Audrey L. Atkin

Quorum Sensing in Dimorphic Fungi: Farnesol and Beyond

Production of farnesol by Candida albicans is the first quorum-sensing system discovered in a eukaryote ([29][1]). In C. albicans , accumulated farnesol affects both dimorphism ([29][1], [50][2]) and biofilm formation ([62][3]). Fungal dimorphism is defined ([64][4]) as an environmentally controlled

Arginine-induced germ tube formation in Candida albicans is essential for escape from murine macrophage line RAW 264.7.

ABSTRACT 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 CO2 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 CO2, 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% CO2, 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.

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

ABSTRACT 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.

Candida albicans Tup1 Is Involved in Farnesol-Mediated Inhibition of Filamentous-Growth Induction

ABSTRACT Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is strongly correlated with virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. The tup1/tup1 and nrg1/nrg1 mutants, but not the rfg1/rfg1 mutant, failed to respond to farnesol. Treatment of C. albicans cells with farnesol caused a small but consistent increase in both TUP1 mRNA and protein levels. Importantly, this increase corresponds with the commitment point, beyond which added farnesol no longer blocks germ tube formation, and it correlates with a strong decrease in the expression of two Tup1-regulated hypha-specific genes, HWP1 and RBT1. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Farnesol did not affect EFG1 (a transcription regulator of filament development), NRG1, or RFG1 mRNA levels, demonstrating specific gene regulation in response to farnesol. Furthermore, the tup1/tup1 and nrg1/nrg1 mutants produced 17- and 19-fold more farnesol, respectively, than the parental strain. These levels of excess farnesol are sufficient to block filamentation in a wild-type strain. Our data are consistent with the role of Tup1 as a crucial component of the response to farnesol in C. albicans.

In Vivo and In Vitro Anaerobic Mating in Candida albicans

ABSTRACT Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37°C, block production of farnesol, and permit in vitro mating at 37°C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous hisG marker from one of the parental strains.

Long 3′-UTRs target wild-type mRNAs for nonsense-mediated mRNA decay in Saccharomyces cerevisiae

The nonsense-mediated mRNA decay (NMD) pathway, present in most eukaryotic cells, is a specialized pathway that leads to the recognition and rapid degradation of mRNAs with premature termination codons and, importantly, some wild-type mRNAs. Earlier studies demonstrated that aberrant mRNAs with artificially extended 3′-untranslated regions (3′-UTRs) are degraded by NMD. However, the extent to which wild-type mRNAs with long 3′-UTRs are degraded by NMD is not known. We used a global approach to identify wild-type mRNAs in Saccharomyces cerevisiae that have longer than expected 3′-UTRs, and of these mRNAs tested, 91% were degraded by NMD. We demonstrate for the first time that replacement of the natural, long 3′-UTR from wild-type PGA1 mRNA, which encodes a protein that is important for cell wall biosynthesis, with a short 3′-UTR renders it immune to NMD. The natural PGA1 3′-UTR is sufficient to target a NMD insensitive mRNA for decay by the NMD pathway. Finally, we show that nmd mutants are sensitive to Calcofluor White, which suggests that the regulation of PGA1 and other cell wall biosynthesis proteins by NMD is physiologically significant.

Regulation of Aromatic Alcohol Production in Candida albicans

ABSTRACT Colonization by the fungal pathogen Candida albicans varies significantly, depending upon the pH and availability of oxygen. Because of our interest in extracellular molecules as potential quorum-sensing molecules, we examined the physiological conditions which regulate the production of the aromatic alcohols, i.e., phenethyl alcohol, tyrosol, and tryptophol. The production of these fusel oils has been well studied for Saccharomyces cerevisiae. Our data show that aromatic alcohol yields for C. albicans are determined by growth conditions. These conditions include the availability of aromatic amino acids, the pH, oxygen levels, and the presence of ammonium salts. For example, for wild-type C. albicans, tyrosol production varied 16-fold merely with the inclusion of tyrosine or ammonium salts in the growth medium. Aromatic alcohol production also depends on the transcription regulator Aro80p. Our results are consistent with aromatic alcohol production—aromatic transaminases (gene products for ARO8 and ARO9), aromatic decarboxylase (ARO10), and alcohol dehydrogenase (ADH)—via the fusel oil pathway. The expression of ARO8, ARO9, and ARO10 is also pH dependent. ARO8 and ARO9 were alkaline upregulated, while ARO10 was alkaline downregulated. The alkaline-dependent change in expression of ARO8 was Rim101 independent, while the expression of ARO9 was Rim101 dependent.

Activity and Toxicity of Farnesol towards Candida albicans Are Dependent on Growth Conditions

ABSTRACT Farnesol interacts with Candida albicans as both a quorum-sensing molecule and toxic agent, but confusion abounds regarding which conditions promote these distinct responses. Farnesol sensitivity was measured when inoculum cell history and size, temperature, and growth media were altered. Parameters for farnesol tolerance/sensitivity were defined, validating previous studies and identifying new variables, such as energy availability. This study clearly defines what farnesol concentrations are lethal to C. albicans, based on environmental conditions.

Genetic background affects relative nonsense mRNA accumulation in wild-type and upf mutant yeast strains

Abstract The Saccharomyces cerevisiae nonsense-mediated mRNA decay (NMD) pathway targets mRNAs with premature stop codons and some wild-type mRNAs for accelerated decay. Upf1p, Upf2p and Upf3p are required for NMD. NMD-targeted mRNAs are degraded rapidly in wild-type cells and stabilized in upf1, upf2 or upf3 mutants. We report here that the relative CYH2 pre-mRNA/mRNA accumulation is enhanced in cells derived from a W303 background, compared with a variety of commonly used strains. The enhanced CYH2 pre-mRNA accumulation phenotype results from a larger difference in mRNA half-lives in the W303 strains than two previously used strains. This phenotype can be selected in crosses and is also seen in upf2 and upf3 mutants. These results suggest there are genes that influence the efficiency of NMD and that yeast strains derived from the W303 background may be useful for measurement of abundance and half-lives of low abundance, short-lived NMD substrates.

Candida albicans Czf1 and Efg1 Coordinate the Response to Farnesol during Quorum Sensing, White-Opaque Thermal Dimorphism, and Cell Death

ABSTRACT Quorum sensing by farnesol in Candida albicans inhibits filamentation and may be directly related to its ability to cause both mucosal and systemic diseases. The Ras1-cyclic AMP signaling pathway is a target for farnesol inhibition. However, a clear understanding of the downstream effectors of the morphological farnesol response has yet to be unraveled. To address this issue, we screened a library for mutants that fail to respond to farnesol. Six mutants were identified, and the czf1Δ/czf1Δ mutant was selected for further characterization. Czf1 is a transcription factor that regulates filamentation in embedded agar and also white-to-opaque switching. We found that Czf1 is required for filament inhibition by farnesol under at least three distinct environmental conditions: on agar surfaces, in liquid medium, and when embedded in a semisolid agar matrix. Since Efg1 is a transcription factor of the Ras1-cyclic AMP signaling pathway that interacts with and regulates Czf1, an efg1Δ/efg1Δ czf1Δ/czf1Δ mutant was tested for filament inhibition by farnesol. It exhibited an opaque-cell-like temperature-dependent morphology, and it was killed by low farnesol levels that are sublethal to wild-type cells and both efg1Δ/efg1Δ and czf1Δ/czf1Δ single mutants. These results highlight a new role for Czf1 as a downstream effector of the morphological response to farnesol, and along with Efg1, Czf1 is involved in the control of farnesol-mediated cell death in C. albicans.