Concentrations of (E,E)-farnesol needed to inhibit germ tube formation were determined for Candida albicans strains A72 and SC5314 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 farnesol was required. Farnesol blocked germ tube formation but did not block elongation of existing germ tubes

Atkin, Audrey L.

Dumitru, Raluca

Hornby, Jacob M.

Mosel, Daniel D.

Nickerson, Kenneth W.

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Microbiology Papers in the Biological Sciences 8-1-2005 Farnesol Concentrations Required To Block Germ Tube Formation in Candida albicans in the Presence and Absence of Serum Daniel D. Mosel University of Nebraska-Lincoln Raluca Dumitru University of Nebraska-Lincoln Jacob M. Hornby University of Nebraska-Lincoln, jmhornby@lcsc.edu Audrey L. Atkin University of Nebraska-Lincoln, aatkin@unl.edu Kenneth W. Nickerson University of Nebraska-Lincoln, knickerson1@unl.edu Follow this and additional works at: https://digitalcommons.unl.edu/bioscimicro  Part of the Microbiology Commons Mosel, Daniel D.; Dumitru, Raluca; Hornby, Jacob M.; Atkin, Audrey L.; and Nickerson, Kenneth W., "Farnesol Concentrations Required To Block Germ Tube Formation in Candida albicans in the Presence and Absence of Serum" (2005). Papers in Microbiology. 38. https://digitalcommons.unl.edu/bioscimicro/38 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Microbiology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2005, p. 4938–4940 Vol. 71, No. 80099-2240/05/$08.000 doi:10.1128/AEM.71.8.4938–4940.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.Farnesol Concentrations Required To Block Germ Tube Formation inCandida albicans in the Presence and Absence of SerumDaniel D. Mosel, Raluca Dumitru, Jacob M. Hornby,† Audrey L. Atkin,and Kenneth W. Nickerson*School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588-0666Received 5 February 2005/Accepted 11 March 2005Concentrations of (E,E)-farnesol needed to inhibit germ tube formation were determined for Candidaalbicans strains A72 and SC5314 by using six different conditions known to trigger germination. For definedmedia, 1 to 2 M farnesol was sufficient. However, with serum at 2 to 20%, up to 250 M farnesol was required.Farnesol blocked germ tube formation but did not block elongation of existing germ tubes.Fungal yeast-mycelium dimorphism is of interest because ofthe economic and medical importance of dimorphic fungi andbecause these organisms may serve as model systems for study-ing differentiation. Farnesol is a fungal quorum-sensing mole-cule with intriguing regulatory properties in Candida albicans(7). This compound is excreted continuously by C. albicans (7),and when it accumulates beyond a threshold level, it blocks theyeast-to-mycelium conversion (7). Farnesol blocks mycelial de-velopment initiated by any of three chemically distinct triggersfor germ tube formation: L-proline, N-acetylglucosamine, andserum (7). Of these, serum (10 to 20% [vol/vol] at 37°C) hasbeen used most commonly (9, 15). Serum has even been de-scribed as the “magic potion” for the induction of germ tubesby C. albicans (5). Thus, both farnesol’s mode of action and thetherapeutic potential of farnesol analogs are of interest.By stationary phase, 2 to 4 M farnesol, which is at a con-centration well above the 1.2 M concentration needed toblock germ tube formation in an N-acetylglucosamine-stimu-lated differentiation assay (17), has accumulated in cultures ofC. albicans (7, 8). However, some recent studies (10, 11, 16, 18,19) with farnesol and C. albicans have used farnesol concen-trations (250 to 500 M) that we believe are much higher thanthose that are physiologically relevant. Farnesol is a lipophilicmolecule that can accumulate in membranes, and the farnesoleffects observed at these concentrations may be nonspecific. Insome cases, these higher concentrations may result in physio-logical artifacts. Indeed, Kim et al. (10) found that 450 Mfarnesol inhibited growth of C. albicans by 35%.Our objectives in this study were threefold: (i) to identify theminimum levels of farnesol needed to block germ tube forma-tion in six media commonly used to trigger germination; (ii) toestablish the physiological relevance of the production levelswe have observed, 2 to 4 M farnesol (7, 8); and (iii) to test ourhypothesis that much higher levels of farnesol are needed toblock germ tube formation in serum. This hypothesis was de-veloped because of the presence of albumins in serum; albu-mins are noted for 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 (SigmaChemical, St. Louis, MO) was freshly prepared as a stocksolution (10 mM) in methanol and stored under nitrogen toprevent 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 2 107 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 wereremoved periodically for microscopic examination.The liquid media used were (i) 2.5 mM N-acetylglucosaminewith imidazole and magnesium (7), (ii) glucose phosphate pro-line (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 bovine sera were obtained from the Meat AnimalResearch Center, Clay Center, Nebr. The solid medium usedwas GPP with 1.5% agar. After being autoclaved, the moltenmedium was cooled to 45°C and then added to 100- by 15-mmplastic petri dishes (20 ml medium/dish) containing appropri-ate aliquots of farnesol. The dishes were swirled to mix thefarnesol into the medium before the agar solidified. Becausethe 20 ml per petri dish was estimated by eye, the resultingfarnesol concentrations also differ somewhat. We believe themedium volumes are accurate to 1 ml, and thus, the farnesolconcentrations would be accurate to 5%. Germ tube forma-tion was quantified on plates by using a Labophot-2 micro-scope (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. Controlcultures with no farnesol always had 98 to 100% germ tubes.For each of the defined media, the level of farnesol needed toreduce germ tube formation to 50% was 1 M for bothstrains of C. albicans (Table 1). Also, the levels of farnesolneeded were the same for liquid and solid GPP media (Table1); they were not influenced by the presence of agar. However,* Corresponding author. Mailing address: School of Biological Sci-ences, University of Nebraska, Lincoln, NE 68588-0666. Phone: (402)472-2253. Fax: (402) 472-8722. E-mail: knickerson1@unl.edu.† Present address: Division of Natural Sciences, Lewis-Clark StateCollege, Lewiston, ID 83501.4938the farnesol levels needed were influenced dramatically by thepresence of serum. Serum increased the amount of farnesolneeded in a dose-dependent manner (Table 1), which reached150 to 250 M farnesol at 10 to 20% serum. Virtually identicalresults were obtained with pig serum, horse serum, sheep se-rum, and bovine serum (Table 1).Commitment. We also sought to clarify the effect of farnesolon preexisting hyphae (Fig. 1). Germ tube formation wasblocked completely when farnesol was added at any time up to30 min after inoculation. However, it was not blocked at allwhen the farnesol was added 90 min after inoculation, and itwas stopped at an intermediate value (ca. 60% germ tubes)when the farnesol was added 60 min after inoculation (Fig. 1).These experiments were done with both strain A72 and strainSC5314 in both the N-acetylglucosamine differentiation me-dium and the GPP growth medium. The data shown in Fig. 1are for SC5314 in GPP. In all cases, farnesol blocked germ tubeformation by cells that did not already have visible germ tubes.These cells formed buds instead. However, farnesol neitherreversed nor altered germ tube formation by cells that alreadyhad visible germ tubes. Those cells were committed to mycelialdevelopment (2, 14). Thus, farnesol does not halt the growth orelongation of existing mycelia, even at concentrations as highas 50 M. When farnesol was added 2 hours after the inocu-lation of C. albicans A72 in N-acetylglucosamine, the germtubes 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.The disparity in farnesol concentrations needed with andwithout serum (Table 1) is consistent with our hypothesis onthe farnesol binding capacity of serum albumins. Albuminsplay an important role in the transport of sparingly solublemetabolic products from one tissue to another (20). Substancessuch as dyes, fatty acids, bilirubin, sulfonamides, and naphtho-quinone derivatives, all of which are sparingly soluble in water,are readily dissolved in albumin solutions (20), and in eachinstance, it has been demonstrated that the dissolved substanceis actually bound to the protein (20). Farnesol is also sparinglysoluble in water (7). These differences with and without serumfurther illustrate the advantages of working with chemicallydefined media. Moreover, the fatty acid binding abilities ofserum albumins can be exploited; adding fatty acid-deficientbovine serum albumin (0.4%) is a common mechanism foravoiding the toxic effects of excess fatty acids. The added al-bumins create a slow-release fatty acid buffer (3). We expectthat the influences of farnesol on cell morphology will differgreatly during pathogenesis depending on location within thebody. Two relevant factors would be the presence of albuminsand the degree of anaerobicity, since C. albicans does notexcrete farnesol under strictly anaerobic conditions (4).This work was supported by grants from the National Science Foun-dation (MCB-0110999) and the University of Nebraska Tobacco Set-tlement Biomedical Research Enhancement Fund. D. D. Mosel, anundergraduate student at Nebraska Wesleyan University, Lincoln, wassupported by a grant from the Howard Hughes Medical Institute toNebraska Wesleyan University.We thank Susan Hassler and Joe Ford, Meat Animal ResearchCenter, Clay Center, Nebr., for providing the serum samples.REFERENCES1. Atlas, R. M. 1993. Handbook of microbiological media. CRC Press, BocaRaton, Fla.2. Chaffin, W. L., and D. E. Wheeler. 1981. Morphological commitment inCandida albicans. Can. J. Microbiol. 27:131–137.3. Das, D. V. M., and G. Weeks. 1979. Effects of polyunsaturated fatty acids onthe growth and differentiation of the cellular slime mould, Dictyosteliumdiscoideum. Exp. Cell Res. 118:237–243.4. Dumitru, R., J. M. Hornby, and K. W. Nickerson. 2004. Defined anaerobicFIG. 1. Kinetics of germ tube formation (GTF) with farnesoladded at various time points. C. albicans SC5314 was grown in GPPmedium at 37°C, and farnesol (30 M) was added at time zero (■), 30min (F), 60 min (Œ), and 90 min (}). X, no farnesol addition. Valuesshown are the averages of duplicate measurements conducted on sep-arate days which agreed within 5%. The values for the samples withfarnesol added at 90 min were the same as those with no farnesoladdition.TABLE 1. Concentration of farnesol needed to reduce germ tubeformation to 50%Germ tube triggerConcn of farnesol (M)a forindicated strainA72 SC5314N-Acetylglucosamine 1 0.9GPP medium 1 1Solid GPP medium 0.75 1Lee’s medium 1 3YNB plus N-acetylglucosamine 1 1RPMI 1640 0.9 1Serum (%)2 9 (P) 17.5 (P), 10 (H)5 ND 25 (P), 50 (B)10 150 (P) 175 (P), 150 (B)20 ND 250 (P), 250 (S)a Concentration of farnesol (in M) needed to reduce germ tube formation to50%. Replicate experiments (two to four) were conducted at 37°C. Germ tubeformation was measured at 4 hours for all media except solid GPP, which wasmeasured at 5 hours. All controls without farnesol had 98% germ tube forma-tion. Values indicated are the means of two to four separate experiments. Allvalues are within 10% of the mean. Sera were pig (P), horse (H), sheep (S), orbovine (B) sera. YNB, yeast nitrogen base without amino acids; ND, not deter-mined.VOL. 71, 2005 FARNESOL CONCENTRATIONS FOR CANDIDA ALBICANS 4939growth medium for studying Candida albicans basic biology and resistance toeight antifungal drugs. Antimicrob. Agents Chemother. 48:2350–2354.5. Ernst, J. F. 2000. Transcription factors in Candida albicans—environmentalcontrol of morphogenesis. Microbiology 146:1763–1774.6. Hornby, J. M. 2003. Quorum sensing and the regulation of morphology inthe dimorphic fungus Candida albicans. Ph.D. thesis. University of Nebras-ka—Lincoln, Lincoln.7. Hornby, J. M., E. C. Jensen, A. D. Lisec, J. J. Tasto, B. Jahnke, R. Shoe-maker, P. Dussault, and K. W. Nickerson. 2001. Quorum sensing in thedimorphic fungus Candida albicans is mediated by farnesol. Appl. Environ.Microbiol. 67:2982–2992.8. Hornby, J. M., and K. W. Nickerson. 2004. Enhanced production of farnesolby Candida albicans treated with four azoles. Antimicrob. Agents Che-mother. 48:2305–2307.9. Hudson, D. A., Q. L. Sciascia, R. J. Sanders, G. E. Norris, P. J. B. Edwards,P. A. Sullivan, and P. C. Farley. 2004. Identification of the dialyzable seruminducer of germ-tube formation in Candida albicans. Microbiology 150:3041–3049.10. Kim, S., E. Kim, D.-S. Shin, H. Kang, and K.-B. Oh. 2002. Evaluation ofmorphogenic regulatory activity of farnesoic acid and its derivatives againstCandida albicans dimorphism. Bioorg. Med. Chem. Lett. 12:895–898.11. Kruppa, M., B. P. Krom, N. Chauchan, A. V. Bambach, R. L. Cihlar, andR. A. Calderone. 2004. The two-component signal transduction proteinChk1p regulates quorum sensing in Candida albicans. Eukaryot. Cell 3:1062–1065.12. Kulkarni, R. K., and K. W. Nickerson. 1981. Nutritional control of dimor-phism in Ceratocystis ulmi. Exp. Mycol. 5:148–154.13. Lee, K. L., H. R. Buckley, and C. C. Campbell. 1975. An amino acid liquidsynthetic medium for the development of mycelial and yeast forms of Can-dida albicans. Sabouraudia 13:148–153.14. Mitchell, L. H., and D. R. Soll. 1979. Commitment to germ tube or budformation during release from stationary phase in Candida albicans. Exp.Cell Res. 120:167–179.15. Odds, F. C. 1988. Candida and candidosis, 2nd ed. Bailliere Tindall, London,United Kingdom.16. Ramage, G., S. P. Saville. B. L. Wickes, and J. L. Lopez-Ribot. 2002. Inhi-bition of Candida albicans biofilm formation by farnesol, a quorum-sensingmolecule. Appl. Environ. Microbiol. 68:5459–5463.17. Schepin, R., J. M. Hornby, E. Burger, T. Niessen, P. Dussault, and K. W.Nickerson. 2003. Quorum sensing in Candida albicans: probing farnesol’smode of action with 40 natural and synthetic farnesol analogs. Chem. Biol.10:743–750.18. Soto, T., T. Watanabe, T. Mikami, and T. Matsumoto. 2004. Farnesol, amorphogenetic autoregulatory substance in the dimorphic fungus Candidaalbicans, inhibits hyphae growth through suppression of a mitogen-activatedprotein kinase cascade. Biol. Pharm. Bull. 27:751–752.19. Uppuluri, P., and L. Chaffin. 2004. Effect of farnesol on expression ofCandida albicans stationary phase genes SNO1 and SNZ1. Abstr. 7th Am.Soc. Microbiol. Candida Candidiasis Conf., Austin, Tex., abstr. 243.20. White, A., P. Handler, and E. L. Smith. 1959. Principles of biochemistry, 3rded. McGraw-Hill, New York, N.Y.4940 MOSEL ET AL. APPL. ENVIRON. MICROBIOL.

Farnesol Concentrations Required To Block Germ Tube Formation in \u3ci\u3eCandida albicans\u3c/i\u3e in the Presence and Absence of Serum

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Papers in Microbiology Papers in the Biological Sciences 
8-1-2005 
Farnesol Concentrations Required To Block Germ Tube Formation 
in Candida albicans in the Presence and Absence of Serum 
Daniel D. Mosel 
University of Nebraska-Lincoln 
Raluca Dumitru 
University of Nebraska-Lincoln 
Jacob M. Hornby 
University of Nebraska-Lincoln, jmhornby@lcsc.edu 
Audrey L. Atkin 
University of Nebraska-Lincoln, aatkin@unl.edu 
Kenneth W. Nickerson 
University of Nebraska-Lincoln, knickerson1@unl.edu 
Follow this and additional works at: https://digitalcommons.unl.edu/bioscimicro 
 Part of the Microbiology Commons 
Mosel, Daniel D.; Dumitru, Raluca; Hornby, Jacob M.; Atkin, Audrey L.; and Nickerson, Kenneth W., 
"Farnesol Concentrations Required To Block Germ Tube Formation in Candida albicans in the Presence 
and Absence of Serum" (2005). Papers in Microbiology. 38. 
https://digitalcommons.unl.edu/bioscimicro/38 
This Article is brought to you for free and open access by the Papers in the Biological Sciences at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Microbiology by an 
authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2005, p. 4938–4940 Vol. 71, No. 8
0099-2240/05/$08.000 doi:10.1128/AEM.71.8.4938–4940.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Farnesol Concentrations Required To Block Germ Tube Formation in
Candida albicans in the Presence and Absence of Serum
Daniel D. Mosel, Raluca Dumitru, Jacob M. Hornby,† Audrey L. Atkin,
and Kenneth W. Nickerson*
School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588-0666
Received 5 February 2005/Accepted 11 March 2005
Concentrations of (E,E)-farnesol needed to inhibit germ tube formation were determined for Candida
albicans strains A72 and SC5314 by using six different conditions known to trigger germination. For defined
media, 1 to 2 M farnesol was sufficient. However, with serum at 2 to 20%, up to 250 M farnesol was required.
Farnesol blocked germ tube formation but did not block elongation of existing germ tubes.
Fungal yeast-mycelium dimorphism is of interest because of
the economic and medical importance of dimorphic fungi and
because these organisms may serve as model systems for study-
ing differentiation. Farnesol is a fungal quorum-sensing mole-
cule with intriguing regulatory properties in Candida albicans
(7). This compound is excreted continuously by C. albicans (7),
and when it accumulates beyond a threshold level, it blocks the
yeast-to-mycelium conversion (7). Farnesol blocks mycelial de-
velopment initiated by any of three 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 de-
scribed as the “magic potion” for the induction of germ tubes
by C. albicans (5). Thus, both farnesol’s mode of action and the
therapeutic potential of farnesol analogs are of interest.
By stationary phase, 2 to 4 M farnesol, which is at a con-
centration well above the 1.2 M concentration needed to
block germ tube formation in an N-acetylglucosamine-stimu-
lated differentiation 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 concen-
trations (250 to 500 M) that we believe are much higher than
those that are physiologically relevant. Farnesol is a lipophilic
molecule that can accumulate in membranes, and the farnesol
effects observed at these concentrations may be nonspecific. In
some cases, these higher concentrations may result in physio-
logical artifacts. Indeed, Kim et al. (10) found that 450 M
farnesol inhibited growth of C. albicans by 35%.
Our objectives in this study were threefold: (i) to identify the
minimum levels of farnesol needed to block germ tube forma-
tion in six media commonly used to trigger germination; (ii) to
establish the physiological relevance of the production levels
we have observed, 2 to 4 M farnesol (7, 8); and (iii) to test our
hypothesis that much higher levels of farnesol are needed to
block germ tube formation in serum. This hypothesis was de-
veloped because of the presence of albumins in serum; albu-
mins are noted for their nonspecific lipid binding capacity (20).
Procedures for the growth and storage of C. albicans were as
described 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 50
mM potassium phosphate buffer (pH 6.5) prior to use. Resting
cells were inoculated into 5 ml of prewarmed (37°C) medium
at a cell density of 2 107 per ml and incubated in 25-ml flasks
at 37°C with aeration by shaking at 200 rpm on a G2 shaker
(New Brunswick Scientific Co., Edison, NJ). Samples were
removed periodically for microscopic examination.
The liquid media used were (i) 2.5 mM N-acetylglucosamine
with imidazole and magnesium (7), (ii) glucose phosphate pro-
line (GPP) (7, 12), (iii) Lee’s medium (13), (iv) yeast nitrogen
base 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 bovine sera were obtained from the Meat Animal
Research Center, Clay Center, Nebr. The solid medium used
was GPP with 1.5% agar. After being autoclaved, the molten
medium was cooled to 45°C and then added to 100- by 15-mm
plastic petri dishes (20 ml medium/dish) containing appropri-
ate aliquots of farnesol. The dishes were swirled to mix the
farnesol into the medium before the agar solidified. Because
the 20 ml per petri dish was estimated by 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 forma-
tion was quantified on plates by using a Labophot-2 micro-
scope (Nikon Inc. Instrument Group, Melville, NY) modified
as a tetrad dissection microscope (Micro Video Instruments,
Avon, MA) using a Plan 10/0.30 objective lens.
Farnesol concentrations with and without serum. Control
cultures with no farnesol always had 98 to 100% germ tubes.
For each of the defined media, the level of farnesol needed to
reduce germ tube formation to 50% was 1 M for both
strains of C. albicans (Table 1). Also, the levels of farnesol
needed were the same for liquid and solid GPP media (Table
1); they were not influenced by the presence of agar. However,
* Corresponding author. Mailing address: School of Biological Sci-
ences, University of Nebraska, Lincoln, NE 68588-0666. Phone: (402)
472-2253. Fax: (402) 472-8722. E-mail: knickerson1@unl.edu.
† Present address: Division of Natural Sciences, Lewis-Clark State
College, Lewiston, ID 83501.
4938
the farnesol levels needed were influenced dramatically by the
presence of serum. Serum increased the amount of farnesol
needed in a dose-dependent manner (Table 1), which reached
150 to 250 M farnesol at 10 to 20% serum. Virtually identical
results were obtained with pig serum, horse serum, sheep se-
rum, and bovine serum (Table 1).
Commitment. We also sought to clarify the effect of farnesol
on preexisting hyphae (Fig. 1). Germ tube formation was
blocked completely when farnesol was added at any time up to
30 min after inoculation. However, it was not blocked at all
when the farnesol was added 90 min after inoculation, and it
was stopped at an intermediate value (ca. 60% germ tubes)
when the farnesol was added 60 min after inoculation (Fig. 1).
These experiments were done with both strain A72 and strain
SC5314 in both the N-acetylglucosamine differentiation me-
dium and the GPP growth medium. The data shown in Fig. 1
are for SC5314 in GPP. In all cases, farnesol blocked germ tube
formation by cells that did not already have visible germ tubes.
These cells formed buds instead. However, farnesol neither
reversed nor altered germ tube formation by cells that already
had visible germ tubes. Those cells were committed to mycelial
development (2, 14). Thus, farnesol does not halt the growth or
elongation of existing mycelia, even at concentrations as high
as 50 M. When farnesol was added 2 hours after the inocu-
lation of C. albicans A72 in N-acetylglucosamine, the germ
tubes continued to elongate for at least the next 3.5 hours (6).
Ramage et al. (16) reported a similar phenomenon during
biofilm formation by C. albicans. Once hyphal formation had
been 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.
The disparity in farnesol concentrations needed with and
without serum (Table 1) is consistent with our hypothesis on
the farnesol binding capacity of serum albumins. Albumins
play an important role in the transport of sparingly soluble
metabolic products from one tissue to another (20). Substances
such as dyes, fatty acids, bilirubin, sulfonamides, and naphtho-
quinone derivatives, all of which are sparingly soluble in water,
are readily dissolved in albumin solutions (20), and in each
instance, it has been demonstrated that the dissolved substance
is actually bound to the protein (20). Farnesol is also sparingly
soluble in water (7). These differences with and without serum
further illustrate the 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 a common mechanism for
avoiding the toxic effects of excess fatty acids. The added al-
bumins create a slow-release fatty acid buffer (3). We expect
that the influences of farnesol on cell morphology will differ
greatly during pathogenesis depending on location within the
body. Two relevant factors would be the presence of albumins
and the degree of anaerobicity, since C. albicans does not
excrete farnesol under strictly anaerobic conditions (4).
This work was supported by grants from the National Science Foun-
dation (MCB-0110999) and the University of Nebraska Tobacco Set-
tlement 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 Institute to
Nebraska Wesleyan University.
We thank Susan Hassler and Joe Ford, Meat Animal Research
Center, Clay Center, Nebr., for providing the serum samples.
REFERENCES
1. Atlas, R. M. 1993. Handbook of microbiological media. CRC Press, Boca
Raton, Fla.
2. Chaffin, W. L., and D. E. Wheeler. 1981. Morphological commitment in
Candida albicans. Can. J. Microbiol. 27:131–137.
3. Das, D. V. M., and G. Weeks. 1979. Effects of polyunsaturated fatty acids on
the growth and differentiation of the cellular slime mould, Dictyostelium
discoideum. Exp. Cell Res. 118:237–243.
4. Dumitru, R., J. M. Hornby, and K. W. Nickerson. 2004. Defined anaerobic
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 M) was added at time zero (■), 30
min (F), 60 min (Œ), and 90 min (}). X, no farnesol addition. Values
shown are the averages of duplicate measurements conducted on sep-
arate 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.
TABLE 1. Concentration of farnesol needed to reduce germ tube
formation to 50%
Germ tube trigger
Concn of farnesol (M)a for
indicated strain
A72 SC5314
N-Acetylglucosamine 1 0.9
GPP medium 1 1
Solid GPP medium 0.75 1
Lee’s medium 1 3
YNB plus N-acetylglucosamine 1 1
RPMI 1640 0.9 1
Serum (%)
2 9 (P) 17.5 (P), 10 (H)
5 ND 25 (P), 50 (B)
10 150 (P) 175 (P), 150 (B)
20 ND 250 (P), 250 (S)
a Concentration of farnesol (in M) needed to reduce germ tube formation to
50%. Replicate experiments (two to four) were conducted at 37°C. Germ tube
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measured at 5 hours. All controls without farnesol had 98% germ tube forma-
tion. Values indicated are the means of two to four separate experiments. All
values are within 10% of the mean. Sera were pig (P), horse (H), sheep (S), or
bovine (B) sera. YNB, yeast nitrogen base without amino acids; ND, not deter-
mined.
VOL. 71, 2005 FARNESOL CONCENTRATIONS FOR CANDIDA ALBICANS 4939
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4940 MOSEL ET AL. APPL. ENVIRON. MICROBIOL.


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Farnesol Concentrations Required To Block Germ Tube Formation in \u3ci\u3eCandida albicans\u3c/i\u3e in the Presence and Absence of Serum

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