Ergosterol Biosynthetic Pathway Research Articles

Efficient 7-Dehydrocholesterol Production by Multiple Metabolic Engineering of Diploid Saccharomyces cerevisiae.

7-Dehydrocholesterol (7-DHC), a direct precursor of vitamin D3, has attracted increasing attention in microbial fermentation recently. In this study, 7-DHC biosynthesis in diploid Saccharomyces cerevisiae with robust ergosterol production was achieved by heterologous 24-dehydrocholesterol reductase expression, generating 44.1 mg/L 7-DHC, whereas the titer of ergosterol decreased by 40.5%. The ergosterol biosynthetic pathway was completely blocked by knocking out ERG6 and ERG5, affording a 4.2-fold increase in the 7-DHC titer. Subsequently, the facilitation of the mevalonate and the postsqualene pathways accompanied by elimination of transcriptional repressors enhanced 7-DHC synthesis, and the 7-DHC titer reached 738.5 mg/L in a shake flask. Further validation in a 50 L fermenter demonstrated that the 7-DHC titer reached 3.80 g/L within just 24 h, with productivity reaching 158.3 mg/L/h, setting a new benchmark as the highest reported to date. This study paves the way toward a large-scale and cost-effective manufacture of 7-DHC.

Ergosterol Biosynthesis and Regulation Impact the Antifungal Resistance and Virulence of Candida spp.

Ergosterol is a key fungal sterol that is mainly found in the plasma membrane and is responsible for the proper membrane structure, rigidity, permeability and activity of membrane proteins. Ergosterol plays a crucial role in the ability of fungi to adapt to environmental stresses. The biosynthesis of ergosterol is also intimately connected with the antifungal resistance and virulence of pathogenic fungi. The most common etiological agents of life-threatening fungal infections are yeasts belonging to the genus Candida. The antifungal agents mostly used to treat Candida spp. infections are azoles, which act as competitive inhibitors of sterol demethylase, a key enzyme in the fungal ergosterol biosynthetic pathway. Although most studies on ergosterol biosynthesis, its regulation and the uptake of sterols are from the baker’s yeast Saccharomyces cerevisiae, the study of ergosterol biosynthesis and its relationship to antifungal drug resistance and virulence in pathogenic fungi is of utmost importance. The increasing antifungal drug resistance of Candida spp. and the limited armamentarium of antimycotics pose a challenge in the development of new therapeutic approaches. This review summarizes the available data on ergosterol biosynthesis and related phenomena in Candida albicans and non-albicans Candida species (Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida auris) with special emphasis on C. albicans and C. glabrata as the most common etiological agents of systemic candidiasis.

Changes in Ergosterol Biosynthesis Alter the Response to Cycloheximide, 4-Nitroquinoline-N-Oxide, Weak Organic Acids, and Virulence in Candida glabrata.

The ERG6 gene encodes the sterol C24-methyltransferase converting zymosterol to fecosterol in the ergosterol biosynthetic pathway. Here, we extend the results of functional analysis of the CgERG6 gene, which was previously shown to modulate drug susceptibility in Candida glabrata mutant cells, by demonstrating that its deletion leads to increased susceptibility to cycloheximide, 4-nitroquinoline-N-oxide and weak organic acids, and such effects are associated with attenuated virulence. Together with abrogated efflux of drug substrates by CgCdr1p and CgPdr12p, the Cgerg6Δ mutation leads to reduced cell surface hydrophobicity and decreased virulence of the mutant cells of C. glabrata. The absence of CgErg6p impacts the lipid organization and function of the plasma membrane, resulting in non-specific permeability and abrogation of normal function of membrane-bound proteins accompanied by decreased virulence in Cgerg6Δ cells. Galleria mellonella larvae were used as a non-vertebrate animal host model to determine differences in the virulence potential of C. glabrata strains (parental strain and the Cgerg6Δ deletion mutant). We found that Cgerg6Δ mutant strain attenuated in virulence caused 25-30% survival of larvae compared with parental strain.

Alternative ergosterol biosynthetic pathways confer antifungal drug resistance in the human pathogens within the Mucor species complex.

The Mucor species complex comprises a variety of opportunistic pathogens known to cause mucormycosis, a potentially lethal fungal infection with limited therapeutic options. The only effective first-line treatments against mucormycosis consist of liposomal formulations of amphotericin B and the triazoles posaconazole and isavuconazole, all of which target components within the ergosterol biosynthetic pathway. This study uncovered M. circinelloides Erg3 and Erg6a as key enzymes to produce ergosterol, a vital constituent of fungal membranes. Absence of any of those enzymes leads to decreased ergosterol and consequently, resistance to ergosterol-binding polyenes such as amphotericin B. Particularly, losing Erg6a function poses a higher threat as the ergosterol pathway is channeled into alternative sterols similar to cholesterol, which maintain membrane permeability. As a result, erg6a mutants survive within the host and disseminate the infection, indicating that Erg6a deficiency may arise during human infections and confer resistance to the most effective treatment against mucormycoses.

The limit to evolutionary rescue depends on ploidy in yeast exposed to nystatin.

The number of copies of each chromosome, or ploidy, of an organism is a major genomic factor affecting adaptation. We set out to determine how ploidy can impact the outcome of evolution, as well as the likelihood of evolutionary rescue, using short-term experiments with yeast (Saccharomyces cerevisiae) in a high concentration of the fungicide nystatin. In similar experiments using haploid yeast, the genetic changes underlying evolutionary rescue were highly repeatable, with all rescued lines containing a single mutation in the ergosterol biosynthetic pathway. All of these beneficial mutations were recessive, which led to the expectation that diploids would find alternative genetic routes to adaptation. To test this, we repeated the experiment using both haploid and diploid strains and found that diploid populations did not evolve resistance. Although diploids are able to adapt at the same rate as haploids to a lower, not fully inhibitory, concentration of nystatin, the present study suggests that diploids are limited in their ability to adapt to an inhibitory concentration of nystatin, while haploids may undergo evolutionary rescue. These results demonstrate that ploidy can tip the balance between adaptation and extinction when organisms face an extreme environmental change.

Azoles activate type I and type II programmed cell death pathways in crop pathogenic fungi

Triazoles are widely used to control pathogenic fungi. They inhibit the ergosterol biosynthetic pathway, but the precise mechanisms leading to fungicidal activities in many fungal pathogens are poorly understood. Here, we elucidate the mode of action of epoxiconazole and metconazole in the wheat pathogen Zymoseptoria tritici and the rice blast fungus Magnaporthe oryzae. We show that both azoles have fungicidal activity and reduce fluidity, but not integrity, of the plasma membrane. This impairs localisation of Cdc15-like F-BAR proteins, resulting in defective actin ring assembly and incomplete septation. However, mutant studies and pharmacological experiments in vitro and in planta show that azole lethality is due to a combination of reactive oxygen species-induced apoptosis and macroautophagy. Simultaneous inhibition of both programmed cell death pathways abolishes azole-induced cell death. Other classes of ergosterol biosynthesis inhibitors also induce apoptosis and macroautophagy, suggesting that activation of these two cell death pathways is a hallmark of ergosterol synthesis-targeting fungicides. This knowledge will inform future crop protection strategies.

Loss of a conserved C-terminal region of the Aspergillus fumigatus AtrR transcriptional regulator leads to a gene-specific defect in target gene expression.

Treatment of fungal infections associated with the filamentous fungus Aspergillus fumigatus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in treatment of aspergillosis with this drug class acting by inhibiting a key step in biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the cyp51A gene. A common route of azole resistance involves an increase in transcription of cyp51A. This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter (abcC/cdr1B here called abcG1). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain in A. fumigatus is required for expression of abcG1 but dispensable for cyp51A transcription. This domain is also found in several other fungal pathogen AtrR homologues consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA-seq, we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced.

Gallic Acid Derived 1, 2-Diarylindole as a Potential Antifungal Agent against Candida Strain.

In recent decades, Candida albicans has become a serious issue for public health. The worldwide rapid rise in drug resistance to conventional therapies is the main contributing reason. Moreover, because of their potent activity at low concentrations and apparent lack of toxicity, compounds originating from plants are used in therapeutic treatments because of their potent activity at low concentrations and apparent lack of toxicity. Particularly in immunocompromised people, Candida species can result in a wide range of ailments. Present manuscript describes antifungal activity of an indole derivative 1-(4-((5- methoxy-2-(3,4,5-trimethoxyphenyl)-1H-indol-1-yl) methyl) phenoxy)-N,N-dimethylethan-1- amine (7, 100DL-6) by using an in-silico and in-vitro anti-candidal activity against two Candida strains; Candida kefyr-DS-02 (ATCC-204093) and Candida albicans (AI-clinical isolate, AIIMS- Delhi). The synthetic strategy for the preparation of indole derivatives was modified through Fischer indole reaction. Antifungal activity of an indole derivative 1-(4-((5-methoxy-2-(3,4,5- trimethoxyphenyl)-1H-indol-1-yl) methyl) phenoxy)-N,N-dimethylethan-1-amine (7, 100DL-6) was done by using an in-silico and in-vitro anti-candidal activity against two Candida strains; Candida kefyr-DS-02 (ATCC-204093) and Candida albicans (AI-clinical isolate, AIIMS-Delhi). Compound 100DL-6 efficacy was determined by Combination synergy study, ergosterol binding assay, MTT toxicity study and Mutagenicity. Compound 100DL-6 was obtained in 65% yield on desired motifs. Docking scores found were 100DL-6 (-8.7 kcal/mol) and Fluconazole (-7.6 kcal/mol). Further, RMSD were shown for 100DL6 (0.26 ± 0.23 nm) and fluconazole (1.2 ± 0.62 nm). Indole derivative 100DL-6 was active against the tested fungal pathogens and the total zone of inhibition was measured between 13-14 mm in diameter and MIC values between 31.25 μg/mL to 250 μg/mL and MFC values between 62.5 μg/mL to 500 μg/mL. In checkerboard assay synergistic mode of interaction of 100DL-6 with known antifungal drugs was observed. In the presence of ergosterol 100DL-6 and standard drug (s) increased their MIC values, demonstrating a considerable affinity for ergosterol. Compound 100DL-6 was considered to be less-cytotoxic to the cells as determined by MTT assay. Lead compound 100DL-6 was found to be non-mutagenic. In the present study, 100DL6 (indole derivatives) significantly abrupted the ergosterol biosynthetic pathway and showed moderate anti-candidal effects. These studies suggest that 100DL6 significantly enhances antifungal effect of clinical drug fluconazole synergistically and may be considered as in clinical trial prior to some extensive in-vivo validations.

Monoterpenes as potential antifungal molecules against Candida cell membranes: in-vitro and in-silico studies

Azoles are the frequently used antifungal drugs that target the enzyme lanosterol 14 α-demethylase (erg11p). This enzyme plays a vital role in ergosterol biosynthesis and hence maintainenance of cell membrane fluidity and integrity. The emergence of resistance to azoles and their fungistatic nature against several fungal pathogens is the major challenge to combat invasive candidiasis. Therefore, there is an urgent need to discover new antifungals with better efficacy. This study targets erg11 protein using in silico approach and identifies the monoterpene compounds (α-terpineol, carveol, and terpinene-4-ol) based on docking score and ligand interaction analysis. Further dynamic behavior of best-docked compounds with erg11p was analyzed by various parameters of MD simulation. The binding free energy of selected compounds towards the definitive pocket was also calculated. To further investigate the antifungal activity of selected compounds, in vitro studies were conducted on C. albicans. Studies thus suggest that the proposed the mechanism of antifungal action of test compounds involves targeting the ergosterol biosynthetic pathway. The compounds were explored for their effect on the disruption of membrane integrity by studying ERG11gene expression analysis, scanning electron microscopy, PI uptake (fluorescence microscopy,) and H+-extrusion. The results suggest that the selected monoterpenes are safer natural antifungals that disrupt membrane integrity by inhibiting ergosterol biosynthesis and other membrane associated structures. Communicated by Ramaswamy H. Sarma

Controlling antifungal activity with light: Optical regulation of fungal ergosterol biosynthetic pathway with photo-responsive CYP51 inhibitors.

Invasive fungal infections (IFIs) have been associated with high mortality, highlighting the urgent need for developing novel antifungal strategies. Herein the first light-responsive antifungal agents were designed by optical control of fungal ergosterol biosynthesis pathway with photocaged triazole lanosterol 14α-demethylase (CYP51) inhibitors. The photocaged triazoles completely shielded the CYP51 inhibition. The content of ergosterol in fungi before photoactivation and after photoactivation was 4.4% and 83.7%, respectively. Importantly, the shielded antifungal activity (MIC80≥64μg/mL) could be efficiently recovered (MIC80=0.5-8μg/mL) by light irradiation. The new chemical tools enable optical control of fungal growth arrest, morphological conversion and biofilm formation. The ability for high-precision antifungal treatment was validated by invivo models. The light-activated compound A1 was comparable to fluconazole in prolonging survival in Galleria mellonella larvae with a median survival of 14 days and reducing fungal burden in the mouse skin infection model. Overall, this study paves the way for precise regulation of antifungal therapy with improved efficacy and safety.

Fingerprint Stimulated Raman Scattering Imaging Unveils Ergosteryl Ester as a Metabolic Signature of Azole-Resistant Candida albicans.

Candida albicans (C. albicans), a major fungal pathogen, causes life-threatening infections in immunocompromised individuals. Fluconazole (FLC) is recommended as first-line therapy for treatment of invasive fungal infections. However, the widespread use of FLC has resulted in increased antifungal resistance among different strains of Candida, especially C. albicans, which is a leading source of hospital-acquired infections. Here, by hyperspectral stimulated Raman scattering imaging of single fungal cells in the fingerprint window and pixel-wise spectral unmixing, we report aberrant ergosteryl ester accumulation in azole-resistant C. albicans compared to azole-susceptible species. This accumulation was a consequence of de novo lipogenesis. Lipid profiling by mass spectroscopy identified ergosterol oleate to be the major species stored in azole-resistant C. albicans. Blocking ergosterol esterification by oleate and suppressing sterol synthesis by FLC synergistically suppressed the viability of C. albicans in vitro and limited the growth of biofilm on mouse skin in vivo. Our findings highlight a metabolic marker and a new therapeutic strategy for targeting azole-resistant C. albicans by interrupting the esterified ergosterol biosynthetic pathway.

Inhibiting Leishmania donovani Sterol Methyltransferase to Identify Lead Compounds Using Molecular Modelling.

The recent outlook of leishmaniasis as a global public health concern coupled with the reportage of resistance and lack of efficacy of most antileishmanial drugs calls for a concerted effort to find new leads. The study combined In silico and in vitro approaches to identify novel potential synthetic small-molecule inhibitors targeting the Leishmania donovani sterol methyltransferase (LdSMT). The LdSMT enzyme in the ergosterol biosynthetic pathway is required for the parasite's membrane fluidity, distribution of membrane proteins, and control of the cell cycle. The lack of LdSMT homologue in the human host and its conserved nature among all Leishmania parasites makes it a viable target for future antileishmanial drugs. Initially, six known inhibitors of LdSMT with IC50 < 10 μM were used to generate a pharmacophore model with a score of 0.9144 using LigandScout. The validated model was used to screen a synthetic library of 95,630 compounds obtained from InterBioScreen limited. Twenty compounds with pharmacophore fit scores above 50 were docked against the modelled three-dimensional structure of LdSMT using AutoDock Vina. Consequently, nine compounds with binding energies ranging from -7.5 to -8.7 kcal/mol were identified as potential hit molecules. Three compounds comprising STOCK6S-06707, STOCK6S-84928, and STOCK6S-65920 with respective binding energies of -8.7, -8.2, and -8.0 kcal/mol, lower than 22,26-azasterol (-7.6 kcal/mol), a known LdSMT inhibitor, were selected as plausible lead molecules. Molecular dynamics simulation studies and molecular mechanics Poisson-Boltzmann surface area calculations showed that the residues Asp25 and Trp208 were critical for ligand binding. The compounds were also predicted to have antileishmanial activity with reasonable pharmacological and toxicity profiles. When the antileishmanial activity of the three hits was evaluated in vitro against the promastigotes of L. donovani, mean half-maximal inhibitory concentrations (IC50) of 21.9 ± 1.5 μM (STOCK6S-06707), 23.5 ± 1.1 μM (STOCK6S-84928), and 118.3 ± 5.8 μM (STOCK6S-65920) were obtained. Furthermore, STOCK6S-84928 and STOCK6S-65920 inhibited the growth of Trypanosoma brucei, with IC50 of 14.3 ± 2.0 μM and 18.1 ± 1.4 μM, respectively. The identified compounds could be optimised to develop potent antileishmanial therapeutic agents.

Modular Inducible Multigene Expression System for Filamentous Fungi.

Inducible promoters are indispensable elements when considering the possibility to modulate gene expression on demand. Desirable traits of conditional expression systems include their capacity for tight downregulation, high overexpression, and in some instances for fine-tuning, to achieve a desired product's stoichiometry. Although the number of inducible systems is slowly increasing, suitable promoters comprising these features are rare. To date, the concomitant use of multiple regulatable promoter platforms for controlled multigene expression has been poorly explored. This work provides pioneer work in the human pathogenic fungus Aspergillus fumigatus, wherein we investigated different inducible systems, elucidated three candidate promoters, and proved for the first time that up to three systems can be used simultaneously without interfering with each other. Proof of concept was obtained by conditionally expressing three antifungal drug targets within the ergosterol biosynthetic pathway under the control of the xylose-inducible PxylP system, the tetracycline-dependent Tet-On system, and the thiamine-repressible PthiA system. IMPORTANCE In recent years, inducible promoters have gained increasing interest for industrial or laboratory use and have become key instruments for protein expression, synthetic biology, and metabolic engineering. Constitutive, high-expressing promoters can be used to achieve high expression yields; however, the continuous overexpression of specific proteins can lead to an unpredictable metabolic burden. To prevent undesirable effects on the expression host's metabolism, the utilization of tunable systems that allow expression of a gene product on demand is indispensable. Here, we elucidated several excellent tunable promoter systems and verified that each can be independently induced in a single strain to ultimately develop a unique conditional multigene expression system. This highly efficient, modular toolbox has the potential to significantly advance applications in fundamental as well as applied research in which regulatable expression of several genes is a key requirement.

Erg6p is essential for antifungal drug resistance, plasma membrane properties and cell wall integrity in Candida glabrata.

ERG6 gene encodes C-24 methyltransferase, one of the specific enzymes that differ in mammalian and yeast sterol biosynthesis. To explore the function of CgErg6p in the yeast pathogen Candida glabrata, we have constructed the Cgerg6Δ deletion mutant. We found that C. glabrata cells lacking CgErg6p exhibit reduced susceptibility to both antifungal azoles and polyenes. The reduced content of ergosterol in the Cgerg6 deletion mutant was accompanied by increased expression of genes encoding the last steps of the ergosterol biosynthetic pathway. The absence of CgErg6p leads to plasma membrane hyperpolarization and decrease in its fluidity compared to the parental C. glabrata strain. The absence of sterols containing C-24 alkyls influenced the susceptibility of Cgerg6Δ mutant cells to alkali metal cations and several other metabolic inhibitors. Our results thus show that sterols lacking C-24 alkyls are not sufficient substitutes for maintaining yeast plasma membrane function. The absence of CgErg6p influences also the cell wall integrity and calcineurin signaling in C. glabrata.

Experimental and in-host evolution of triazole resistance in human pathogenic fungi.

The leading fungal pathogens causing systemic infections in humans are Candida spp., Aspergillus fumigatus, and Cryptococcus neoformans. The major class of antifungals used to treat such infections are the triazoles, which target the cytochrome P450 lanosterol 14-α-demethylase, encoded by the ERG11 (yeasts)/cyp51A (molds) genes, catalyzing a key step in the ergosterol biosynthetic pathway. Triazole resistance in clinical fungi is a rising concern worldwide, causing increasing mortality in immunocompromised patients. This review describes the use of serial clinical isolates and in-vitro evolution toward understanding the mechanisms of triazole resistance. We outline, compare, and discuss how these approaches have helped identify the evolutionary pathways taken by pathogenic fungi to acquire triazole resistance. While they all share a core mechanism (mutation and overexpression of ERG11/cyp51A and efflux transporters), their timing and mechanism differs: Candida and Cryptococcus spp. exhibit resistance-conferring aneuploidies and copy number variants not seen in A. fumigatus. Candida spp. have a proclivity to develop resistance by undergoing mutations in transcription factors (TAC1, MRR1, PDR5) that increase the expression of efflux transporters. A. fumigatus is especially prone to accumulate resistance mutations in cyp51A early during the evolution of resistance. Recently, examination of serial clinical isolates and experimental lab-evolved triazole-resistant strains using modern omics and gene editing tools has begun to realize the full potential of these approaches. As a result, triazole-resistance mechanisms can now be analyzed at increasingly finer resolutions. This newfound knowledge will be instrumental in formulating new molecular approaches to fight the rapidly emerging epidemic of antifungal resistant fungi.

Sterol profiling of Leishmania parasites using a new HPLC-tandem mass spectrometry-based method and antifungal azoles as chemical probes reveals a key intermediate sterol that supports a branched ergosterol biosynthetic pathway

Human leishmaniasis is an infectious disease caused by Leishmania protozoan parasites. Current chemotherapeutic options against the deadly disease have significant limitations. The ergosterol biosynthetic pathway has been identified as a drug target in Leishmania. However, remarkable differences in the efficacy of antifungal azoles that inhibit ergosterol biosynthesis have been reported for the treatment of leishmaniasis. To better understand the sterol biosynthetic pathway in Leishmania and elucidate the mechanism underlying the differential efficacy of antifungal azoles, we developed a new LC-MS/MS method to study sterol profiles in promastigotes of three Leishmania species, including two L. donovani, one L. major and one L. tarentolae strains. A combination of distinct precursor ion masses and LC retention times allowed for specific detection of sixteen intermediate sterols between lanosterol and ergosterol using the newly developed LC-MS/MS method. Although both posaconazole and fluconazole are known inhibitors of fungal lanosterol 14α-demethylase (CYP51), only posaconazole led to a substantial accumulation of lanosterol in azole-treated L. donovani promastigotes. Furthermore, a key intermediate sterol accumulated by 40- and 7-fold when these parasites were treated with posaconazole and fluconazole, respectively, which was determined as 4α,14α-dimethylzymosterol by high resolution mass spectrometry and NMR spectroscopy. The identification of 4α,14α-dimethylzymosterol supports a branched ergosterol biosynthetic pathway in Leishmania, where lanosterol C4- and C14-demethylation reactions occur in parallel rather than sequentially. Our results suggest that selective inhibition of leishmanial CYP51 is insufficient to effectively prevent parasite growth and dual inhibitors of both CYP51 and the unknown sterol C4-demethylase may be required for optimal antiparasitic effect.

Transcriptional regulation of ergosterol biosynthesis genes in response to iron deficiency.

Iron participates as an essential cofactor in the biosynthesis of critical cellular components, including DNA, proteins and lipids. The ergosterol biosynthetic pathway, which is an important target of antifungal treatments, depends on iron in four enzymatic steps. Our results in the model yeast Saccharomyces cerevisiae show that the expression of ergosterol biosynthesis (ERG) genes is tightly modulated by iron availability probably through the iron-dependent variation of sterol and heme levels. Whereas the transcription factors Upc2 and Ecm22 are responsible for the activation of ERG genes upon iron deficiency, the heme-dependent factor Hap1 triggers their Tup1-mediated transcriptional repression. The combined regulation by both activating and repressing regulatory factors allows for the fine-tuning of ERG transcript levels along the progress of iron deficiency, avoiding the accumulation of toxic sterol intermediates and enabling efficient adaptation to rapidly changing conditions. The lack of these regulatory factors leads to changes in the yeast sterol profile upon iron-deficient conditions. Both environmental iron availability and specific regulatory factors should be considered in ergosterol antifungal treatments.

Applying the auxin-based degron system for the inducible, reversible and complete protein degradation in Komagataella phaffii

SummaryThe auxin-inducible degron (AID) system is a useful technique to rapidly deplete any protein of interest “on-demand.” In this study, we successfully established the AID system for the “biotech” yeast Komagataella phaffii. First, we tested different expression levels of TIR1 for auxin-induced degradation of the glycerol kinase Gut1. Moderate expression of TIR1 resulted in complete degradation of the target protein within several minutes. Second, we show that the absence of all three Wsc type sensors is detrimental to cell growth, which indicates that these are the dominant cell wall sensors this yeast. Third, down-regulation of Erg1, an essential enzyme of the ergosterol biosynthetic pathway, resulted in quick and efficient accumulation of squalene, a pharmaceutically relevant reagent. We conclude that AID is an extremely powerful tool that, for the first time, enables the analysis of gene essentiality and function in K. phaffii.

Modulation of yeast Erg1 expression and terbinafine susceptibility by iron bioavailability.

Ergosterol is a specific sterol component of yeast and fungal membranes. Its biosynthesis is one of the most effective targets for antifungal treatments. However, the emergent resistance to multiple sterol-based antifungal drugs emphasizes the need for new therapeutic approaches. The allylamine terbinafine, which selectively inhibits squalene epoxidase Erg1 within the ergosterol biosynthetic pathway, is mainly used to treat dermatomycoses, whereas its effectiveness in other fungal infections is limited. Given that ergosterol biosynthesis depends on iron as an essential cofactor, in this report, we used the yeast Saccharomyces cerevisiae to investigate how iron bioavailability influences Erg1 expression and terbinafine susceptibility. We observed that both chemical and genetic depletion of iron decrease ERG1 expression, leading to an increase in terbinafine susceptibility. Deletion of either ROX1 transcriptional repressor or CTH1 and CTH2 post-transcriptional repressors of ERG1 expression led to an increase in Erg1 protein levels and terbinafine resistance. On the contrary, overexpression of CTH2 led to the opposite effect, lowering Erg1 levels and increasing terbinafine susceptibility. Although strain-specific particularities exist, opportunistic pathogenic strains of S.cerevisiae displayed a response similar to the laboratory strain. These data indicate that iron bioavailability and particular regulatory factors could be used to modulate susceptibility to terbinafine.

Population genomics confirms acquisition of drug-resistant Aspergillus fumigatus infection by humans from the environment

Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.