Physiological adventures in Candida albicans: farnesol and ubiquinones.

SUMMARYFarnesol was first identified as a quorum-sensing molecule, which blocked the yeast to hyphal transition in Candida albicans, 22 years ago. However, its interactions with Candida biology are surprisingly complex. Exogenous (secreted or supplied) farnesol can also act as a virulence factor during pathogenesis and as a fungicidal agent triggering apoptosis in other competing fungi. Farnesol synthesis is turned off both during anaerobic growth and in opaque cells. Distinctly different cellular responses are observed as exogenous farnesol levels are increased from 0.1 to 100 µM. Reported changes include altered morphology, stress response, pathogenicity, antibiotic sensitivity/resistance, and even cell lysis. Throughout, there has been a dearth of mechanisms associated with these observations, in part due to the absence of accurate measurement of intracellular farnesol levels (Fi). This obstacle has recently been overcome, and the above phenomena can now be viewed in terms of changing Fi levels and the percentage of farnesol secreted. Critically, two aspects of isoprenoid metabolism present in higher organisms are absent in C. albicans and likely in other yeasts. These are pathways for farnesol salvage (converting farnesol to farnesyl pyrophosphate) and farnesylcysteine cleavage, a necessary step in the turnover of farnesylated proteins. Together, these developments suggest a unifying model, whereby high, threshold levels of Fi regulate which target proteins are farnesylated or the extent to which they are farnesylated. Thus, we suggest that the diversity of cellular responses to farnesol reflects the diversity of the proteins that are or are not farnesylated.

Farnesol as an antifungal agent: comparisons among MTLa and MTLα haploid and diploid Candida albicans and Saccharomyces cerevisiae.

Aims: Farnesol was identified 20 years ago in a search for Candida albicans quorum sensing molecules (QSM), but there is still uncertainty regarding many aspects of its mode of action including whether it employs farnesol transport mechanisms other than diffusion. Based on the structural similarity between farnesol and the farnesylated portion of the MTL a pheromone, we explored the effects of ploidy and mating type locus (MTL) on the antifungal activity of exogenous farnesol. Methods and results: We approached this question by examining five MTL a and five MTLα haploid strains with regard to their farnesol sensitivity in comparison to six heterozygous MTL a/ α diploids. We examined the haploid and diploid strains for percent cell death after exposure of exponentially growing cells to 0-200 µM farnesol. The heterozygous (MTL a/α) diploids were tolerant of exogenous farnesol whereas the MTL a and MTLα haploids were on average 2- and 4-times more sensitive, respectively. In the critical range from 10-40 µM farnesol their cell death values were in the ratio of 1:2:4. Very similar results were obtained with two matched sets of MAT a, MATα, and MAT a/α Saccharomyces cerevisiae strains. Conclusion: We propose that the observed MTL dependence of farnesol is based on differentially regulated mechanisms of entry and efflux which determine the actual cellular concentration of farnesol. The mechanisms by which pathogens such as C. albicans tolerate the otherwise lethal effects of farnesol embrace a wide range of physiological functions, including MTL type, ubiquinone type (UQ6-UQ9), energy availability, and aerobic/anaerobic status.

Farnesol remodels the peritoneal cavity immune environment influencing Candida albicans pathogenesis during intra-abdominal infection.

Candida albicans is a lifelong member of the mycobiome causing mucosal candidiasis and life-threatening, systemic, and intra-abdominal disease in immunocompromised and transplant patients. Despite the clinical importance of intra-abdominal candidiasis with mortality rates between 40% and 70%, the contribution of fungal virulence factors and host immune responses to disease has not been extensively studied. Secretion of the quorum-sensing molecule, farnesol, acts as a virulence factor for C. albicans during systemic infection, while inducing local, protective innate immune responses in oral models of infection. Previously, we reported that farnesol recruits macrophages to the peritoneal cavity in mice, suggesting a role for farnesol in innate immune responses. Here, we expand on our initial findings, showing that farnesol profoundly alters the peritoneal cavity microenvironment promoting innate inflammation. Intra-peritoneal injection of farnesol stimulates rapid local death of resident peritoneal cells followed by recruitment of neutrophils and inflammatory macrophages into the peritoneal cavity and peritoneal mesothelium associated with an early increase in chemokines followed by proinflammatory cytokines. These rapid inflammatory responses to farnesol significantly increase morbidity and mortality of mice with intra-abdominal candidiasis associated with increased formation of peritoneal adhesions, despite similar rates of fungal clearance from the peritoneal cavity and retro-peritoneal organs. C. albicans ddp3Δ/ddp3Δ knockout and reconstituted strains recapitulate these findings. This indicates that farnesol may be detrimental to the host during intra-abdominal infections. Importantly, our results highlight a need to understand how C. albicans virulence factors modulate the host immune response within the peritoneum, an exceedingly common site of Candida infection.

Transcriptional regulation of the synthesis and secretion of farnesol in the fungus Candida albicans: examination of the Homann transcription regulator knockout collection.

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 that is produced by C. albicans throughout growth. However, the synthesis, secretion, regulation, and turnover of farnesol are 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 9.2× 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.

Quantitative assay for farnesol and the aromatic fusel alcohols from the fungus Candida albicans.

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.

Deciphering fungal dimorphism: Farnesol's unanswered questions.

Candida albicans excretes E,E-farnesol as a virulence factor and quorum sensing molecule that prevents the yeast to hyphal conversion. Polke et al. (2016) identified eed1Δ/Δ as the first farnesol hypersensitive mutant of C. albicans. eed1Δ/Δ also excretes 10X more farnesol and while able to form hyphae, it cannot maintain hyphae. This mutant enables new research into unanswered questions, including the existence of potential farnesol receptors and transporters, regulation of farnesol synthesis, and relationships among farnesol, germ tube formation and hyphal maintenance. The eed1 farnesol hypersensitivity can be explained by higher internal concentrations of farnesol or lower thresholds for response. One possibility invokes misexpression of a transporter. Saccharomyces cerevisiae and C. albicans have transporters for farnesylated peptides, like the a-factor pheromone, which could potentially also transport farnesol for virulence and quorum sensing. Significantly, these transporters are repressed in MTLa/MTLα C. albicans. An evolutionary pressure for C. albicans to become diploid could derive from its use of farnesol. Alternatively, maintenance of hyphal growth may increase the farnesol response threshold. Finally, Dpp1p, Dpp2p and Dpp3p are non-specific pyrophosphatases responsible for farnesol synthesis. Changes in expression of these enzymes do not explain differences in farnesol levels implicating involvement of additional factors like a scaffolding molecule.

Candida albicans Quorum Sensing Molecules Stimulate Mouse Macrophage Migration.

The polymorphic commensal fungus Candida albicans causes life-threatening disease via bloodstream and intra-abdominal infections in immunocompromised and transplant patients. Although host immune evasion is a common strategy used by successful human fungal pathogens, C. albicans provokes recognition by host immune cells less capable of destroying it. To accomplish this, C. albicans white cells secrete a low-molecular-weight chemoattractive stimulant(s) of macrophages, a phagocyte that they are able to survive within and eventually escape from. C. albicans opaque cells do not secrete this chemoattractive stimulant(s). We report here a physiological mechanism that contributes to the differences in the interaction of C. albicans white and opaque cells with macrophages. E,E-Farnesol, which is secreted by white cells only, is a potent stimulator of macrophage chemokinesis, whose activity is enhanced by yeast cell wall components and aromatic alcohols. E,E-farnesol results in up to an 8.5-fold increase in macrophage migration in vitro and promotes a 3-fold increase in the peritoneal infiltration of macrophages in vivo. Therefore, modulation of farnesol secretion to stimulate host immune recognition by macrophages may help explain why this commensal is such a successful pathogen.

Immunity of the Saccharomyces cerevisiae SSY5 mRNA to nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway is a specialized pathway that triggers the rapid degradation of select mRNAs. Initially, identified as a pathway that degrades mRNAs with premature termination codons, NMD is now recognized as a pathway that also regulates some natural mRNAs. Since natural mRNAs do not typically contain premature termination codons, these mRNAs contain features that target them to NMD. In Saccharomyces cerevisiae mRNAs with atypically long 3'-UTRs are usually degraded by NMD, however in some conditions a constitutively expressed SSY5 mRNA with multiple NMD targeting signals including an atypically long 3'-UTR is an exception. We investigated the features of the SSY5 mRNAs that confer immunity to NMD. We found that the SSY5 mRNA 3'-UTRs are sufficient to target NMD insensitive mRNA to the pathway. Replacing the SSY5 3'-UTRs with the cyc1-512 3'-UTRs, known to target mRNAs to NMD or with the CYC1 3'-UTR, known not to target mRNAs to NMD, resulted in production of SSY5 mRNAs that were regulated by NMD. These observations suggest that the SSY5 mRNAs require sequences both within the 5'-UTR and/or ORF as well as the 3'-UTR to escape decay by NMD.

Candida albicans Czf1 and Efg1 coordinate the response to farnesol during quorum sensing, white-opaque thermal dimorphism, and cell death.

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.

Reducing odorous VOC emissions from swine manure using soybean peroxidase and peroxides.

The objective of the research was to determine the optimum application rates of soybean peroxidase (SBP) plus peroxide (SBPP) for reducing odorous VOC emissions from swine manure. Industrial-grade SBP was applied in combination with liquid hydrogen peroxide (H(2)O(2)) or powdered calcium peroxide (CaO(2)) to standard phenolic solutions and swine manure, and emissions were measured in a wind tunnel. The primary odorant in the untreated manure was 4-methylphenol, which accounted for 68-81% of the odor activity value. At the optimum application rate of SBPP (50 g L(-1)), 4-methylphenol emissions were reduced from the swine manure by 62% (H(2)O(2)) and 98% (CaO(2)) after 24h (P<0.0001). The CaO(2) had a longer residence time, remaining effective for 48 h with 92% reduction in emission rates (P<0.0001), while H(2)O(2) was similar to the control at 48 h (P=0.28).

Analysis of nonsense-mediated mRNA decay in Saccharomyces cerevisiae.

Nonsense-mediated mRNA decay is a highly conserved pathway that degrades mRNAs with premature termination codons. These mRNAs include mRNAs transcribed from nonsense or frameshift alleles as well as wild-type mRNA with signals that direct ribosomes to terminate prematurely. This unit describes techniques to monitor steady-state mRNA levels, decay rates, and structural features of mRNAs targeted by this pathway, as well as in vivo analysis of nonsense suppression and allosuppression in the yeast Saccharomyces cerevisiae. Protocols for the structural features of mRNA include analysis of cap status, 5' and 3' untranslated region (UTR) lengths, and poly(A) tail length.

Copper tolerance of Saccharomyces cerevisiae nonsense-mediated mRNA decay mutants.

The eukaryotic nonsense-mediated mRNA (NMD) is a specialized pathway that leads to the recognition and rapid degradation of mRNAs with premature termination codons, and importantly some natural mRNAs as well. Natural mRNAs with atypically long 3'-untranslated regions (UTRs) are degraded by NMD in Saccharomyces cerevisiae. A number of S. cerevisiae mRNAs undergo alternative 3'-end processing producing mRNA isoforms that differ in their 3'-UTR lengths. Some of these alternatively 3'-end processed mRNA isoforms have atypically long 3'-UTRs and would be likely targets for NMD-mediated degradation. Here, we investigated the role NMD plays in the regulation of expression of CTR2, which encodes a vacuolar membrane copper transporter. CTR2 pre-mRNA undergoes alternative 3'-end processing to produce two mRNA isoforms with 300-nt and 2-kb 3'-UTRs. We show that both CTR2 mRNA isoforms are differentially regulated by NMD. The regulation of CTR2 mRNA by NMD has physiological consequences, since nmd mutants are more tolerant to toxic levels of copper relative to wild-type yeast cells and the copper tolerance of nmd mutants is dependent on the presence of CTR2.

Yeast bioinformatics and strain engineering resources.

Saccharomyces cerevisiae, commonly known as baker's or budding yeast, is an attractive organism for design-based engineering because it is an industrially important organism with a well-annotated genome sequence and an extensive collection of resources for molecular analyses. This chapter describes the utility of Saccharomyces Genome Database for analysis of S. cerevisiae genes and identification of homologs, strategies for integration and analysis of gene expression data, and the genetic resources available for doing experiments using S. cerevisiae.

N5-phosphonoacetyl-L-ornithine (PALO): a convenient synthesis and investigation of influence on regulation of amino acid biosynthetic genes in Saccharomyces cerevisiae.

A scalable four-step synthesis of the ornithine transcarbamylase inhibitor N(5)-phosphonoacetyl-l-ornithine (PALO) is achieved through boroxazolidinone protection of ornithine. Investigations in the model organism Saccharomyces cerevisiae found that, in contrast to a previous report, PALO did not influence growth rate or expression of genes involved in arginine metabolism.

Activity and toxicity of farnesol towards Candida albicans are dependent on growth conditions.

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.

Cellular interactions of farnesol, a quorum-sensing molecule produced by Candida albicans.

Farnesol is a quorum-sensing molecule produced by Candida albicans that has many effects, including filament inhibition of this polymorphic fungus. In the past 9 years, the effect of farnesol on C. albicans has been reported in nearly 160 publications, with early work examining its influence on morphology. This article presents an update on the literature published since 2006, focusing on points that still need to be resolved as well as identifying possible artifacts that might interfere with this goal. In addition, the regulation of C. albicans farnesol production, C. albicans' resistance/sensitivity to farnesol and the influence of farnesol on other species as well as the host are discussed. It is intriguing that we still do not know precisely how farnesol works, but interference with the Ras1-cAMP pathway is part of the story.

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.

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

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% 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.

Regulation of aromatic alcohol production in Candida albicans.

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.

Candida albicans Tup1 is involved in farnesol-mediated inhibition of filamentous-growth induction.

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.