may serve as model systems for studying differentiation.Farnesol is a fungal quorum-sensing molecule with intriguingregulatory properties in Candida albicans (7). This compoundis excreted continuously by C. albicans (7), and when it accumulatesbeyond a threshold level, it blocks the yeast-to-mycelium conversion(7). Farnesol blocks mycelial development initiated by any ofthree chemically distinct triggers for germ tube formation:L-proline, N-acetylglucosamine, and serum (7). Of these, serum(10 to 20% [vol/vol] at 37°C) has been used most commonly(9, 15). Serum has even been described as the "magic potion"for the induction of germ tubes by C. albicans (5). Thus, bothfarnesol's mode of action and the therapeutic potential of farnesolanalogs are of interest.

By stationary phase, 2 to 4 µM farnesol, which is at aconcentration well above the 1.2 µM concentration neededto block germ tube formation in an N-acetylglucosamine-stimulateddifferentiation assay (17), has accumulated in cultures of C.albicans (7, 8). However, some recent studies (10, 11, 16, 18,19) with farnesol and C. albicans have used farnesol concentrations(250 to 500 µM) that we believe are much higher than thosethat are physiologically relevant. Farnesol is a lipophilicmolecule that can accumulate in membranes, and the farnesoleffects observed at these concentrations may be nonspecific.In some cases, these higher concentrations may result in physiologicalartifacts. Indeed, Kim et al. (10) found that 450 µM farnesolinhibited growth of C. albicans by 35%.

Our objectives in this study were threefold: (i) to identifythe minimum levels of farnesol needed to block germ tube formationin six media commonly used to trigger germination; (ii) to establishthe physiological relevance of the production levels we haveobserved, 2 to 4 µM farnesol (7, 8); and (iii) to testour hypothesis that much higher levels of farnesol are neededto block germ tube formation in serum. This hypothesis was developedbecause of the presence of albumins in serum; albumins are notedfor their nonspecific lipid binding capacity (20).

Procedures for the growth and storage of C. albicans were asdescribed previously (7, 8, 17). Strain A72 is also ATCC MYA-2430.Strain SC5314 was used for the genomic sequence for C. albicans(www.candidagenome.org). (E,E)-Farnesol (Sigma Chemical, St.Louis, MO) was freshly prepared as a stock solution (10 mM)in methanol and stored under nitrogen to prevent oxidation (17).

All resting cell preparations were washed three times in 50mM potassium phosphate buffer (pH 6.5) prior to use. Restingcells were inoculated into 5 ml of prewarmed (37°C) mediumat a cell density of 2 x 10⁷ per ml and incubated in 25-ml flasksat 37°C with aeration by shaking at 200 rpm on a G2 shaker(New Brunswick Scientific Co., Edison, NJ). Samples were removedperiodically for microscopic examination.

The liquid media used were (i) 2.5 mM N-acetylglucosamine withimidazole and magnesium (7), (ii) glucose phosphate proline(GPP) (7, 12), (iii) Lee's medium (13), (iv) yeast nitrogenbase without amino acids but supplemented with 2.5 mM N-acetylglucosamine(1), (v) RPMI 1640 (1) (Invitrogen), and (vi) serum (2 to 20%)in distilled, deionized water. Pig, horse, sheep, and bovinesera were obtained from the Meat Animal Research Center, ClayCenter, Nebr. The solid medium used was GPP with 1.5% agar.After being autoclaved, the molten medium was cooled to 45°Cand then added to 100- by 15-mm plastic petri dishes (20 mlmedium/dish) containing appropriate aliquots of farnesol. Thedishes were swirled to mix the farnesol into the medium beforethe agar solidified. Because the 20 ml per petri dish was estimatedby eye, the resulting farnesol concentrations also differ somewhat.We believe the medium volumes are accurate to ±1 ml,and thus, the farnesol concentrations would be accurate to ±5%.Germ tube formation was quantified on plates by using a Labophot-2microscope (Nikon Inc. Instrument Group, Melville, NY) modifiedas a tetrad dissection microscope (Micro Video Instruments,Avon, MA) using a Plan 10/0.30 objective lens.

Farnesol concentrations with and without serum.

Top

Farnesol concentrations with and...

Control cultures with no farnesol always had 98 to 100% germtubes. For each of the defined media, the level of farnesolneeded to reduce germ tube formation to 50% was $~$ 1 µM forboth strains of C. albicans (Table 1). Also, the levels of farnesolneeded were the same for liquid and solid GPP media (Table 1);they were not influenced by the presence of agar. However, thefarnesol levels needed were influenced dramatically by the presenceof serum. Serum increased the amount of farnesol needed in adose-dependent manner (Table 1), which reached 150 to 250 µMfarnesol at 10 to 20% serum. Virtually identical results wereobtained with pig serum, horse serum, sheep serum, and bovineserum (Table 1).

View this table:
[in this window]
[in a new window]
TABLE 1. Concentration of farnesol needed to reduce germ tube formation to 50%

Commitment.

Top

Commitment.

We also sought to clarify the effect of farnesol on preexistinghyphae (Fig. 1). Germ tube formation was blocked completelywhen farnesol was added at any time up to 30 min after inoculation.However, it was not blocked at all when the farnesol was added90 min after inoculation, and it was stopped at an intermediatevalue (ca. 60% germ tubes) when the farnesol was added 60 minafter inoculation (Fig. 1). These experiments were done withboth strain A72 and strain SC5314 in both the N-acetylglucosaminedifferentiation medium and the GPP growth medium. The data shownin Fig. 1 are for SC5314 in GPP. In all cases, farnesol blockedgerm tube formation by cells that did not already have visiblegerm tubes. These cells formed buds instead. However, farnesolneither reversed nor altered germ tube formation by cells thatalready had visible germ tubes. Those cells were committed tomycelial development (2, 14). Thus, farnesol does not halt thegrowth or elongation of existing mycelia, even at concentrationsas high as 50 µM. When farnesol was added 2 hours afterthe inoculation of C. albicans A72 in N-acetylglucosamine, thegerm tubes continued to elongate for at least the next 3.5 hours(6). Ramage et al. (16) reported a similar phenomenon duringbiofilm formation by C. albicans. Once hyphal formation hadbeen initiated, it was not inhibited by the addition of farnesol(16). The present work extends their finding to include other,nonbiofilm germ tube-forming conditions.

View larger version (11K):
[in this window]
[in a new window]
FIG. 1. Kinetics of germ tube formation (GTF) with farnesol added at various time points. C. albicans SC5314 was grown in GPP medium at 37°C, and farnesol (30 µM) was added at time zero ( ${blacksquare}$ ), 30 min (•), 60 min ( ${blacktriangleup}$ ), and 90 min ( ${blacklozenge}$ ). X, no farnesol addition. Values shown are the averages of duplicate measurements conducted on separate days which agreed within ±5%. The values for the samples with farnesol added at 90 min were the same as those with no farnesol addition.

The disparity in farnesol concentrations needed with and withoutserum (Table 1) is consistent with our hypothesis on the farnesolbinding capacity of serum albumins. Albumins play an importantrole in the transport of sparingly soluble metabolic productsfrom one tissue to another (20). Substances such as dyes, fattyacids, bilirubin, sulfonamides, and naphthoquinone derivatives,all of which are sparingly soluble in water, are readily dissolvedin albumin solutions (20), and in each instance, it has beendemonstrated that the dissolved substance is actually boundto the protein (20). Farnesol is also sparingly soluble in water(7). These differences with and without serum further illustratethe advantages of working with chemically defined media. Moreover,the fatty acid binding abilities of serum albumins can be exploited;adding fatty acid-deficient bovine serum albumin (0.4%) is acommon mechanism for avoiding the toxic effects of excess fattyacids. The added albumins create a slow-release fatty acid buffer(3). We expect that the influences of farnesol on cell morphologywill differ greatly during pathogenesis depending on locationwithin the body. Two relevant factors would be the presenceof albumins and the degree of anaerobicity, since C. albicansdoes not excrete farnesol under strictly anaerobic conditions(4).

ACKNOWLEDGMENTS

This work was supported by grants from the National ScienceFoundation (MCB-0110999) and the University of Nebraska TobaccoSettlement Biomedical Research Enhancement Fund. D. D. Mosel,an undergraduate student at Nebraska Wesleyan University, Lincoln,was supported by a grant from the Howard Hughes Medical Instituteto Nebraska Wesleyan University.

We thank Susan Hassler and Joe Ford, Meat Animal Research Center,Clay Center, Nebr., for providing the serum samples.

FOOTNOTES

* Corresponding author. Mailing address: School of Biological Sciences, University of Nebraska, Lincoln, NE 68588-0666. Phone: (402) 472-2253. Fax: (402) 472-8722. E-mail: knickerson1{at}unl.edu.

Present address: Division of Natural Sciences, Lewis-Clark StateCollege, Lewiston, ID 83501.

REFERENCES

Top