Effects of trehalose and ergosterol on pinene stress of Candida glycerinogenes.

Pinene is a commercially important monoterpene that can be prepared using engineered bacterial and yeast species; however, high pinene levels can adversely affect the stability and permeability of microbial membranes leading to significantly reduced growth yields. This study reports that the fluidities and permeabilities of cell membranes of Candida glycerinogenes decrease as pinene levels increase resulting in adverse effects on cell growth. Exposure of cells to pinene results in upregulation of the genes encoding ergosterol and trehalose whose production helps stabilize their cell membranes. Exogenous addition of ergosterol and trehalose to pinene-treated cells also reduces the fluidity and permeability of the cell membrane, whilst also reducing production of intracellular reactive oxygen species. This led to the finding that the biomass of yeast cells cultivated in shake flask systems are improved by exogenous addition of trehalose and ergosterol. Overexpression of genes that encode trehalose and ergosterol produced a recombinant C. glycerinogenes strain that was found to tolerate higher concentrations of  pinene.

Metabolic engineering of Saccharomyces cerevisiae for gram-scale diosgenin production.

Diosgenin (DSG) is a naturally occurring steroidal saponin with a variety of biological activities that is also an important precursor for the synthesis of various steroidal drugs. The traditional industrial production of DSG is based on natural plant extraction and chemical processing. However, the whole process is time-consuming, laborious, and accompanied by severe environmental pollution. Therefore, it is necessary to develop a more convenient and environmentally-friendly process to realize the green production of DSG. In our previous work, we achieved de novo synthesis of DSG in Saccharomyces cerevisiae using glucose as the carbon source. However, DSG production was only at the milligram level, which is too low for industrial production. In this work, we further developed yeast strains for DSG overproduction by optimizing the synthesis pathway, fine-tuning pathway gene expression, and eliminating competing pathways. Cholesterol 22-hydroxylase was used to construct the DSG biosynthesis pathway. The optimal ratio of cytochrome P450 (CYP) to cytochrome P450 reductase (CPR) associated with DSG synthesis was screened to increase DSG production. Weakening the expression of the ERG6 gene further increased DSG synthesis and reduced the formation of by-products. In addition, we investigated the impact of DSG accumulation on yeast cell physiology and growth by transcriptome analysis and found that the multidrug transporter PDR5 and the sterol-binding protein PRY1 contributed to DSG production. Finally, we obtained a DSG titer of 2.03 g/L after 288 h of high-cell-density fed-batch fermentation using the engineered strain LP118, which represents the highest DSG titer reported to date for a yeast de novo synthesis system.

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.

Virtual Screening of Alkaloid and Terpenoid Inhibitors of SMT Expressed in Naegleria sp.

The pathogenic form of thermophilic Naegleria sp. i.e., Naegleria fowleri, also known as brain eating amoeba, causes primary amoebic encephalitis (PAM) with a >97% fatality rate. To date, there are no specific drugs identified to treat this disease specifically. The present antimicrobial combinatorial chemotherapy is hard on many patients, especially children. Interestingly, Naegleria fowleri has complex lipid biosynthesis pathways like other protists and also has a strong preference to utilize absorbed host lipids for generating energy. The ergosterol biosynthesis pathway provides a unique drug target opportunity, as some of the key enzymes involved in this pathway are absent in humans. Sterol 24-C Methyltransferase (SMT) is one such enzyme that is not found in humans. To select novel inhibitors for this enzyme, alkaloids and terpenoids inhibitors were screened and tested against two isozymes of SMT identified in N. gruberi (non-pathogenic) as well as its homolog found in yeast, i.e., ERG6. Five natural product derived inhibitors i.e., Cyclopamine, Chelerythrine, Berberine, Tanshinone 2A, and Catharanthine have been identified as potential drug candidates based on multiple criteria including binding affinity, ADME scores, absorption, and, most importantly, its ability to cross the blood brain barrier. This study provides multiple leads for future drug exploration against Naegleria fowleri.

Antifungal Activities of cis-trans Citral Isomers against Trichophyton rubrum with ERG6 as a Potential Target.

Trichophyton rubrum causes ringworm worldwide. Citral (CIT), extracted from Pectis plants, is a monoterpene and naturally composed of geometric isomers neral (cis-citral) and geranial (trans-citral). CIT has promising antifungal activities and ergosterol biosynthesis inhibition effects against several pathogenic fungi. However, no study has focused on neral and geranial against T. rubrum, which hinders the clinical application of CIT. This study aimed to compare antifungal activities of neral and geranial and preliminarily elucidate their ergosterol biosynthesis inhibition mechanism against T. rubrum. Herein, the disc diffusion assays, cellular leakage measurement, flow cytometry, SEM/TEM observation, sterol quantification, and sterol pattern change analyses were employed. The results showed geranial exhibited larger inhibition zones (p < 0.01 or 0.05), higher cellular leakage rates (p < 0.01), increased conidia with damaged membranes (p < 0.01) within 24 h, more distinct shriveled mycelium in SEM, prominent cellular material leakage, membrane damage, and morphological changes in TEM. Furthermore, geranial possessed more promising ergosterol biosynthesis inhibition effects than neral, and both induced the synthesis of 7-Dehydrodesmosterol and Cholesta-5,7,22,24-tetraen-3ß-ol, which represented marker sterols when ERG6 was affected. These results suggest geranial is more potent than neral against T. rubrum, and both inhibit ergosterol biosynthesis by affecting ERG6.

ERG6 and ERG2 Are Major Targets Conferring Reduced Susceptibility to Amphotericin B in Clinical Candida glabrata Isolates in Kuwait.

Candida glabrata is intrinsically less susceptible to azoles, and resistance to echinocandins and reduced susceptibility (RS) to amphotericin B (AMB) have also been detected. The molecular mechanisms of RS to AMB were investigated in C. glabrata strains in Kuwait by sequence analyses of genes involved in ergosterol biosynthesis. A total of 1,646 C. glabrata isolates were tested by Etest, and results for 12 selected isolates were confirmed by reference broth microdilution. PCR sequencing of three genes (ERG2, ERG6, and ERG11) was performed for all isolates with RS to AMB (RS-AMB isolates) and 5 selected wild-type C. glabrata isolates by using gene-specific primers. The total cell sterol content was analyzed by gas chromatography-mass spectrometry. The phylogenetic relationship among the isolates was investigated by multilocus sequence typing. Wild-type isolates contained only synonymous mutations in ERG2, ERG6, or ERG11, and the total sterol content was similar to that of the reference strains. A nonsynonymous ERG6 mutation (AGA48AAA, R48K) was found in both RS-AMB and wild-type isolates. Four RS-AMB isolates contained novel nonsense mutations at Trp286, Tyr192, and Leu341, and 2 isolates contained a nonsynonymous mutation in ERG6 (V126F or C198F); and the sterol content of these isolates was consistent with ERG6 deficiency. Two other RS-AMB isolates contained a novel nonsynonymous ERG2 mutation (G119S or G122S), and their sterol content was consistent with ERG2 deficiency. Of 8 RS-AMB isolates, 1 fluconazole-resistant isolate also contained nonsynonymous Y141H plus L381M mutations, while 7 isolates contained only synonymous mutations in ERG11 All isolates with ERG6, ERG2, and ERG11 mutations were genotypically distinct strains. Our data show that ERG6 and ERG2 are major targets conferring RS-AMB in clinical C. glabrata isolates.

Genomic perspective of triazole resistance in clinical and environmental Aspergillus fumigatus isolates without cyp51A mutations.

Aspergillus fumigatus is the most common etiologic agent of primarily all clinical manifestations of aspergillosis. A steady increase in the number of azole resistant A. fumigatus (ARAF) isolates from environment and clinical samples leading to therapeutic failures in clinical settings have alarmed the mycologists and clinicians worldwide. Although mutations in azole target cyp51A gene have been implicated in conferring azole resistance in A. fumigatus, recent studies have demonstrated occurrence of azole resistant strains without cyp51A mutations. In this study, next generation sequencing techniques and the expression profiling of transporter genes with single nucleotide polymorphisms (SNPs) in clinical and environmental ARAF isolates with (G54E) and without known cyp51A mutations was undertaken to understand the genetic background and role of transporters in azole resistance. The raw reads of four ARAF strains when mapped to Af293 reference genome (>100X depth) covered at least 93.1% of the reference genome. Among all four strains, a total of 212,711 SNPs was identified with 37,829 were common in at least two isolates. The expression analysis suggested the overexpression of MFS transporter, namely, mfsC in all ARAF isolates. None of the resistant strain showed significant upregulation of cyp51A and cyp51B gene. On the other hand, abcD was upregulated (5-fold) in the isolates with cyp 51A mutation (G54E). The whole genome sequence analysis showed the presence of two previously described amino acid substitutions S269F and F390Y in HMG1 gene in a clinical panazole resistant strain without cyp51A mutations. These mutations have been previously associated with azole resistance in A. fumigatus strains without cyp51A mutations. Further, several punctual mutations and a large-segment deletion among different strains were observed suggesting the involvement of resistance mechanisms other than cyp51A.

ERG6 gene deletion modifies Kluyveromyces lactis susceptibility to various growth inhibitors.

The ERG6 gene encodes an S-adenosylmethionine dependent sterol C-24 methyltransferase in the ergosterol biosynthetic pathway. In this work we report the results of functional analysis of the Kluyveromyces lactis ERG6 gene. We cloned the KlERG6 gene, which was able to complement the erg6Δ mutation in both K. lactis and Saccharomyces cerevisiae. The lack of ergosterol in the Klerg6 deletion mutant was accompanied by increased expression of genes encoding the last steps of the ergosterol biosynthesis pathway as well as the KlPDR5 gene encoding an ABC transporter. The Klerg6Δ mutation resulted in reduced cell susceptibility to amphotericin B, nystatin and pimaricin and increased susceptibility to azole antifungals, fluphenazine, terbinafine, brefeldin A and caffeine. The susceptibility phenotype was suppressed by the KlPDR16 gene encoding one of the phosphatidylinositol transfer proteins belonging to the Sec14 family. Decreased activity of KlPdr5p in Klerg6Δ mutant (measured as the ability to efflux rhodamine 6G) together with increased amount of KlPDR5 mRNA suggest that the zymosterol which accumulates in the Klerg6Δ mutant may not fully compensate for ergosterol in the membrane targeting of efflux pumps. These results point to the fact that defects in sterol transmethylation appear to cause a multitude of physiological effects in K. lactis cells. Copyright © 2016 John Wiley & Sons, Ltd.

Identification and characterization of a drug-sensitive strain enables puromycin-based translational assays in Saccharomyces cerevisiae.

Puromycin is an aminonucleoside antibiotic with structural similarity to aminoacyl tRNA. This structure allows the drug to bind the ribosomal A site and incorporate into nascent polypeptides, causing chain termination, ribosomal subunit dissociation and widespread translational arrest at high concentrations. In contrast, at sufficiently low concentrations, puromycin incorporates primarily at the C-terminus of proteins. While a number of techniques utilize puromycin incorporation as a tool for probing translational activity in vivo, these methods cannot be applied in yeasts that are insensitive to puromycin. Here, we describe a mutant strain of the yeast Saccharomyces cerevisiae that is sensitive to puromycin and characterize the cellular response to the drug. Puromycin inhibits the growth of yeast cells mutant for erg6∆, pdr1∆ and pdr3∆ (EPP) on both solid and liquid media. Puromycin also induces the aggregation of the cytoplasmic processing body component Edc3 in the mutant strain. We establish that puromycin is rapidly incorporated into yeast proteins and test the effects of puromycin on translation in vivo. This study establishes the EPP strain as a valuable tool for implementing puromycin-based assays in yeast, which will enable new avenues of inquiry into protein production and maturation.

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

Mucormycoses are emerging fungal infections caused by a variety of heterogeneous species within the Mucorales order. Among the Mucor species complex, Mucor circinelloides is the most frequently isolated pathogen in mucormycosis patients and despite its clinical significance, there is an absence of established genome manipulation techniques to conduct molecular pathogenesis studies. In this study, we generated a spontaneous uracil auxotrophic strain and developed a genetic transformation procedure to analyze molecular mechanisms conferring antifungal drug resistance. With this new model, phenotypic analyses of gene deletion mutants were conducted to define Erg3 and Erg6a as key biosynthetic enzymes in the M. circinelloides ergosterol pathway. Erg3 is a C-5 sterol desaturase involved in growth, sporulation, virulence, and azole susceptibility. In other fungal pathogens, erg3 mutations confer azole resistance because Erg3 catalyzes the production of a toxic diol upon azole exposure. Surprisingly, M. circinelloides produces only trace amounts of this toxic diol and yet, it is still susceptible to posaconazole and isavuconazole due to alterations in membrane sterol composition. These alterations are severely aggravated by erg3Δ mutations, resulting in ergosterol depletion and, consequently, hypersusceptibility to azoles. We also identified Erg6a as the main C-24 sterol methyltransferase, whose activity may be partially rescued by the paralogs Erg6b and Erg6c. Loss of Erg6a function diverts ergosterol synthesis to the production of cholesta-type sterols, resulting in resistance to amphotericin B. Our findings suggest that mutations or epimutations causing loss of Erg6 function may arise during human infections, resulting in antifungal drug resistance to first-line treatments against mucormycosis. IMPORTANCE: 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.

Essential Role of CgErg6p in Maintaining Oxidative Stress Tolerance and Iron Homeostasis in Candida glabrata.

The human pathogenic fungus Candida glabrata is the second leading cause of candidemia, a life-threatening invasive mycosis. Clinical outcomes are complicated by reduced susceptibility of C. glabrata to azoles together with its ability to evolve stable resistance to both azoles and echinocandins following drug exposure. Compared to other Candida spp., C. glabrata displays robust oxidative stress resistance. In this study, we investigated the impact of CgERG6 gene deletion on the oxidative stress response in C. glabrata. CgERG6 gene encodes sterol-24-C-methyltransferase, which is involved in the final steps of ergosterol biosynthesis. Our previous results showed that the Cgerg6Δ mutant has a lower ergosterol content in its membranes. Here, we show that the Cgerg6Δ mutant displays increased susceptibility to oxidative stress inducing agents, such as menadione, hydrogen peroxide and diamide, accompanied with increased intracellular ROS production. The Cgerg6Δ mutant is not able to tolerate higher concentrations of iron in the growth media. We observed increased expression of transcription factors, CgYap1p, CgMsn4p and CgYap5p, together with increased expression of catalase encoding the CgCTA1 gene and vacuolar iron transporter CgCCC1 in the Cgerg6Δ mutant cells. However, it seems that the CgERG6 gene deletion does not influence the function of mitochondria.

Characterization of the Sterol 24-C-Methyltransferase Genes Reveals a Network of Alternative Sterol Biosynthetic Pathways in Mucor lusitanicus.

Certain members of the order Mucorales can cause a life-threatening, often-fatal systemic infection called mucormycosis. Mucormycosis has a high mortality rate, which can reach 96 to 100% depending on the underlying condition of the patient. Mucorales species are intrinsically resistant to most antifungal agents, such as most of the azoles, which makes mucormycosis treatment challenging. The main target of azoles is the lanosterol 14α-demethylase (Erg11), which is responsible for an essential step in the biosynthesis of ergosterol, the main sterol component of the fungal membrane. Mutations in the erg11 gene can be associated with azole resistance; however, resistance can also be mediated by loss of function or mutation of other ergosterol biosynthetic enzymes, such as the sterol 24-C-methyltransferase (Erg6). The genome of Mucor lusitanicus encodes three putative erg6 genes (i.e., erg6a, erg6b, and erg6c). In this study, the role of erg6 genes in azole resistance of Mucor was analyzed by generating and analyzing knockout mutants constructed using the CRISPR-Cas9 technique. Susceptibility testing of the mutants suggested that one of the three genes, erg6b, plays a crucial role in the azole resistance of Mucor. The sterol composition of erg6b knockout mutants was significantly altered compared to that of the original strain, and it revealed the presence of at least four alternative sterol biosynthesis pathways leading to formation of ergosterol and other alternative, nontoxic sterol products. Dynamic operation of these pathways and the switching of biosynthesis from one to the other in response to azole treatment could significantly contribute to avoiding the effects of azoles by these fungi. IMPORTANCE The fungal membrane contains ergosterol instead of cholesterol, which offers a specific point of attack for the defense against pathogenic fungi. Indeed, most antifungal agents target ergosterol or its biosynthesis. Mucormycoses-causing fungi are resistant to most antifungal agents, including most of the azoles. For this reason, the drugs of choice to treat such infections are limited. The exploration of ergosterol biosynthesis is therefore of fundamental importance to understand the azole resistance of mucormycosis-causing fungi and to develop possible new control strategies. Characterization of sterol 24-C-methyltransferase demonstrated its role in the azole resistance and virulence of M. lusitanicus. Moreover, our experiments suggest that there are at least four alternative pathways for the biosynthesis of sterols in Mucor. Switching between pathways may contribute to the maintenance of azole resistance.

Characterization of an allosteric inhibitor of fungal-specific C-24 sterol methyltransferase to treat Candida albicans infections.

Filamentation is an important virulence factor of the pathogenic fungus Candida albicans. The abolition of Candida albicans hyphal formation by disrupting sterol synthesis is an important concept for the development of antifungal drugs with high safety. Here, we conduct a high-throughput screen using a C. albicans strain expressing green fluorescent protein-labeled Dpp3 to identify anti-hypha agents by interfering with ergosterol synthesis. The antipyrine derivative H55 is characterized to have minimal cytotoxicity and potent inhibition of C. albicans hyphal formation in multiple cultural conditions. H55 monotherapy exhibits therapeutic efficacy in mouse models of azole-resistant candidiasis. H55 treatment increases the accumulation of zymosterol, the substrate of C-24 sterol methyltransferase (Erg6). The results of enzyme assays, photoaffinity labeling, molecular simulation, mutagenesis, and cellular thermal shift assays support H55 as an allosteric inhibitor of Erg6. Collectively, H55, an inhibitor of the fungal-specific enzyme Erg6, holds potential to treat C. albicans infections.

Erg6 Acts as a Downstream Effector of the Transcription Factor Flo8 To Regulate Biofilm Formation in Candida albicans.

The yeast-to-hyphal morphotype transition and subsequent biofilm formation are important virulence factors of Candida albicans and are closely associated with ergosterol biosynthesis. Flo8 is an important transcription factor that determines filamentous growth and biofilm formation in C. albicans. However, the relationship between Flo8 and regulation of the ergosterol biosynthesis pathway remains elusive. Here, we analyzed the sterol composition of a flo8-deficient C. albicans strain by gas chromatography-mass spectrometry and observed the accumulation of the sterol intermediate zymosterol, the substrate of Erg6 (C-24 sterol methyltransferase). Accordingly, the transcription level of ERG6 was reduced in the flo8-deficient strain. Yeast one-hybrid experiments revealed that Flo8 physically interacted with the ERG6 promoter. Ectopic overexpression of ERG6 in the flo8-deficient strain partially restored biofilm formation and in vivo virulence in a Galleria mellonella infection model. These findings suggest that Erg6 is a downstream effector of the transcription factor Flo8 that mediates the cross talk between sterol synthesis and virulence factors in C. albicans. IMPORTANCE Biofilm formation by C. albicans hinders its eradication by immune cells and antifungal drugs. Flo8 is an important morphogenetic transcription factor that regulates the biofilm formation and in vivo virulence of C. albicans. However, little is known about how Flo8 regulates biofilm formation and fungal pathogenicity. Here, we determined that Flo8 directly binds to the promoter of ERG6 to positively regulate its transcriptional expression. Consistently, loss of flo8 results in the accumulation of the substrate of Erg6. Moreover, ectopic overexpression of ERG6 at least partially restores the biofilm formation and virulence of the flo8-deficient strain both in vitro and in vivo. This work provides a new perspective on the metabolic link between transcription factors and morphotypes in C. albicans.

In vivo emergence of high-level resistance during treatment reveals the first identified mechanism of amphotericin B resistance in Candida auris.

OBJECTIVE: Candida auris has emerged as a health-care-associated and multidrug-resistant fungal pathogen of great clinical concern. As many as 50% of C. auris clinical isolates are reported to be resistant to amphotericin B, but no mechanisms contributing to this resistance have been identified. Here we describe a clinical case in which high-level amphotericin B resistance was acquired in vivo during therapy and undertake molecular and genetic studies to identify and characterize the genetic determinant of resistance. METHODS: Whole-genome sequencing was performed on four C. auris isolates obtained from a single patient case. Cas9-mediated genetic manipulations were then used to generate mutant strains harbouring mutations of interest, and these strains were subsequently subjected to amphotericin B susceptibility testing and comprehensive sterol profiling. RESULTS: A novel mutation in the C. auris sterol-methyltransferase gene ERG6 was found to be associated with amphotericin B resistance, and this mutation alone conferred a >32-fold increase in amphotericin B resistance. Comprehensive sterol profiling revealed an abrogation of ergosterol biosynthesis and a corresponding accumulation of cholesta-type sterols in isolates and strains harbouring the clinically derived ERG6 mutation. CONCLUSIONS: Together these findings definitively demonstrate mutations in C. auris ERG6 as the first identified mechanism of clinical amphotericin B resistance in C. auris and represent a significant step forward in the understanding of antifungal resistance in this emerging public health threat.

Voriconazole Treatment Induces a Conserved Sterol/Pleiotropic Drug Resistance Regulatory Network, including an Alternative Ergosterol Biosynthesis Pathway, in the Clinically Important FSSC Species, Fusarium keratoplasticum.

Fusarium keratoplasticum is the Fusarium species most commonly associated with human infections (fusariosis). Antifungal treatment of fusariosis is often hampered by limited treatment options due to resistance towards azole antifungals. The mechanisms of antifungal resistance and sterol biosynthesis in fusaria are poorly understood. Therefore, in this study we assessed the transcriptional response of F. keratoplasticum when exposed to voriconazole. Our results revealed a group of dramatically upregulated ergosterol biosynthesis gene duplicates, most notably erg6A (912-fold), cyp51A (52-fold) and ebp1 (20-fold), which are likely part of an alternative ergosterol biosynthesis salvage pathway. The presence of human cholesterol biosynthesis gene homologs in F. keratoplasticum (ebp1, dhcr7 and dhcr24_1, dhcr24_2 and dhcr24_3) suggests that additional sterol biosynthesis pathways may be induced in fusaria under other growth conditions or during host invasion. Voriconazole also induced the expression of a number of ABC efflux pumps. Further investigations suggested that the highly conserved master regulator of ergosterol biosynthesis, FkSR, and the pleiotropic drug resistance network that induces zinc-cluster transcription factor FkAtrR coordinate the response of FSSC species to azole antifungal exposure. In-depth genome mining also helped clarify the ergosterol biosynthesis pathways of moulds and provided a better understanding of antifungal drug resistance mechanisms in fusaria.

Effect of Carum carvi essential oil on ERG6 gene expression and virulence factors in Candida albicans.

BACKGROUND AND PURPOSE: The present study was conducted to investigate the inhibitory effects of Carum carvi essential oil (EO) against ERG6 gene expression in relation to fungal growth and some important virulence factors in Candida albicans. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) of C. carvi EO against C. albicans was determined by the Clinical and Laboratory Standards Institute M27-A4 method at a concentration range of 20-1280 µg/ml. Furthermore, the expression of ERG6 gene was studied at the 0.5× MIC concentration of C. carvi EO using real-time polymerase chain reaction. The proteinase and phospholipase activities, cell surface hydrophobicity (CSH), and cell membrane ergosterol (CME) content of C. albicans were also assessed at the 0.5× MIC concentration of the plant EO using the approved methods. In addition, fluconazole (FLC) was used as a control antifungal drug. RESULTS: The results indicated that the MIC and minimum fungicidal concentration of C. carvi EO for C. albicans growth were 320 and 640 µg/ml, respectively. The expression of fungal ERG6 at an mRNA level and ergosterol content of yeast cells were significantly decreased by both C. carvi EO (640 µg/ml) and FLC (2 µg/ml). The proteinase and phospholipase activities were also reduced in C. carvi EO by 49.82% and 53.26%, respectively, while they were inhibited in FLC-treated cultures by 27.72% and 34.67%, respectively. Furthermore, the CSH was inhibited in EO- and FLC-treated cultures by 12.75% and 20.80%, respectively. CONCLUSION: Our findings revealed that C. carvi EO can be considered a potential natural compound in the development of an efficient antifungal agent against C. albicans.

Dual RNA-Seq Analysis of Trichophyton rubrum and HaCat Keratinocyte Co-Culture Highlights Important Genes for Fungal-Host Interaction.

The dermatophyte Trichophyton rubrum is the major fungal pathogen of skin, hair, and nails that uses keratinized substrates as the primary nutrients during infection. Few strategies are available that permit a better understanding of the molecular mechanisms involved in the interaction of T. rubrum with the host because of the limitations of models mimicking this interaction. Dual RNA-seq is a powerful tool to unravel this complex interaction since it enables simultaneous evaluation of the transcriptome of two organisms. Using this technology in an in vitro model of co-culture, this study evaluated the transcriptional profile of genes involved in fungus-host interactions in 24 h. Our data demonstrated the induction of glyoxylate cycle genes, ERG6 and TERG_00916, which encodes a carboxylic acid transporter that may improve the assimilation of nutrients and fungal survival in the host. Furthermore, genes encoding keratinolytic proteases were also induced. In human keratinocytes (HaCat) cells, the SLC11A1, RNASE7, and CSF2 genes were induced and the products of these genes are known to have antimicrobial activity. In addition, the FLG and KRT1 genes involved in the epithelial barrier integrity were inhibited. This analysis showed the modulation of important genes involved in T. rubrum⁻host interaction, which could represent potential antifungal targets for the treatment of dermatophytoses.

Characterization of a S-adenosyl-l-methionine (SAM)-accumulating strain of Scheffersomyces stipitis.

S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in greater SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism.

A Screen for Germination Mutants in Saccharomyces cerevisiae.

Spore germination in Saccharomyces cerevisiae is a process in which a quiescent cell begins to divide. During germination, the cell undergoes dramatic changes in cell wall and membrane composition, as well as in gene expression. To understand germination in greater detail, we screened the S. cerevisiae deletion set for germination mutants. Our results identified two genes, TRF4 and ERG6, that are required for normal germination on solid media. TRF4 is a member of the TRAMP complex that, together with the exosome, degrades RNA polymerase II transcripts. ERG6 encodes a key step in ergosterol biosynthesis. Taken together, these results demonstrate the complex nature of germination and two genes important in the process.