The Effects of Tormentic Acid and Extracts from Callistemon citrinus on Candida albicans and Candida tropicalis Growth and Inhibition of Ergosterol Biosynthesis in Candida albicans

Candida albicans and Candida tropicalis are the leading causes of human fungal infections worldwide. There is an increase in resistance of Candida pathogens to existing antifungal drugs leading to a need to find new sources of antifungal agents. Tormentic acid has been isolated from different plants including Callistemon citrinus and has been found to possess antimicrobial properties, including antifungal activity. The study aimed to determine the effects of tormentic and extracts from C. citrinus on C. albicans and C. tropicalis and a possible mode of action. The extracts and tormentic acid were screened for antifungal activity using the broth microdilution method. The growth of both species was inhibited by the extracts, and C. albicans was more susceptible to the extract compared to C. tropicalis. The growth of C. albicans was inhibited by 80% at 100 μg/ml of both the DCM: methanol extract and the ethanol: water extract. Tormentic acid reduced the growth of C. albicans by 72% at 100 μg/ml. The effects of the extracts and tormentic acid on ergosterol content in C. albicans were determined using a UV/Vis scanning spectrophotometer. At concentrations of tormentic acid of 25 μg/ml, 50 μg/ml, 100 μg/ml, and 200 μg/ml, the content of ergosterol was decreased by 22%, 36%, 48%, and 78%, respectively. Similarly, the DCM: methanol extract at 100 μg/ml and 200 μg/ml decreased the content by 78% and 88%, respectively. A dose-dependent decrease in ergosterol content was observed in cells exposed to miconazole with a 25 μg/ml concentration causing a 100% decrease in ergosterol content. Therefore, tormentic acid inhibits the synthesis of ergosterol in C. albicans. Modifications of the structure of tormentic acid to increase its antifungal potency may be explored in further studies.

Systematic Investigation of Ergosterol Fermentation by Kluyveromyces marxianus Y.00243 via Statistical Design

Ergosterol, an important pharmaceutical intermediate, is the precursor of liposoluble vitamin D2 and cortisone. It is also a main sterol in yeast cells and responsible for structural features of membranes such as the integrity, fluidity, permeability and activity of membrane-bound enzymes. Kluyveromyces marxianus is able to utilize various sugars such as lactose, xylose and arabinose against Saccharomyces cerevisiae and is also thermotolerant. Based on these aforementioned characteristics, K. marxianus can be of great importance in the utilization of whey and lignocellulosic biomass. In this paper, the effect of four factors on the specific ergosterol content and yeast growth was investigated using two statistical experimental designs. The factors examined were initially added alcohol, temperature, salt concentration and pH. The initially added alcohol had a positive effect on the specific ergosterol content, resulted in 37 % specific ergosterol content increasement. The temperature had a negative effect on yeast growth reducing the biomass concentration by 50 % when increased from 25 °C to 30 °C. The pH had a significant effect only on the specific ergosterol content, having an optimum at pH 5.5. The salt concentration had no significant effect in either case. Based on the results, it is suggested that the setup which facilitates higher ergosterol content but does not slow down the growth of the yeast remarkably should be selected, which are 25 °C, pH 5.3 and 3 % of initial ethanol content.

Combined Biosynthetic Pathway Engineering and Storage Pool Expansion for High-Level Production of Ergosterol in Industrial Saccharomyces cerevisiae

Ergosterol, a terpenoid compound produced by fungi, is an economically important metabolite serving as the direct precursor of steroid drugs. Herein, ergsosterol biosynthetic pathway modification combined with storage capacity enhancement was proposed to synergistically improve the production of ergosterol in Saccharomyces cerevisiae. S. cerevisiae strain S1 accumulated the highest amount of ergosterol [7.8 mg/g dry cell weight (DCW)] among the wild-type yeast strains tested and was first selected as the host for subsequent metabolic engineering studies. Then, the push and pull of ergosterol biosynthesis were engineered to increase the metabolic flux, overexpression of the sterol acyltransferase gene ARE2 increased ergosterol content to 10 mg/g DCW and additional overexpression of a global regulatory factor allele (UPC2-1) increased the ergosterol content to 16.7 mg/g DCW. Furthermore, considering the hydrophobicity sterol esters and accumulation in lipid droplets, the fatty acid biosynthetic pathway was enhanced to expand the storage pool for ergosterol. Overexpression of ACC1 coding for the acetyl-CoA carboxylase increased ergosterol content from 16.7 to 20.7 mg/g DCW. To address growth inhibition resulted from premature accumulation of ergosterol, auto-inducible promoters were employed to dynamically control the expression of ARE2, UPC2-1, and ACC1. Consequently, better cell growth led to an increase of ergosterol content to 40.6 mg/g DCW, which is 4.2-fold higher than that of the starting strain. Finally, a two-stage feeding strategy was employed for high-density cell fermentation, with an ergosterol yield of 2986.7 mg/L and content of 29.5 mg/g DCW. This study provided an effective approach for the production of ergosterol and other related terpenoid molecules.

Effect of Allium cepa on LAC1 gene expression and physiological activities in Cryptococcus neoformans

Background and Purpose: This study aimed to investigate the effects of Allium cepa ethanolic extract (EAC) on Cryptococcus neoformans biological activities and LAC1 gene expression. Materials and Methods: The minimum inhibitory concentration (MIC) of EAC was determined based on the Clinical and Laboratory Standards Institute M27-A4 method at a concentration range of 125- 4000 µg/ml. The EAC synergism activity was determined in combination with fluconazole (FCZ) as an antifungal azole. Laccase activity, melanin production, and cell membrane ergosterol content of C. neoformans were assessed at the 0.5× MIC concentration of EAC (1000 μg/ml) and FCZ (64μg/ml) by approved methods. The expression of the LAC1 gene was studied in the fungus exposed to 0.5× MIC concentration of EAC and FCZ using the real-time polymerase chain reaction. Results: Based on obtained results, MIC of EAC and FCZ were 2000 and 128 μg/ml,respectively. A combinatory effect was reported for FCZ and EAC by a fractional inhibitory concentration index of 0.25. The cell membrane ergosterol content was inhibited in EAC- and FCZ-treated C. neoformans by 58.25% and 49.85%, respectively.The laccase activity and melanin production were reduced in EAC-treated C. neoformans by 45.37% and 51.57%, and in FCZ-treated fungus by 54.64% and 53.68%, respectively.The expression of fungal LAC1 at messenger RNA (mRNA) level was measured 0.46 and 0.58 folds and significantly decreased in both EAC- and FCZ-treated C. neoformans at the 0.5×MIC concentration, respectively (p <0.05). Conclusion: The findings revealed that EAC contains inhibitory compounds which interact with biological activities in C. neoformans and thereby, it could be considered as a potential source for the development of novel antifungal drugs.

Lactate Like Fluconazole Reduces Ergosterol Content in the Plasma Membrane and Synergistically Kills Candida albicans

Candida albicans is an opportunistic pathogen that induces vulvovaginal candidiasis (VVC), among other diseases. In the vaginal environment, the source of carbon for C. albicans can be either lactic acid or its dissociated form, lactate. It has been shown that lactate, similar to the popular antifungal drug fluconazole (FLC), reduces the expression of the ERG11 gene and hence the amount of ergosterol in the plasma membrane. The Cdr1 transporter that effluxes xenobiotics from C. albicans cells, including FLC, is delocalized from the plasma membrane to a vacuole under the influence of lactate. Despite the overexpression of the CDR1 gene and the increased activity of Cdr1p, C. albicans is fourfold more sensitive to FLC in the presence of lactate than when glucose is the source of carbon. We propose synergistic effects of lactate and FLC in that they block Cdr1 activity by delocalization due to changes in the ergosterol content of the plasma membrane.

Whole genome sequencing of a clinical drug resistant Candida albicans isolate reveals known and novel mutations in genes involved in resistance acquisition mechanisms

Candida albicans is an opportunistic pathogen accounting for the majority of cases of Candida infections. Currently, C. albicans are developing resistance towards different classes of antifungal drugs and this has become a global health burden that does not spare Lebanon. This study aims at determining point mutations in genes known to be involved in resistance acquisition and correlating resistance to virulence and ergosterol content in the azole resistant C. albicans isolate CA77 from Lebanon. This pilot study is the first of its kind to be implemented in Lebanon. We carried out whole genome sequencing of the azole resistant C. albicans isolate CA77 and examined 18 genes involved in antifungal resistance. To correlate genotype to phenotype, we evaluated the virulence potential of this isolate by injecting it into BALB/c mice and we quantified membrane ergosterol. Whole genome sequencing revealed that eight out of 18 genes involved in antifungal resistance were mutated in previously reported and novel residues. These genotypic changes were associated with an increase in ergosterol content but no discrepancy in virulence potential was observed between our isolate and the susceptible C. albicans control strain SC5314. This suggests that antifungal resistance and virulence potential in this antifungal resistant isolate are not correlated and that resistance is a result of an increase in membrane ergosterol content and the occurrence of point mutations in genes involved in the ergosterol biosynthesis pathway.

A Novel Sulfone Derivative Containing 1,3,4-Oxadiazole Controls Tea Plant Disease through Inhibiting Lasiodiplodia theobromae Proliferation by Reducing the Ergosterol Content

Diseases caused by fungi can affect the quality and yield of the leaves of tea plants [Camellia sinensis (L.) Kuntze]. At present, the availability of highly effective and safe fungicides for controlling tea plants remains limited. The objectives of this study were to identify novel compounds with anti-fungal activities and to determine their molecular mechanisms. A series of sulfone compounds containing 1,3,4-oxadiazole were evaluated in China for their anti-fungal activities against several pathogens causing foliar diseases and high production losses. Transcriptomics and bioinformatics were used to analyze the differentially expressed genes of Lasiodiplodia theobromae treated with a representative compound, Jiahuangxianjunzuo (JHXJZ). Moreover, the effects of JHXJZ on ergosterol content, membrane permeability, cell structure and seven key genes involved in the ergosterol biosynthetic pathway were investigated. JHXJZ had a strong anti-fungal activity against L. theobromae in vitro, with an EC50 of 3.54 ± 0.55 µg/mL, and its curative efficacies on detached tea leaves reached 41.78% at 100 µg/mL, respectively. JHXJZ up-regulated 899 genes (P < 0.05) and down-regulated 1,185 genes (P < 0.05) in L. theobromae. These genes were found to be associated with carbohydrate metabolic processes, which are closely related to steroid biosynthesis in the KEGG pathways. Because JHXJZ regulates the key genes of sterol biosynthesis, it decreased the ergosterol content, increased cell-membrane permeability, changed the cellular structure, the enhanced roughness of surface of the hyphae, and resulted in degradation of the hyphal nuclei and necrosis of the hyphal cytoplasm.

Analysis of the Contribution of cyp51 Genes to Azole Resistance in Aspergillus Section Nigri with the CRISPR-Cas9 Technique

ABSTRACT Cyp51 contribution to azole resistance has been broadly studied and characterized in Aspergillus fumigatus, whereas it remains poorly investigated in other clinically relevant species of the genus, such as those of section Nigri. In this work, we aimed to analyze the impact of cyp51 genes (cyp51A and cyp51B) on the voriconazole (VRC) response and resistance of Aspergillus niger and Aspergillus tubingensis. We generated CRISPR-Cas9 cyp51A and cyp51B knockout mutants from strains with different genetic backgrounds and diverse patterns of azole susceptibility. Single-gene deletions of cyp51 genes resulted in 2- to 16-fold decreases of the VRC MIC values, which were below the VRC epidemiological cutoff value (ECV) established by the Clinical and Laboratory Standards Institute (CLSI), irrespective of their parental strains’ susceptibilities. Gene expression studies in the tested species confirmed that cyp51A participates more actively than cyp51B in the transcriptional response of azole stress. However, ergosterol quantification revealed that both enzymes comparably impact the total ergosterol content within the cell, as basal- and VRC-induced changes to ergosterol content were similar in all cases. These data contribute to our understanding of Aspergillus azole resistance, especially in non-A. fumigatus species.

Development of a Methodology for Estimating the Ergosterol in Meat Product-Borne Toxigenic Moulds to Evaluate Antifungal Agents

Antifungal agents are commonly used in the meat industry to prevent the growth of unwanted moulds, such as toxigenic ones, on dry-cured meat products. For enhancing the application of antifungals, their mode of action must be evaluated. Their effect on the mould ergosterol content is one of the most studied ones, since it is the target site of some commercialised antifungals or of those that are in development. The aim of this study was to develop a methodology for determining how the antifungal agents used in the meat industry work. A method for analysing ergosterol was firstly developed using high-performance liquid chromatography with fluorescence detection coupled to a diode array detector (HPLC-FLD/DAD). The chromatographically optimised conditions (gradient and mobile phases) allowed us to reduce the time per analysis with respect to previously published methods up to 22 min. Withing the six checked extraction methods, method 5, showing the best mean recovery values (99.51%), the shortest retention time (15.8 min), and the lowest standard deviation values (9.92) and working temperature (60 °C), was selected. The limit of detection and limit of quantification were 0.03 and 0.1 µg/mL, respectively. All the validation parameters corroborated the method’s suitability. Finally, its feasibility for evaluating the effect of a commercial antifungal preparation (AP) and different herbs that are frequently added to meat products on the ergosterol content of several toxigenic moulds was studied. Differences at the strain level were obtained in the presence of AP. Moreover, the addition of herbs significantly reduced the ergosterol content in Penicillium nordicum up to 83.91%. The developed methodology is thus suitable for screening the antifungals’ role in altering mould ergosterol biosynthesis before their application in real meat products.