Purification and characterization of an autoregulatory substance capable of regulating the morphological transition in Candida albicans

Abstract

The yeast Candida albicans has a distinguishing feature, dimorphism, which is the ability to switch between two morphological forms: a budding yeast form and a multicellular invasive filamentous form. This ability has been postulated to contribute to the virulence of this organism. Studies on the morphological transition from a filamentous to a budding yeast form in C. albicans have shown that this organism excretes an autoregulatory substance into the culture medium. This substance was extracted and purified by normal-phase and reversed-phase HPLC. The autoregulatory substance was structurally identified as 3,7,11-trimethyl-2,6,10-dodecatrienoate (farnesoic acid) by NMR and mass spectrometry. Growth experiments suggest that this substance does not inhibit yeast cell growth but inhibits filamentous growth. These findings have implications for developmental signaling by the fungus and might have medicinal value in the development of antifungal therapies.

Figure 1

Demonstration of the essential role of secreted extracellular factor(s) by continuous-culture flow assay. A continuous-culture flow cell was used to monitor hyphal growth with (●) or without (○) medium flow (400 μl/min). To investigate the possibility of nutrient depletion or change in pH in the growth medium, 24-h culture filtrates were neutralized by pH adjustment (7.0, ▴) or nutritionally restored by the addition of 20% fresh GS medium to neutralized filtrate (▵). To ascertain the production of regulatory substances, culture filtrate was passed through a charcoal filter, and the filtrate was nutritionally restored by the addition of 20% fresh GS medium (■). The charcoal-absorbed substances also were eluted with methanol-chloroform (1:1, vol/vol) and concentrated, and the residue was dissolved in fresh GS medium (□). Those medium samples were then subjected to continuous-culture flow assay.

Figure 2

Reversed-phase HPLC elute profile of ARS. A linear gradient of acetonitrile in water was used to obtain the elution of ARS activity at about 80% acetonitrile (column: TSKgel ODS-80; flow rate: 1 ml/min; UV detector: 240 nm).

Figure 3

¹³C NMR spectrum of the purified ARS, 3,7,11-trimethyl-2,6,10-dodecatrienoate (or farnesoic acid), in CDCl₃. ¹H NMR (500 MHz, CDCl₃) δ : 1.60 (s, 6, C-7 CH₃ and C-11 CH₃), 1.68 (s, 3, C-11 CH₃), 1.98 (m, 2, H-9), 2.05 (m, 2, H-8), 2.18 (d, 3, J = 1.5 Hz, C-3 CH₃), 2.20 (s, 4, H-4 and H-5), 5.08 (m, 2, H-6 and H-10), and 5.70 ppm (s, 1, H-2). MS m/z (rel. int) 236 (M⁺ 0.8%), 193 (23), 136 (40), 121 (43), 100 (72), 81 (75), and 69 (100).

Figure 4

Effect of purified ARS on cell growth of C. albicans. (A) Yeast-to-hypha transition. ○, GS medium (control); ▴, GS medium + 0.5% DMSO; ●, GS medium + 0.5% DMSO + 3.12 μg/ml of ARS. (B) Yeast cells were incubated without (a) or with (b) 3.12 μg/ml of ARS in GS medium for 4 h at 37°C. (The bar indicates 35 μm.)

Figure 5

Effect of purified ARS on the hypha-to-yeast transition of C. albicans. At the time indicated by the arrow, GS medium was replaced by GS medium containing 3.12 μg/ml of purified ARS, and the morphology of hyphal tips was counted microscopically for 200 cells from each one of triplicate dishes. ○, GS medium + 0.5% DMSO (control); ●, GS medium + 0.5% DMSO + 3.12 μg/ml of ARS.

Figure 6

Time course of ARS appearance in the large-scale production system. Culture filtrates of C. albicans were collected every 2 h and lyophilized. A crude sample obtained by methanol extract (see Materials and Methods) was loaded onto a C₁₈ column (TSKgel ODS-80, 150 × 4.6 mm, 5 μm) and analyzed by reversed-phase HPLC (flow rate: 1 ml/min; detector: UV 240 nm). A linear gradient of acetonitrile in water was used to obtain elution of ARS at about 80% acetonitrile.

Figure 7

Dose response of C. albicans morphology to purified ARS. Data were obtained with our standard assay as inhibition of yeast cells converted to hyphae after 4 h of incubation at 37°C in the GS medium.