The dimorphic fungus Candida albicans is a commensal and opportunistic fungal pathogen of humans. It secretes at least four small lipophilic molecules, farnesol and three aromatic fusel alcohols. Farnesol has been identified as both a quorum sensing molecule (QSM) and a virulence factor. Our gas chromatography (GC)-based assay for these molecules exhibits high throughput, prevention of analyte loss by avoiding filtration and rotary evaporation, simultaneous cell lysis and analyte extraction by ethyl acetate, and the ability to compare whole cultures with their cell pellets and supernatants. Farnesol synthesis and secretion were separable phenomena and pellet:supernatant ratios for farnesol were high, up to 12:1. The assay was validated in terms of precision, specificity, ruggedness, accuracy, solution stability, detection limits (DL), quantitation limits (QL), and dynamic range. The DL for farnesol was 0.02 ng/µl (0.09 µM). Measurement quality was assessed by the relative error of the whole culture versus the sum of pellet and supernatant fractions (WPS). C. albicans strain SC5314 grown at 30 °C in complex and defined media (YPD and mRPMI) was assayed in biological triplicate 17 times over 3 days. Farnesol and the three aromatic fusel alcohols can be measured in the same assay. The levels of all four are greatly altered by the growth medium chosen. Significantly, the three fusel alcohols are synthesized during stationary phase, not during growth. They are secreted quickly without being retained in the cell pellet and may accumulate up to mM concentrations. Key points• Quantitative analysis of both intra-and extracellular farnesol, and aromatic fusel oils.• High throughput, whole culture assay with simultaneous lysis and extraction.• Farnesol secretion and synthesis are distinct and separate events.

Candida albicans is an efficient colonizer of human gastrointestinal tracts and skin and is an opportunistic pathogen. C. albicans exhibits morphological plasticity and the ability to switch between yeast and filamentous morphologies is associated with virulence. One regulator of this switch is the quorum sensing molecule farnesol which is produced by C. albicans throughout growth. However, the synthesis, secretion, regulation, and turnover of farnesol is not fully understood. To address this, we used our improved farnesol assay to screen a transcription regulator knockout library for differences in farnesol accumulation in whole cultures, pellets, and supernatants. All screened mutants produced farnesol and they averaged 9.2X more farnesol in the pellet than the supernatant. Nineteen mutants had significant differences with ten mutants producing more farnesol than their SN152+ wild-type control strain while nine produced less. Seven mutants exhibited greater secretion of farnesol while two exhibited less. We examined the time course for farnesol accumulation in six mutants with the greatest accumulation differences and found that those differences persisted throughout growth and they were not time dependent. Significantly, two high accumulating mutants did not exhibit the decay in farnesol levels during stationary phase characteristic of wild type C. albicans, suggesting that a farnesol modification/degradation mechanism is absent in these mutants. Identifying these transcriptional regulators provides new insight into farnesol’s physiological functions regarding cell cycle progression, white-opaque switching, yeast-mycelial dimorphism, and response to cellular stress.

Candida albicansis an efficient colonizer of human gastrointestinal tracts and skin and is an opportunistic pathogen.C. albicansexhibits morphological plasticity and the ability to switch between yeast and filamentous morphologies is associated with virulence. One regulator of this switch is the quorum sensing molecule farnesol which is produced inC. albicanscells throughout growth. In addition, aromatic fusel alcohols accumulate inC. albicanssupernatants and are implicated in regulating morphogenesis. The synthesis, secretion, and turnover of these molecules is not fully understood. To address this, we used our improved assay for farnesol and aromatic fusel alcohol quantification of whole cultures, pellets, and supernatants, to screen a transcription regulator knockout library of 164 mutants for differences in farnesol and aromatic fusel alcohol accumulation and localization. We found that all mutants produced farnesol with an average of 9.2X more farnesol localizing to the pellet in most mutants. Nineteen mutants had significant differences in accumulation and nine differed in localization. Ten mutants produced more farnesol than their SN152+parent, while nine produced less. Seven mutants exhibited greater secretion of farnesol while two exhibited less. Several of these mutants have known roles in white/opaque switching and cell cycle progression such as the farnesol overproducing mutantsahr1andswi4. The aromatic fusel alcohols were similarly characterized by four over- and eleven under-producing mutants. Phenethyl alcohol and tryptophol accumulation were closely proportional to one another, but despite their structural similarity, phenethyl alcohol and tyrosol accumulation were often regulated differently.