The inoculum size effect in the dimorphic fungus Candida albicans results from production of an extracellular quorum-sensing molecule (QSM). This molecule prevents mycelial development in both a growth morphology assay and a differentiation assay using three chemically distinct triggers for germ tube formation (GTF): l -proline, N -acetylglucosamine, and serum (either pig or fetal bovine). In all cases, the presence of QSM prevents the yeast-to-mycelium conversion, resulting in actively budding yeasts without influencing cellular growth rates. QSM exhibits general cross-reactivity within C. albicans in that supernatants from strain A72 are active on five other strains of C. albicans and vice versa. The QSM excreted by C. albicans is farnesol (C ₁₅ H ₂₆ O; molecular weight, 222.37). QSM is extracellular, and is produced continuously during growth and over a temperature range from 23 to 43°C, in amounts roughly proportional to the CFU/milliliter. Production is not dependent on the type of carbon source nor nitrogen source or on the chemical nature of the growth medium. Both commercial mixed isomer and ( E,E )-farnesol exhibited QSM activity (the ability to prevent GTF) at a level sufficient to account for all the QSM activity present in C. albicans supernatants, i.e., 50% GTF at ca. 30 to 35 μM. Nerolidol was ca. two times less active than farnesol. Neither geraniol (C ₁₀ ), geranylgeraniol (C ₂₀ ), nor farnesyl pyrophosphate had any QSM activity.

The opportunistic fungal pathogen Candida albicans is a part of the normal flora but it also causes systemic candidiasis if it reaches the bloodstream. Upon being phagocytized by macrophages, an important component of innate immunity, C. albicans rapidly upregulates a set of arginine biosynthetic genes. Arginine, urea, and CO ₂ induced hyphae in a density-dependent manner in wild-type, cph1/cph1 , and rim101/rim101 strains but not in efg1/efg1 or cph1/cph1 efg1/efg1 strains. Arginase (Car1p) converts arginine to urea, which in turn is degraded by urea amidolyase (Dur1,2p) to produce CO ₂ , a signal for hyphal switching. We used a dur1,2/dur1,2 mutant (KWN6) and the complemented strain, KWN8 ( dur1,2/dur1,2 :: DUR1,2/DUR1,2 ) to study germ tube formation. KWN6 could not make germ tubes in the presence of arginine or urea but did in the presence of 5% CO ₂ , which bypasses Dur1,2p. We also tested the effect of arginine on the interaction between the macrophage line RAW 264.7 and several strains of C. albicans . Arginine activated an Efg1p-dependent yeast-to-hypha switch, enabling wild-type C. albicans and KWN8 to escape from macrophages within 6 h, whereas KWN6 was defective in this regard. Additionally, two mutants that cannot synthesize arginine, BWP17 and SN152, were defective in making hyphae inside the macrophages, whereas the corresponding arginine prototrophs, DAY286 and SN87, formed germ tubes and escaped from macrophages. Therefore, metabolism of arginine by C. albicans controls hyphal switching and provides an important mechanism for escaping host defense.

The polymorphic fungus Candida albicans is one of the most versatile opportunistic pathogens in humans. Many organs of the human body are potential targets for infection by this pathogen, but infection is commonly localized in the gastrointestinal tract, an environment providing anaerobic growth conditions. We describe a chemically defined anaerobic growth medium for four strains of Candida albicans (A72, SC5314, MEN, and 10261). It is a defined liquid glucose-phosphate-proline growth medium supplemented with oleic acid, nicotinic acid, and ammonium chloride. The cells did not require or respond to added ergosterol. Oleic acid and nicotinic acid are growth factors which are required only for the anaerobic growth of C. albicans . An important technical feature of this study was the use of anaerobically grown inocula to study anaerobic growth. Anaerobically, the cells grew exclusively as mycelia at 25, 30, and 37°C. The doubling time at 30°C was ca. 20 h. The cells did not produce farnesol and did not respond to exogenous farnesol, and they were resistant to the highest tested levels of amphotericin B and four of the azole antifungals. We suggest that the anaerobic growth of C. albicans may contribute to the trailing end point phenomenon and the resistance of C. albicans biofilms to antifungal drugs.

This work extends our previous observation that the fungus Candida albicans secretes micromolar levels of farnesol and that accumulation of farnesol in vitro prevents the yeast-to-mycelium conversion in a quorum-sensing manner. What does farnesol do in vivo? The purpose of this study was to determine the role of farnesol during infection with a well-established mouse model of systemic candidiasis with C. albicans A72 administered by tail vein injection. This question was addressed by altering both endogenous and exogenous farnesol. For endogenous farnesol, we created a knockout mutation in DPP3 , the gene encoding a phosphatase which converts farnesyl pyrophosphate to farnesol. This mutant (KWN2) produced six times less farnesol and was ca. 4.2 times less pathogenic than its SN152 parent. The strain with DPP3 reconstituted (KWN4) regained both its farnesol production levels and pathogenicity. These mutants (KWN1 to KWN4) retained their full dimorphic capability. With regard to exogenous farnesol, farnesol was administered either intraperitoneally (i.p.) or orally in the drinking water. Mice receiving C. albicans intravenously and farnesol (20 mM) orally had enhanced mortality ( P < 0.03). Similarly, mice ( n = 40) injected with 1.0 ml of 20 mM farnesol i.p. had enhanced mortality ( P < 0.03), and the onset of mortality was 30 h sooner than for mice which received a control injection without farnesol. The effect of i.p. farnesol was more pronounced ( P < 0.04) when mice were inoculated with a sublethal dose of C. albicans . These mice started to die 4 days earlier, and the percent survival on day 6 postinoculation (p.i.) was five times lower than for mice receiving C. albicans with control i.p. injections. In all experiments, mice administered farnesol alone or Tween 80 alone remained normal throughout a 14-day observation period. Finally, beginning at 12 h p.i., higher numbers of C. albicans cells were detected in kidneys from mice receiving i.p. farnesol than in those from mice receiving control i.p. injections. Thus, reduced endogenous farnesol decreased virulence, while providing exogenous farnesol increased virulence. Taken together, these data suggest that farnesol may play a role in disease pathogenesis, either directly or indirectly, and thus may represent a newly identified virulence factor.

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.