Farnesol Production Research Articles

Basic Research – Significance of Detection and Clinical Impact of <i>Candida albicans</i> in Non-Immunosuppressed Patients

Background: The clinical significance of the detection of Candida albicans on mucous membranes of the respiratory or intestinal tract from patients in intensive care units is still not finally clarified. Many patients reveal colonization, although, despite increased risk, there are only a few invasive infections detectable. Therefore, antimycotic therapy in this setting is strongly discouraged. In reality, however, many patients receive antimycotics as a pre-emptive therapy. To elucidate this point, a literature research was performed. Results: In the light of new results on the pathogenicity of C.albicans, the recommendation not to treat should be discussed anew. Without becoming invasive, C.albicans influences the immune system negatively in an anti-inflammatory sense (Th2) by means of at least two distinct mechanisms [action on toll like receptors (TLR), production of farnesol], which will be discussed. Conclusion: It is believed that patients in the phase of CARS or MARS can be further endangered by concomitant colonization of mucous membranes by C.albicans, i.e., in the sense of an anti-inflammatory immune response. Treatment with azole preparations, like fluconazole, which interacts with ergosterol synthesis in this phase of the disease, may trigger an additional effect on the patient, through increase of farnesol concentration by way of a negative feedback. Results of animal experiments on the immune system and concomitant therapeutic consequences indicate the need for verification through clinical trials.

Farnesol production from Escherichia coli by harnessing the exogenous mevalonate pathway

Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two-phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway-only control.

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.

Prenyl Alcohol Production by Expression of Exogenous Isopentenyl Diphosphate Isomerase and Farnesyl Diphosphate Synthase Genes inEscherichia coli

Isopentenyl diphosphate isomerase (idi) and farnesyl diphosphate synthase (ispA) genes were overexpressed in Escherichia coli. The resulting transformant showed 6.8-fold higher production of farnesol (389 microg/l). In a similar manner, overexpression of idi and mutated ispA led to high production of geranylgeraniol (128 microg/l).

Accumulation of prenyl alcohols by terpenoid biosynthesis inhibitors in various microorganisms

Squalene synthase inhibitors significantly accelerate the production of farnesol by various microorganisms. However, farnesol production by Saccharomyces cerevisiae ATCC 64031, in which the squalene synthase gene is deleted, was not affected by the inhibitors, indicating that farnesol accumulation is enhanced in the absence of squalene synthase activity. The combination of diphenylamine as an inhibitor of carotenoid biosynthesis and a squalene synthase inhibitor increases geranylgeraniol production by a yeast, Rhodotorula rubra NBRC 0870. An ent-kauren synthase inhibitor also enhances the production of farnesol and geranylgeraniol by a filamentous fungus, Gibberella fujikuroi NBRC 30336. These results indicate that the inhibition of downstream enzymes from prenyl diphosphate synthase leads to the production of farnesol and geranylgeraniol.

Various oils and detergents enhance the microbial production of farnesol and related prenyl alcohols

The object of this research was improvement of prenyl alcohol production with squalene synthase-deficient mutant Saccharomyces cerevisiae ATCC 64031. On screening of many kinds of additives, we found that oils and detergents significantly enhanced the extracellular production of prenyl alcohols. Soybean oil showed the most prominent effect among the additives tested. Its effect was accelerated by a high concentration of glucose in the medium. The combination of these cultivation conditions led to the production of more than 28 mg/l of farnesol in the soluble fraction of the broth. The addition of these compounds to the medium was an effective method for large-scale production of prenyl alcohols with microorganisms.

Effect of Farnesol on a Mouse Model of Systemic Candidiasis, Determined by Use of a DPP3 Knockout Mutant of Candida albicans

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.

In Vivo and In Vitro Anaerobic Mating in Candida albicans

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 degrees C, block production of farnesol, and permit in vitro mating at 37 degrees 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.

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

Farnesol restores wild-type colony morphology to 96% ofCandida albicanscolony morphology variants recovered following treatment with mutagens

Candida albicans is a diploid fungus that undergoes a morphological transition between budding yeast, hyphal, and pseudohyphal forms. The morphological transition is strongly correlated with virulence and is regulated in part by quorum sensing. Candida albicans produces and secretes farnesol that regulates the yeast to mycelia morphological transition. Mutants that fail to synthesize or respond to farnesol could be locked in the filamentous mode. To test this hypothesis, a collection of C. albicans mutants were isolated that have altered colony morphologies indicative of the presence of hyphal cells under environmental conditions where C. albicans normally grows only as yeasts. All mutants were characterized for their ability to respond to farnesol. Of these, 95.9% fully or partially reverted to wild-type morphology on yeast malt (YM) agar plates supplemented with farnesol. All mutants that respond to farnesol regained their hyphal morphology when restreaked on YM plates without farnesol. The observation that farnesol remedial mutants are so common (95.9%) relative to mutants that fail to respond to farnesol (4.1%) suggests that farnesol activates and (or) induces a pathway that can override many of the morphogenesis defects in these mutants. Additionally, 9 mutants chosen at random were screened for farnesol production. Two mutants failed to produce detectable levels of farnesol.

Enhanced pathogenicity of Candida albicans pre-treated with subinhibitory concentrations of fluconazole in a mouse model of disseminated candidiasis

To investigate the relative pathogenicity of Candida albicans treated with subinhibitory concentrations of fluconazole in a mouse model of disseminated candidiasis. Previous studies indicate that these cells secrete 10 times more farnesol than do untreated cells. In our usage, subinhibitory means a concentration which causes a prominent decrease in turbidity but still allows some cell growth. C. albicans A72 cells were grown overnight in 0-5.0 microM fluconazole, washed, and inoculated in mice by tail vein injection. Groups of 15 or 16 mice were injected with 1.3 x 10(6) cells and mortality was recorded for 7 days post-inoculation. The levels of farnesol in control and treated C. albicans were determined by GC/MS. The MIC50 for strain A72 was 0.125 mg/L (0.4 microM). Mice administered C. albicans pre-treated with 0.5 to 1.0 microM fluconazole died 2.5 to 4 days earlier and had 2 to 4 times higher mortality rates than mice given untreated C. albicans. Fluconazole (0.5 to 1.0 microM) pre-treated cells were 4.2 to 8.5 times more lethal (P < 0.001) than untreated cells. The extracellular, membrane bound, and intracellular farnesol concentrations of cells pre-treated with 1.0 muM fluconazole were 12-, 2- and 6-times those of untreated cells. The effects of fluconazole on C. albicans are very concentration-dependent. The enhanced pathogenicity of fluconazole pre-treated C. albicans in mice should be relevant to the therapeutic and prophylactic use of fluconazole. Further research is needed to explore whether farnesol production by C. albicans is a virulence factor.

Enhanced production of farnesol by Candida albicans treated with four azoles.

The dimorphic fungus Candida albicans excretes farnesol, which is produced enzymatically from the sterol biosynthetic intermediate farnesyl pyrophosphate. Inhibition of C. albicans by four azole antifungals, fluconazole, ketoconazole, miconazole, and clotrimazole, caused elevated farnesol production (10- to 45-fold). Furthermore, farnesol production occurs in both laboratory strains and clinical isolates (J. M. Hornby et al., Appl. Environ. Microbiol. 67:2982-2992, 2001) of C. albicans.

The characterization of transgenic rice plants expressing a pepper 5- epi aristolochene synthase, the first committed step enzyme for capsidiol synthesis in isoprenoid pathway

5- Epi-aristolochene synthase (EAS) from Capsicum annuum was introduced into the rice genome under the control of a maize ubiquitin promoter by Agrobacterium-mediated transformation. Fifteen independent transgenic rice plants were isolated and characterized. The integration and expression of the transgene were verified through the use of Southern, Northern, and Western blot analyses. The heterologous expression of EAS in transgenic rice did not interfere with the activities of endogenous squalene synthase (SS) or farnesyl diphosphatase (FDPase), in which both enzymes used the same substrate-farnesyl diphosphate (FDP), for the production of squalene and farnesol, respectively. Transgenic rice cells exhibited the induction of EAS enzyme activity upon elicitor ( N-acetylchitohexaose) and jasmonic acid (JA) treatments. The induction of EAS enzyme activity was accompanied by an increase in EAS mRNA when challenged by the elicitor. Results indicated that the maize ubiquitin promoter was upregulated upon jasmonic acid and elicitor treatments. Results also show that the EAS ectopic expression in transgenic rice plants resulted in the synthesis of 5- epi-aristolochene in vivo upon elicitor treatment, suggesting that the heterologous expression of pepper EAS is coupled with the endogenous biosynthetic pathway channel of isoprenoid in rice cells.

Farnesol as a Regulator of HMG-CoA Reductase Degradation: Characterization and Role of Farnesyl Pyrophosphatase

We have recently reported that the isoprenoid compound farnesol accelerates degradation of the cholesterologenic enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, when added to cultured cells. We have thus proposed that farnesol is a required nonsterol regulator of this degradation event (T. E. Meigs, D. S. Roseman, and R. D. Simoni, 1996,J. Biol. Chem.271, 7916–7922). In this report, we have studied the enzyme farnesyl pyrophosphatase (FPPase) in Chinese hamster ovary cells. We demonstrate that FPPase activity increases under conditions of increased metabolic flow through the isoprenoid pathway. Also, we show that a nonhydrolyzable analog of farnesyl pyrophosphate, an isoprenoid (phosphinylmethyl)phosphonate, inhibits FPPasein vitro,and when added to cells this inhibitor blocks the mevalonate-dependent, sterol-induced degradation of HMG-CoA reductase. Furthermore, exogenous farnesol overcomes the effect of this inhibitor. These results suggest an isoprenoid-mediated regulatory mechanism governing intracellular farnesol production and support the hypothesis that farnesol is a nonsterol regulator of reductase degradation.

Growth Retardant - Induced Inhibition of Neutral Terpene Biosynthesis Using Plant and Animal Cell-Free Systems1

Abstract Nine plant growth retardants were tested for their ability to inhibit neutral terpene biosynthesis by enzymes in cell-free extracts from rat liver, iris (Iris hollandica Hoog. cv. ‘Wedgwood’) sprouts, and castor bean (Ricinus communis L. cv. ‘Baker Hybrid 66’) seeds. Succinic acid 2,2-dimethylhydrazide (SADH), α-cyclopropyl-α (4-methoxyphenyl)-5-pyrimidine-methanol (A-Rest), n,n-dimethylmorpholinium chloride (BAS 0660-W), and 2-chloroethyl trimethyl-ammonium chloride (Cycocel) showed little or no inhibitory effects regardless of concentration or the test system used. Using rat liver enzymes, 2-isopropyl-4-dimethylamino-5-methylphenyl-l-piperidine-carboxy-late methyl chloride (AMO-1618) at 0.01 mM, inhibited cholesterol biosynthesis after the farnesyl-pyrophosphate step but had no inhibitory effect in the bean and iris cell-free systems. CHE-9064 (structure not released), Phosfon-D (2,4-dichlorobenzyl tributylphosphonium chloride) and 3-trifluoromethyl sulfonamido-p-acetotoluidide (Sustar) also inhibited cholesterol biosynthesis in rat liver enzymes at concentrations as low as 0.01 mM while 5-chloro-2-thenyl-tri-n-butyl chloride (CHE-8728) lost its effectiveness at concentrations below 0.1 mM. In the iris system, CHE-8728, Phosfon-D and Sustar inhibited cholesterol and farnesol production at 0.1 mM but were ineffective at lower concentrations. With the castor bean system, Sustar was the only retardant to inhibit terpene biosynthesis. Results indicate that CHE-8728, CHE-9064, Phosfon-D, and Sustar inhibit the terpene biosynthetic pathway prior to the formation of the farnesyl-pyrophosphate (rat liver and iris system) or geranylgeranyl-pyrophosphate (castor bean system).