ERG11 Gene Research Articles

Cytostatic effects of structurally different ginsenosides on yeast cells with altered sterol biosynthesis and transport

Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene glycosides show membranolytic activity, and, therefore, are considered to be promising antimicrobial drugs. However, the interrelation between their structure, biological activities, and target membrane lipid composition remains elusive. Here we studied the antifungal effects of four Panax triterpene glycosides (ginsenosides) with sugar moieties at the C-3 (ginsenosides Rg3, Rh2), C-20 (compound K), and both (ginsenoside F2) positions in Saccharomyces cerevisiae mutants with altered sterol plasma membrane composition. We observed reduced cytostatic activity of the Rg3 and compound K in the UPC2-1 strain with high membrane sterol content. Moreover, LAM gene deletion reduced yeast resistance to Rg3 and digitonin, another saponin with glycosylated aglycon in the C-3 position. LAM genes encode plasma membrane-anchored StARkin superfamily-member sterol transporters. We also showed that the deletion of the ERG6 gene that inhibits ergosterol biosynthesis at the stage of zymosterol increased the cytostatic effects of Rg3 and Rh2, but not the other two tested ginsenosides. At the same time, in silico simulation revealed that the substitution of ergosterol with zymosterol in the membrane changes the spatial orientation of Rg3 and Rh2 in the membranes. These results imply that the plasma membrane sterol composition defines its interaction with triterpene glycoside depending on their glycoside group position. Our results also suggest that the biological role of membrane-anchored StARkin family protein is to protect eukaryotic cells from triterpenes glycosylated at the C-3 position.

A New Variant of Mutational and Polymorphic Signatures in the ERG11 Gene of Fluconazole-Resistant Candida albicans.

BackgroundResistance to antifungal drugs for treating Candida infections remains a major concern globally despite the range of medications available. Most of these drugs target key proteins essential to the life cycle of the organism. An enzyme essential for fungal cell membrane integrity, lanosterol 14–α demethylase (CYP51), is encoded by the ERG11 gene in Candida species. This enzyme is the target of azole–based drugs. The organism has, however, devised molecular adaptations to evade the activity of these drugs.Materials and MethodsClassical methods were employed to characterize clinical isolates sampled from women and dogs of reproductive age. For fluconazole efficacy studies, CLSI guidelines on drug susceptibility testing were used. To understand the susceptibility pattern, various molecular and structural analytic approaches, including sequencing, in silico site-directed mutagenesis, and protein-ligand profiling, were applied to the ERG11 gene and CYP51 protein sequences. Several platforms, comprising Clustal Omega, Pymol plugin manager, Pymol molecular visualizer, Chimera–curated Dynameomics rotamer library, protein–ligand interaction profiler, Charmm36 force field, GROMACS, Geneious, and Mega7, were employed for this analysis.ResultsThe following Candida species distribution was obtained: 37.84% C. albicans, 8.12% C. glabrata, 10.81% C. krusei, 5.41% C. tropicalis, and 37.84% of other unidentified Candida species. Two codons in the nucleotide sequence of the wild-type (CTC and CCA) coding for LEU–370 and PRO–375, respectively, were mutated to L370S and P375H in the resistant strain. The mutation stabilized the protein at the expense of the heme moiety. We found that the susceptible isolate from dogs (Can–iso–029/dog) is closely related to the most resistant isolate from humans.ConclusionTaken together, our results showed new mutations in the heme-binding pocket of caCYP51 that explain the resistance to fluconazole exhibited by the Candida isolates. So far, the L370S and P375H resistance-linked mutations have not been previously reported.

Encephalartos villosus Lem. Displays a Strong In Vivo and In Vitro Antifungal Potential against Candida glabrata Clinical Isolates.

Recently, Candida glabrata has been recognized as one of the most common fungal species that is highly associated with invasive candidiasis. Its spread could be attributed to its increasing resistance to antifungal drugs. Thus, there is a high need for safer and more efficient therapeutic alternatives such as plant extracts. Here, we investigated the antifungal potential of Encephalartos villosus leaves methanol extract (EVME) against C. glabrata clinical isolates. Tentative phytochemical identification of 51 metabolites was conducted in EVME using LC–MS/MS. EVME demonstrated antifungal activity with minimum inhibitory concentrations that ranged from 32 to 256 µg/mL. The mechanism of the antifungal action was studied by investigating the impact of EVME on nucleotide leakage. Additionally, a sorbitol bioassay was performed, and we found that EVME affected the fungal cell wall. In addition, the effect of EVME was elucidated on the efflux activity of C. glabrata isolates using acridine orange assay and quantitative real-time PCR. EVME resulted in downregulation of the expression of the efflux pump genes CDR1, CDR2, and ERG11 in the tested isolates with percentages of 33.33%, 41.67%, and 33.33%, respectively. Moreover, we investigated the in vivo antifungal activity of EVME using a murine model with systemic infection. The fungal burden was determined in the kidney tissues. Histological and immunohistochemical studies were carried out to investigate the effect of EVME. We noticed that EVME reduced the congestion of the glomeruli and tubules of the kidney tissues of the rats infected with C. glabrata. Furthermore, it decreased both the proinflammatory cytokine tumor necrosis factor-alpha and the abnormal collagen fibers. Our results reveal, for the first time, the potential in vitro (by inhibition of the efflux activity) and in vivo (by decreasing the congestion and inflammation of the kidney tissues) antifungal activity of EVME against C. glabrata isolates.

Lack of Association between Fluconazole Susceptibility and ERG11 Nucleotide Polymorphisms in Cryptococcus neoformans Clinical Isolates from Uganda.

Fluconazole is the drug of choice for cryptococcal meningitis (CM) monoprophylaxis in resource-limited settings such as Uganda. Emerging fluconazole resistance linked to mutations in the Cryptococcus neoformans ERG11 gene (CYP51) has been observed in clinical isolates. Currently, the single nucleotide polymorphisms [SNPs] in the Cryptococcus spp. ERG11 gene that could be responsible for fluconazole resistance are poorly characterized within Ugandan C. neoformans clinical isolates. If available, this information would be useful in the management of cryptococcosis among HIV patients. This cross-sectional study investigates the SNPs present in the coding region of the C. neoformans ERG11 gene to determine the relationship between the SNPs identified and fluconazole susceptibility of the clinical isolates. 310 C. neoformans isolates recovered from the Cerebrospinal Fluid (CSF) of patients with HIV and cryptococcal meningitis were examined. The fluconazole half-maximal inhibitory concentrations (IC50 range: 0.25–32 μg/mL) was determined using the microbroth dilution method. A total of 56.1% of the isolates had low IC50 values of <8 μg/mL while 43.9% had high IC50 values ≥ 8 μg/mL. We amplified and sequenced 600 bp of the ERG11 coding sequence from 40 of the clinical isolates. Novel synonymous and 2 missense mutations, S460T and A457V, were identified in the ERG11 gene. The identified SNPs were not associated with differences in fluconazole IC50 values in vitro (p = 0.179).

Functional Analysis of the C-5 Sterol Desaturase PcErg3 in the Sterol Auxotrophic Oomycete Pathogen Phytophthora capsici

Although sterols play an important role in most eukaryotes, some oomycetes, including Phytophthora spp., have lost the sterol synthesis pathway. Nevertheless, the ERG3 gene encoding C-5 sterol desaturase in the sterol synthesis pathway is still present in the genomes of Phytophthora spp. Phytophthora capsici, a destructive pathogen with a broad range of plant hosts, poses a significant threat to the production of agriculture. This study focused on the ERG3 gene in P. capsici (PcERG3) and explored its function in this pathogen. It showed that the PcERG3 gene could be expressed in all tested developmental stages of P. capsici, with sporangium and mycelium displaying higher expression levels. A potential substrate of Erg3 (stellasterol) was used to treat the P. capsici wild-type strain and a PcERG3Δ transformant, and their sterol profiles were determined by GC-MS. The wild-type strain could convert stellasterol into the down-stream product while the transformant could not, indicating that PcErg3 retains the C-5 sterol desaturase activity. By comparing the biological characteristics of different strains, it was found that PcERG3 is not important for the development of P. capsici. The pathogenicity of the PcERG3Δ transformants and the wild-type strain was comparable, suggesting that PcERG3 is not necessary for the interaction between P. capsici and its hosts. Further investigations revealed that the PcERG3Δ transformants and the wild-type strain displayed a similar level of tolerance to external adversities such as unsuitable temperatures, high osmotic pressures, and intemperate pH, signifying that PcERG3 is not essential for P. capsici to cope with these environmental stresses.

Green synthesis of selenium nanoparticles and evaluate their effect on the expression of ERG3, ERG11 and FKS1 antifungal resistance genes in Candida albicans and Candida glabrata.

Drug resistance in Candida species has been considerably increased in the last decades. Given the opposition to antifungal agents, toxicity and interactions of the antimicrobial drugs, identifying new antifungal agents seems essential. This study assessed the antifungal effects of nanoparticles (NPs) on the standard strains of Candida albicans and Candida glabrata and determined the expression genes, including ERG3, ERG11 and FKS1. Selenium nanoparticles (Se-NPs) were biosynthesized with a standard strain of C. albicans and approved by several methods including, ultraviolet-visible spectrophotometer, X-ray diffraction technique, Fourier-transform infrared analysis, field-emission scanning electron microscopy and EDX diagram. The antifungal susceptibility testing performed the minimum inhibitory concentrations (MICs) using the CLSI M27-A3 and M27-S4 broth microdilution method. The expression of the desired genes was examined by the real-time PCR assay between untreated and treated by antifungal drugs and Se-NPs. The MICs of itraconazole, amphotericin B and anidulafungin against C. albicans and C. glabrata were 64, 16 and 4 µg ml-1 . In comparison, reduced the MIC values for samples treated with Se-NPs to 1 and 0·5 µg ml-1 . The results obtained from real-time PCR and analysis of the ∆∆Cq values showed that the expression of ERG3, ERG11 and FKS1 genes was significantly down-regulated in Se-NPs concentrations (P < 0·05). This study's evidence implies biosafety Se-NPs have favourable effects on the reducing expression of ERG3, ERG11 and FKS1 antifungal resistance genes in C. albicans and C. glabrata.

Impact of the COVID-19 Pandemic on the Clinical Profile of Candidemia and the Incidence of Fungemia Due to Fluconazole-Resistant Candida parapsilosis.

Severely ill COVID-19 patients are at high risk of nosocomial infections. The aim of the study was to describe the characteristics of candidemia during the pre-pandemic period (January 2019–February 2020) compared to the pandemic period (March 2020–September 2021). Antifungal susceptibilities were assessed using the EUCAST E.Def 7.3.2 broth dilution method. Fluconazole-resistant C. parapsilosis isolates (FRCP) were studied for sequencing of the ERG11 gene. The incidence of candidemia and C. parapsilosis bloodstream infection increased significantly in the pandemic period (p = 0.021). ICU admission, mechanical ventilation, parenteral nutrition and corticosteroids administration were more frequent in patients with candidemia who had been admitted due to COVID-19. Fifteen cases of FRCP fungemia were detected. The first case was recorded 10 months before the pandemic in a patient transferred from another hospital. The incidence of FRCP in patients admitted for COVID-19 was 1.34 and 0.16 in all other patients (p < 0.001). ICU admission, previous Candida spp. colonization, arterial catheter use, parenteral nutrition and renal function replacement therapy were more frequent in patients with candidemia due to FRCP. All FRCP isolates showed the Y132F mutation. In conclusion, the incidence of candidemia experienced an increase during the COVID-19 pandemic and FRCP fungemia was more frequent in patients admitted due to COVID-19.

Fluconazole-resistant Candida parapsilosis clonally related genotypes: first report proving the presence of endemic isolates harbouring the Y132F ERG11 gene substitution in Spain

ObjectivesWe report here for the first time the presence of Candida parapsilosis isolates harbouring the Y132F ERG11 gene substitution in patients admitted to a Spanish hospital. MethodsWe studied the available (n = 104) C parapsilosis isolates from patients admitted to the Son Espases reference hospital in the Balearic Islands from 1 April 2019 to 30 November 2020. Isolates were sourced from 70 patients: catheter (n = 41), blood cultures (n = 37), lower respiratory tract (n = 15), intra-abdominal (n = 8), and other samples (n = 3). Isolates were genotyped and tested for antifungal susceptibilities to amphotericin B, triazoles, anidulafungin, and micafungin using EUCAST E.Def 7.3.2. The ERG11 gene was sequenced in fluconazole-resistant isolates. ResultsAmong the 104 isolates, fluconazole and voriconazole resistance was found in 87 (84%) and 30 (29%) isolates, respectively; all isolates were fully susceptible to echinocandins and amphotericin B. All fluconazole-resistant isolates harboured the Y132F substitution in the ERG11 gene and were grouped into 11 clonally related genotypes. A genotype accounted for 70% (61/87) of fluconazole-resistant isolates. Genotypes involving the fluconazole-resistant isolates were different from those found in the remaining fluconazole-susceptible genotypes. Fifty-six patients harboured fluconazole-resistant genotypes, and 35 of the 56 had candidaemia (48%), abdominal candidiasis (17%), or other forms of candidiasis (35%). Only 20% of the study patients infected by fluconazole-resistant genotypes had a history of azole use. DiscussionFluconazole resistance in C parapsilosis isolates from patients admitted to this reference hospital is not attributable to prior azole use, but rather to the presence of a group of fluconazole-resistant C parapsilosis genotypes that have become endemic.

Novel culture chamber to evaluate in vitro plant-microbe volatile interactions: Effects of Trichoderma harzianum volatiles on wheat plantlets

The field of plant-microbe interactions mediated by Biogenic Volatile Organic Compounds (BVOCs) still faces several limitations due to the lack of reliable equipment. We present a novel device designed to evaluate in vitro plant-microbe volatile interactions, the plant-microbe VOC Chamber. It was tested by evaluating the effects exerted on wheat development by volatiles from three Trichoderma harzianum strains, a wild type and two genetically modified strains; one expressing the tri5 gene, which leads to the synthesis and emission of the volatile trichodiene, and the other by silencing the erg1 gene, impairing ergosterol production. The wild type and the erg1-silenced strain enhanced fresh weight and length of the aerial part, but reduced root dry weight. Interestingly, no differences were found between them. Conversely, the tri5-transformant strain reduced root and aerial growth compared to the control and the other strains. No differences were observed regarding chlorophyll fluorescence quantum yield and leaf chlorophyll content, suggesting that the released BVOCs do not interfere with photosynthesis. The plant-microbe VOC Chamber proved to be a simple and reliable method to evaluate the in vitro effects of microbial BVOCs on plant development, perfect for the screening of microorganisms with interesting volatile traits.

Anu taila, an herbal nasal drop, suppresses mucormycosis by regulating host TNF-α response and fungal ergosterol biosynthesis.

The intractable, mucormycosis, caused by Mucorales primarily targets immunocompromised individuals. The first-line therapy, intravenous liposomal amphotericin B and surgical debridement of necrotic tissue, is contraindicative in individuals with compromised kidneys. This invokes a pressing need to identify safer treatment options. The antifungal effect of the classical nasal drop, Anu taila, against Mucor spp. was investigated through microbiological, cytological, analytical chemical (HPLC and GS-MS/MS) and scanning electron microscopic (SEM) approaches. Anu taila-pretreated spores germinated late, resulting in reduced infectivity, observed as milder monocytic immune response. Conversely, Anu taila-pretreated human THP-1 cells exhibited an improved immune response against Mucor spores, through TNF-α. Repeated Anu taila application rapidly abolished fungal microarchitectures than amphotericin B, evident from swift replacement of hyphae, sporangiophores and sporangia with fused biomass, in the SEM images. HPLC analysis showed that Anu taila treatment significantly reduced overall ergosterol content in Mucor biomass. Anu taila also downregulated sterol-C5-desaturase-coding ERG3 gene, crucial for ergosterol biosynthesis and resultant structural integrity, in Mucor spp. Taken together, Anu taila was found effective against Mucor spp., with both prophylactic and curative implications, which is attributable to the phytochemical composition of this classical nasal drop. The potential remedial effects of a classical nasal drop against an obdurate and challenging fungal infection are identified.

Molecular mechanisms underlying the emergence of polygenetic antifungal drug resistance in msh2 mismatch repair mutants of Cryptococcus.

BackgroundFungal infections are common life-threatening diseases amongst immunodeficient individuals. Invasive fungal disease is commonly treated with an azole antifungal agent, resulting in selection pressure and the emergence of drug resistance. Antifungal resistance is associated with higher mortality rates and treatment failure, making the current clinical management of fungal disease very challenging. Clinical isolates from a variety of fungi have been shown to contain mutations in the MSH2 gene, encoding a component of the DNA mismatch repair pathway. Mutation of MSH2 results in an elevated mutation rate that can increase the opportunity for selectively advantageous mutations to occur, accelerating the development of antifungal resistance.ObjectivesTo characterize the molecular mechanisms causing the microevolutionary emergence of antifungal resistance in msh2 mismatch repair mutants of Cryptococcus neoformans.MethodsThe mechanisms resulting in the emergence of antifungal resistance were investigated using WGS, characterization of deletion mutants and measuring ploidy changes.ResultsThe genomes of resistant strains did not possess mutations in ERG11 or other genes of the ergosterol biosynthesis pathway. Antifungal resistance was due to small contributions from mutations in many genes. MSH2 does not directly affect ploidy changes.ConclusionsThis study provides evidence that resistance to fluconazole can evolve independently of ERG11 mutations. A common microevolutionary route to the emergence of antifungal resistance involves the accumulation of mutations that alter stress signalling, cellular efflux, membrane trafficking, epigenetic modification and aneuploidy. This complex pattern of microevolution highlights the significant challenges posed both to diagnosis and treatment of drug-resistant fungal pathogens.

Volatile-mediated interactions between Trichoderma harzianum and Acanthoscelides obtectus: A novel in vitro methodology to evaluate the impact of microbial volatile compounds on dry grain storage pests

Biological interactions mediated by Biogenic Volatile Organic Compounds (BVOCs) is a well-established field that has been researched for decades. Although extensive focus is currently given to the control of insect pests using natural molecules, the study of volatile interactions between microorganisms and insects has been largely neglected and has only begun to attract more attention in recent years. In this work, we developed a novel protocol to assess the effects of microbial BVOCs directly produced by growing microbial strains on dry grain insect pests and the seed damage they cause, using VOC Chambers to evaluate both sealed and unsealed conditions. Four Trichoderma harzianum strains were tested against Acanthoscelides obtectus, a wild type and three of its transformants. These had been previously obtained by introducing the tri5 gene and thus overproducing the volatile trichodiene, or by silencing the erg1 gene, which encodes for a squalene epoxidase, therefore reducing ergosterol levels and increasing squalene ones in the fungus. Results demonstrated that ventilation plays a key role in these interactions. All fungal strains significantly increased adult mortality in sealed conditions, while this effect was barely noticeable in unsealed ones. Nevertheless, subsequent insect emergence from bean seeds and bean damage were still significantly reduced in both conditions. The erg1 silenced strains caused significantly higher levels of adult mortality than the rest in sealed conditions and lower insect emergence in both sealed and unsealed ones. Bean damage produced by insects was lower also when exposed to BVOCs from these strains in sealed conditions. Conversely, trichodiene overproduction did not show enhanced toxicity or significant reduction of insect emergence and bean damage in the tested conditions. Therefore, T. harzianum BVOCs, especially those from erg1 silenced strains, should be further researched for their potential use in the biological control of A. obtectus infestation in dry grain storing facilities. VOC Chambers have shown themselves to be a reliable method in the screening of in vitro volatile mediated interactions between growing microbial strains and insect pests.

Engineered yeast for efficient de novo synthesis of 7-dehydrocholesterol.

The synthesis of vitamin D3 precursor 7-dehydrocholesterol (7-DHC) by microbial fermentation has much attracted attention owing to its advantages of environmental protection. In this study, Saccharomyces cerevisiae was engineered for a de novo biosynthesis of 7-DHC. First, seven essential genes (six endogenous genes and one heterologous gene) were overexpressed, and the ROX1 gene (heme-dependent repressor of hypoxic genes) was knocked out. The resulting strain produced 82.6 mg/L 7-DHC from glucose. Then, we predicted five gene knockout targets for 7-DHC overproduction by the reconstruction of genome-scale metabolic model. GDH1 gene knockout increased the 7-DHC titer from 82.6 to 101.5 mg/L, and the specific growth rate of the ΔGDH1 mutant was also increased by 28%. Next, Ty1 transposon in S. cerevisiae was applied to increase the copies of the ERG1 gene and DHCR24 gene, resulting in a 120% increase in 7-DHC titer to 223.3 mg/L. Besides, to optimize the metabolic flux distribution, Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system was used to dynamically inhibit the competitive pathway, and the best binding site of ERG6 (delta (24)-sterol C-methyltransferase) promoter was screened out. The OD600 value of ERG6 regulated cells increased by 43% than knocking out ERG6 directly, and 7-DHC titer increased to 365.5 mg/L in a shake flask. Finally, the 7-DHC titer reached 1328 mg/L in 3-L bioreactor and the specific titer of 7-DHC reached up to 114.7 mg/g dry cell weight). Overall, this study constructed a yeast chassis for the highly efficient production of 7-DHC by systems metabolic engineering.

Dynamic control of ERG20 expression to improve production of monoterpenes by engineering Saccharomyces cerevisiae

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.

Molecular Characterization of Candida auris Isolates at a Major Tertiary Care Center in Lebanon.

BackgroundThe globally emerging Candida auris pathogens poses heavy burden to the healthcare system. Their molecular analyses assist in understanding their epidemiology, dissemination, treatment, and control. This study was warranted to describe the genomic features and drug resistance profiles using whole genome sequencing (WGS) among C. auris isolates from Lebanon.MethodsA total of 28 C. auris clinical isolates, from different hospital units, were phenotypically identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and tested for antifungal resistance using Vitek-2 system and E test. The complete genomes were determined by WGS using long reads sequencing (PacBio) to reveal the clade distribution and antifungal resistance genes.ResultsCandida auris revealed uniform resistance to fluconazole and amphotericin B, with full susceptibility to echinocandins. Among key resistance genes studied, only two mutations were detected: Y132F in ERG11 gene and a novel mutation, D709E, found in CDR1 gene encoding for an ABC efflux pump. Phylogenetically, C. auris genomes belonged to South Asian clade I and showed limited genetic diversity, suggesting person to person transmission.ConclusionThis characterization of C. auris isolates from Lebanon revealed the exclusivity of clade I lineage together with uniform resistance to fluconazole and amphotericin B. The control of such highly resistant pathogen necessitates an appropriate and rapid recovery and identification to contain spread and outbreaks.

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.

A Brazilian Inter-Hospital Candidemia Outbreak Caused by Fluconazole-Resistant Candida parapsilosis in the COVID-19 Era.

Horizontal transmission of fluconazole-resistant Candida parapsilosis (FRCP) through healthcare workers’ hands has contributed to the occurrence of candidemia outbreaks worldwide. Since the first COVID-19 case in Brazil was detected in early 2020, hospitals have reinforced hand hygiene and disinfection practices to minimize SARS-CoV-2 contamination. However, a Brazilian cardiology center, which shares ICU patients with a cancer center under a FRCP outbreak since 2019, reported an increased FRCP candidemia incidence in May 2020. Therefore, the purpose of this study was to investigate an inter-hospital candidemia outbreak caused by FRCP isolates during the first year of the COVID-19 pandemic in Brazil. C. parapsilosis bloodstream isolates obtained from the cancer (n = 35) and cardiology (n = 30) centers in 2020 were submitted to microsatellite genotyping and fluconazole susceptibility testing. The ERG11 gene of all isolates from the cardiology center was sequenced and compared to the corresponding sequences of the FRCP genotype responsible for the cancer center outbreak in 2019. Unprecedentedly, most of the FRCP isolates from the cardiology center presented the same genetic profile and Erg11-Y132F mutation detected in the strain that has been causing the persistent outbreak in the cancer center, highlighting the uninterrupted horizontal transmission of clonal isolates in our hospitals during the COVID-19 pandemic.

Kangbainian Lotion Ameliorates Vulvovaginal Candidiasis in Mice by Inhibiting the Growth of Fluconazole-Resistant Candida albicans and the Dectin-1 Signaling Pathway Activation.

Vulvovaginal candidiasis (VVC) is an infectious disease caused by Candida species, which affects millions of women worldwide every year. The resistance to available antifungal drugs for clinical treatment is a growing problem. The treatment of refractory VVC caused by azole-resistant Candida is still facing challenges. However, research on new antifungal drugs is progressing slowly. Although a lot of reports on new antifungal drugs, only three new antifungal drugs (Isavuconazole, ibrexafungerp, and rezafungin) and two new formulations of posaconazole were marketed over the last decade. Chinese botanical medicine has advantages in the treatment of drug-resistant VVC, such as outstanding curative effects and low adverse reactions, which can improve patients’ comfort and adherence to therapy. Kangbainian lotion (KBN), a Chinese botanical formulation, has achieved very good clinical effects in the treatment of VVC. In this study, we investigated the antifungal and anti-inflammatory effects of KBN at different doses in fluconazole-resistant (FLC-resistant) VVC model mice. We further studied the antifungal mechanism of KBN against FLC-resistant Candida albicans (C. albicans) and the anti-inflammatory mechanism correlated with the Dectin-1 signaling pathway. In vivo and in vitro results showed that KBN had strong antifungal and anti-inflammatory effects in FLC-resistant VVC, such as inhibiting the growth of C. albicans and vaginal inflammation. Further studies showed that KBN inhibited the biofilm and hypha formation, reduced adhesion, inhibited ergosterol synthesis and the expression of ergosterol synthesis-related genes ERG11, and reduced the expression of drug-resistant efflux pump genes MDR1 and CDR2 of FLC-resistant C. albicans in vitro. In addition, in vivo results showed that KBN reduced the expression of inflammatory factor proteins TNF-α, IL-1β, and IL-6 in vaginal tissues, and inhibited the expression of proteins related to the Dectin-1 signaling pathway. In conclusion, our study revealed that KBN could ameliorate vaginal inflammation in VVC mice caused by FLC-resistance C. albicans. This effect may be related to inhibiting the growth of FLC-resistance C. albicans and Dectin-1 signaling pathway activation.

Azole-Resistant Alleles of ERG11 in Candida glabrata Trigger Activation of the Pdr1 and Upc2A Transcription Factors.

Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol α-14 demethylase enzyme, which is referred to as Erg11. Inhibition of Erg11 ultimately leads to a reduction in ergosterol production, an essential fungal membrane sterol. Many Candida species, such as Candida albicans, develop mutations in this enzyme which reduces the azole binding affinity and results in increased resistance. Candida glabrata is also a pathogenic yeast that has low intrinsic susceptibility to azole drugs and easily develops elevated resistance. In C. glabrata, these azole resistant mutations typically cause hyperactivity of the Pdr1 transcription factor and rarely lie within the ERG11 gene. Here, we generated C. glabrata ERG11 mutations that were analogous to azole resistance alleles from C. albicans ERG11. Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. Resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. Crucially, the presence of the DM ERG11 allele was sufficient to activate the Pdr1 transcription factor in the absence of azole drugs. Our data indicate that azole resistance linked to changes in ERG11 activity can involve cellular effects beyond an alteration in this key azole target enzyme. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata.

Inhibitory effects of Allium cepa L. ethanolic extract on biological activities and expression of ERG11 in Candida albicans

BackgroundAllium cepa L. is a well-known traditional medicinal plant with different biological activities distributed worldwide. In the present study, the efficacy of Allium cepa ethanolic extract (EAC) is investigated on biological activity, virulence factors and ERG11 gene expression in C. albicans. Material and methodsEAC was tested against C. albicans growth at the concentrations of 0.125–32 mg/mL. Proteinase and phospholipase activity, cell membrane integrity, hydrophobicity and ergosterol content were determined in EAC-treated and non-treated C. albicans by standard methods. The expression of fungal ERG11gene was studied using Real-Time PCR. ResultsThe results indicated that EAC strongly inhibited C. albicans growth, reduced proteinase and phospholipase activity, diminished cell membrane integrity, hydrophobicity and affected ergosterol content at the MIC50 concentration. The expression of ERG11gene was suppressed in EAC-treated C. albicans by 47.25%, while it was suppressed by 16.48% for fluconazole-treated fungus in comparison with the non-treated controls. ConclusionsTaken together, the results demonstrated that A. cepa is an effective natural agent capable of inhibiting the growth of C. albicans and suppressing major fungal virulence factors; hence it could be considered as being developed as a phytomedicine in drug discovery against life-threatening candidiasis for therapeutic purposes.