Characterization of the cholesterol biosynthetic pathway in Dioscorea transversa.

Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1ß-hydroxyprotoneogracillin and protoneogracillin. Here, we used D. transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D. transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28 double bonds required for phytosterol biogenesis as well as Δ24,25 double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that the D. transversa sterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites.

The CCAAT-binding complex mediates azole susceptibility of Aspergillus fumigatus by suppressing SrbA expression and cleavage.

In fungal pathogens, the transcription factor SrbA (a sterol regulatory element-binding protein, SREBP) and CBC (CCAAT binding complex) have been reported to regulate azole resistance by competitively binding the TR34 region (34 mer) in the promoter of the drug target gene, erg11A. However, current knowledge about how the SrbA and CBC coordinately mediate erg11A expression remains limited. In this study, we uncovered a novel relationship between HapB (a subunit of CBC) and SrbA in which deletion of hapB significantly prolongs the nuclear retention of SrbA by increasing its expression and cleavage under azole treatment conditions, thereby enhancing Erg11A expression for drug resistance. Furthermore, we verified that loss of HapB significantly induces the expression of the rhomboid protease RbdB, Dsc ubiquitin E3 ligase complex, and signal peptide peptidase SppA, which are required for the cleavage of SrbA, suggesting that HapB acts as a repressor for these genes which contribute to the activation of SrbA by proteolytic cleavage. Together, our study reveals that CBC functions not only to compete with SrbA for binding to erg11A promoter region but also to affect SrbA expression, cleavage, and translocation to nuclei for the function, which ultimately regulate Erg11A expression and azole resistance.

Mechanism of validamycin A inhibiting DON biosynthesis and synergizing with DMI fungicides against Fusarium graminearum.

Deoxynivalenol (DON) is a vital virulence factor of Fusarium graminearum, which causes Fusarium head blight (FHB). We recently found that validamycin A (VMA), an aminoglycoside antibiotic, can be used to control FHB and inhibit DON contamination, but its molecular mechanism is still unclear. In this study, we found that both neutral and acid trehalase (FgNTH and FgATH) are the targets of VMA in F. graminearum, and the deficiency of FgNTH and FgATH reduces the sensitivity to VMA by 2.12- and 1.79-fold, respectively, indicating that FgNTH is the main target of VMA. We found FgNTH is responsible for vegetative growth, FgATH is critical to sexual reproduction, and both of them play an important role in conidiation and virulence in F. graminearum. We found that FgNTH resided in the cytoplasm, affected the localization of FgATH, and positively regulated DON biosynthesis; however, FgATH resided in vacuole and negatively regulated DON biosynthesis. FgNTH interacted with FgPK (pyruvate kinase), a key enzyme in glycolysis, and the interaction was reduced by VMA; the deficiency of FgNTH affected the localization of FgPK under DON induction condition. Strains with a deficiency of FgNTH were more sensitive to demethylation inhibitor (DMI) fungicides. FgNTH regulated the expression level of FgCYP51A and FgCYP51B by interacting with FgCYP51B. Taken together, VMA inhibits DON biosynthesis by targeting FgNTH and reducing the interaction between FgNTH and FgPK, and synergizes with DMI fungicides against F. graminearum by decreasing FgCYP51A and FgCYP51B expression.

Targeted Genome Mining Reveals the Biosynthetic Gene Clusters of Natural Product CYP51 Inhibitors.

Lanosterol 14α-demethylase (CYP51) is an important target in the development of antifungal drugs. The fungal-derived restricticin 1 and related molecules are the only examples of natural products that inhibit CYP51. Here, using colocalizations of genes encoding self-resistant CYP51 as the query, we identified and validated the biosynthetic gene cluster (BGC) of 1. Additional genome mining of related BGCs with CYP51 led to production of the related lanomycin 2. The pathways for both 1 and 2 were identified from fungi not known to produce these compounds, highlighting the promise of the self-resistance enzyme (SRE) guided approach to bioactive natural product discovery.

Antifungal susceptibility profile and molecular identification of Cyp51C mutations in clinical and environmental isolates of Aspergillus flavus from Argentina.

BACKGROUND: The emergence of azole resistance in non-fumigatus Aspergillus strains is on the raise. OBJECTIVES: To study the susceptibility profiles and the molecular mechanisms of azole resistance of environmental and clinical strains of Aspergillus flavus from Argentina. METHODS: Thirty-five A flavus isolates (18 from soybean seeds and chickpea seeds and 17 from the clinic) were analysed for amphotericin B and azole resistance using the standard microbroth dilution method according to CLSI M38-A2 guidelines. Sequencing analysis of the cyp51 genes was conducted in those isolates displaying high MICs values to itraconazole, voriconazole and/or posaconazole. RESULTS: Among the environmental isolates, 33.3% of them showed high MIC values for at least one triazole whereas 23.5% of the clinical isolates displayed high MIC values for amphotericin B. Point mutations in the Cyp51C gene were recorded in most environmental isolates with non-wild-type MIC values. CONCLUSIONS: Susceptibility differences among environmental A flavus isolates might suggest the possibility of native resistance to certain triazole antifungals used in the clinic. To the best of our knowledge, this is the first report of antifungal screening of environmental strains of A flavus in soybean seeds and chickpea seeds from Argentina that showed increased resistance to voriconazole and itraconazole in comparison to clinical strains.

Azole resistance mechanisms in Aspergillus: update and recent advances.

Aspergillus fumigatus is the main causal agent of invasive aspergillosis (IA), however other species of the genus can also cause IA, such as Aspergillus flavus, Aspergillus terreus, Aspergillus niger and related cryptic species. This infectious disease mainly affects immunosuppressed patients and is linked to elevated mortality rates. As voriconazole is the treatment of choice for this condition, the relevant increase in the number of azole-resistant isolates in recent years has gathered alarming attention, as it also translates into an increase in clinical failures. In this review, we summarise and discuss the azole resistance molecular data described to date in the most clinically prevalent sections of Aspergillus, including mechanisms involving the target proteins Cyp51 and ATP-binding cassette (ABC) or major facilitator superfamily (MFS) efflux pumps. Other resistance mechanisms proposed but not yet fully characterised are also discussed.

Study on the efficiency of dsRNAs with increasing length in RNA-based silencing of the Fusarium CYP51 genes.

Previously, we have demonstrated that transgenic Arabidopsis and barley plants, expressing a 791 nucleotide (nt) dsRNA (CYP3RNA) that targets all three CYP51 genes (FgCYP51A, FgCYP51B, FgCYP51C) in Fusarium graminearum (Fg), inhibited fungal infection via a process designated as host-induced gene silencing (HIGS). More recently, we have shown that spray applications of CYP3RNA also protect barley from fungal infection via a process termed spray-induced gene silencing (SIGS). Thus, RNAi technology may have the potential to revolutionize plant protection in agriculture. Therefore, successful field application will require optimization of RNAi design necessary to maximize the efficacy of the RNA silencing construct for making RNAi-based strategies a realistic and sustainable approach in agriculture. Previous studies indicate that silencing is correlated with the number of siRNAs generated from a dsRNA precursor. To prove the hypothesis that silencing efficiency is correlated with the number of siRNAs processed out of the dsRNA precursor, we tested in a HIGS and SIGS approach dsRNA precursors of increasing length ranging from 400 nt to 1500 nt to assess gene silencing efficiency of individual FgCYP51 genes. Concerning HIGS-mediated disease control, we found that there is no significant correlation between the length of the dsRNA precursor and the reduction of Fg infection on CYP51-dsRNA-expressing Arabidopsis plants. Importantly and in clear contrast to HIGS, we measured a decrease in SIGS-mediated Fg disease resistance that significantly correlates with the length of the dsRNA construct that was sprayed, indicating that the size of the dsRNA interferes with a sufficient uptake of dsRNAs by the fungus.

Distribution and Diversity of Cytochrome P450 Monooxygenases in the Fungal Class Tremellomycetes.

Tremellomycetes, a fungal class in the subphylum Agaricomycotina, contain well-known opportunistic and emerging human pathogens. The azole drug fluconazole, used in the treatment of diseases caused by some species of Tremellomycetes, inhibits cytochrome P450 monooxygenase CYP51, an enzyme that converts lanosterol into an essential component of the fungal cell membrane ergosterol. Studies indicate that mutations and over-expression of CYP51 in species of Tremellomycetes are one of the reasons for fluconazole resistance. Moreover, the novel drug, VT-1129, that is in the pipeline is reported to exert its effect by binding and inhibiting CYP51. Despite the importance of CYPs, the CYP repertoire in species of Tremellomycetes has not been reported to date. This study intends to address this research gap. Comprehensive genome-wide CYP analysis revealed the presence of 203 CYPs (excluding 16 pseudo-CYPs) in 23 species of Tremellomycetes that can be grouped into 38 CYP families and 72 CYP subfamilies. Twenty-three CYP families are new and three CYP families (CYP5139, CYP51 and CYP61) were conserved across 23 species of Tremellomycetes. Pathogenic cryptococcal species have 50% fewer CYP genes than non-pathogenic species. The results of this study will serve as reference for future annotation and characterization of CYPs in species of Tremellomycetes.

Effects of omega-3 polyunsaturated fatty acids on steroidogenesis and cellular development in PCOS rats.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder, which is characterized by hyperandrogenism. Polyunsaturated fatty acids (PUFAs) are necessary for the body's metabolism, growth and development. Despite the well-known benefits of omega-3 polyunsaturated fatty acid supplementation on the modulation of PCOS ovarian function, relatively little is known about the precise regulation mechanism. The objective of this study was to determine the cellular and molecular mechanisms by which omega-3 regulates CYP51 expression and steroid biosynthesis during follicle growth in PCOS. The results indicated that the CYP51 expression was up-regulated in granulosa cells by omega-3. Moreover, the knockdown of CYP51 blocked omega-3 induced estradiol (E2) and progesterone (P4) synthesis as well as cellular viability and proliferation. These changes were accompanied by the up-regulation of the p-Akt level. Furthermore, the PI3K/Akt pathway was required for the regulation of CYP51 expression, steroidogenesis and cell development by omega-3 in PCOS granulosa cells. Our data demonstrate that omega-3 potentiates the cellular development and steroid biosynthesis via CYP51 up-regulation in PCOS, which are mediated through the activation of the PI3K/Akt pathway.

Rapid Parallel Evolution of Azole Fungicide Resistance in Australian Populations of the Wheat Pathogen Zymoseptoria tritici.

Zymoseptoria tritici is a globally distributed fungal pathogen which causes Septoria tritici blotch on wheat. Management of the disease is attempted through the deployment of resistant wheat cultivars and the application of fungicides. However, fungicide resistance is commonly observed in Z. tritici populations, and continuous monitoring is required to detect breakdowns in fungicide efficacy. We recently reported azole-resistant isolates in Australia; however, it remained unknown whether resistance was brought into the continent through gene flow or whether resistance emerged independently. To address this question, we screened 43 isolates across five Australian locations for azole sensitivity and performed whole-genome sequencing on 58 isolates from seven locations to determine the genetic basis of resistance. Population genomic analyses showed extremely strong differentiation between the Australian population recovered after azoles began to be used and both Australian populations recovered before azoles began to be used and populations on different continents. The apparent absence of recent gene flow between Australia and other continents suggests that azole fungicide resistance has evolved de novo and subsequently spread within Tasmania. Despite the isolates being distinct at the whole-genome level, we observed combinations of nonsynonymous substitutions at the CYP51 locus identical to those observed elsewhere in the world. We observed nine previously reported nonsynonymous mutations as well as isolates that carried a combination of the previously reported L50S, S188N, A379G, I381V, Y459DEL, G460DEL, and N513K substitutions. Assays for the 50% effective concentration against a subset of isolates exposed to the tebuconazole and epoxiconazole fungicides showed high levels of azole resistance. The rapid, parallel evolution of a complex CYP51 haplotype that matches a dominant European haplotype demonstrates the enormous potential for de novo resistance emergence in pathogenic fungi.IMPORTANCE Fungicides are essential to control diseases in agriculture because many crops are highly susceptible to pathogens. However, many pathogens rapidly evolve resistance to fungicides. A large body of studies have described specific mutations conferring resistance and have often made inferences about the origins of resistance based on sequencing data from the target gene alone. Here, we show the de novo acquisition of resistance to the ubiquitously used azole fungicides in genetically isolated populations of the wheat pathogen Zymoseptoria tritici in Tasmania, Australia. We confirm evidence for parallel evolution through genome-scale analyses of representative worldwide populations. The emergence of complex resistance haplotypes following a well-documented recent introduction of azoles into Australian farming practices demonstrates how rapidly chemical resistance evolves in agricultural ecosystems.

Development and Application of a New PCR Method for Detection of Blumeria graminis f. sp. tritici.

We developed new PCR assays that target beta-tubulin (TUB2) and 14 alpha-demethylase (CYP51) genes and used them for the species-specific detection of Blumeria graminis f. sp. tritici (Bgt). Based on fungi DNA sequences available in the NCBI (National Center for Biotechnology Information) GenBank database we developed simplex and duplex PCR assays. The specificities of the primer sets were evaluated using environmental samples of wheat leaves collected during the 2015/2016 growing season across Poland. Primer sets LidBg17/18 and LidBg21/22 strongly amplified fragments of the expected length for all 67 tested samples. Primer specificity was confirmed using field samples of Zymoseptoria tri-tici, Puccinia triticina (syn. P. recondita f. sp. tritici), P. striiformis f. sp. tritici, and Pyrenophora tritici-repentis.

Molecular and biological characteristics of laboratory metconazole-resistant mutants in Fusarium graminearum.

The Fusarium graminearum species complex (FGSC), the causal agents of Fusarium head blight (FHB) in wheat, has the different geographically distributed species. Our previous study suggested that a DMI fungicide metconazole exhibits a strong fungicidal activity in mycelial growth of Chinese FHB pathogens and metconazole is currently a most effective compound of commercial fungicides for controlling FHB in China. In the current study, metconazole-resistant F. graminearum mutants were induced by chemical taming and their molecular and biological characteristics were determined. Compared to the corresponding parental strains, three mutation genotypes (two single mutations G443S and D243N, and a combined mutation E103Q&V157 L) were observed in the FgCYP51A of metconazole-resistant mutants. In addition to FgCYP51A mutation, all the mutants had no change on sequences of FgCYP51B and FgCYP51C and promotor sequences of FgCYP51s, but expression patterns of FgCYP51s were different. Compared to the corresponding parental strains, overexpression of FgCYP51A, FgCYP51B and FgCYP51C was observed in the mutant conferring D243N mutation, overexpression of FgCYP51A and FgCYP51B was observed in the mutant conferring E103Q&V157L mutations, and overexpression of FgCYP51A was observed in the mutant conferring G443S mutation. Biological fitness of the mutants conferring D243N mutation or E103Q&V157 L mutations significantly decreased in comparison to the corresponding parental strains, suggesting a fitness penalty. The mutants conferring G443S mutation had no change in biological fitness as compared with the parental strain, indicating that the G443S mutation may emerge in field resistant populations of F. graminearum in the future. In addition, a positive cross resistance between metconazole and other tested DMI fungicides was observed in the mutants conferring D243N mutation or E103Q&V157L mutations, but no cross resistance between metconazole and ipconazole or prochloraz was observed in the mutants conferring G443S mutation. Therefore, we concluded that the mutation genotype of FgCYP51A may cause the differences of biological fitness, cross-resistance and FgCYP51s overexpression patterns. Such information will increase our understanding of resistance mechanism of F. graminearum to DMIs and could provide new reference data for the management of FHB.

Molecular Modeling and Structural Stability of Wild-Type and Mutant CYP51 from Leishmania major: In Vitro and In Silico Analysis of a Laboratory Strain.

Cutaneous leishmaniasis is a neglected tropical disease and a major public health in the most countries. Leishmania major is the most common cause of cutaneous leishmaniasis. In the Leishmania parasites, sterol 14α-demethylase (CYP51), which is involved in the biosynthesis of sterols, has been identified as an attractive target for development of new therapeutic agents. In this study, the sequence and structure of CYP51 in a laboratory strain (MRHO/IR/75/ER) of L. major were determined and compared to the wild-type strain. The results showed 19 mutations including seven non-synonymous and 12 synonymous ones in the CYP51 sequence of strain MRHO/IR/75/ER. Importantly, an arginine to lysine substitution at position of 474 resulted in destabilization of CYP51 (ΔΔG = 1.17 kcal/mol) in the laboratory strain; however, when the overall effects of all substitutions were evaluated by 100 ns molecular dynamics simulation, the final structure did not show any significant changes (p-value < 0.05) in stability parameter of the strain MRHO/IR/75/ER compared to the wild-type protein. The energy level for the CYP51 of wild-type and MRHO/IR/75/ER strain were -40,027.1 and -39,706.48 Kcal/mol respectively. The overall Root-mean-square deviation (RMSD) deviation between two proteins was less than 1 Å throughout the simulation and Root-mean-square fluctuation (RMSF) plot also showed no substantial differences between amino acids fluctuation of the both protein. The results also showed that, these mutations were located on the protein periphery that neither interferes with protein folding nor with substrate/inhibitor binding. Therefore, L. major strain MRHO/IR/75/ER is suggested as a suitable laboratory model for studying biological role of CYP51 and inhibitory effects of sterol 14α-demethylase inhibitors.

The Point Mutation G461S in the MfCYP51 Gene is Associated with Tebuconazole Resistance in Monilinia fructicola Populations in Brazil.

The ascomycete Monilinia fructicola is the causal agent of brown rot of stone fruit in Brazil, causing major pre- and postharvest losses. For many years, the demethylation inhibitor (DMI) fungicide tebuconazole has been used as the most effective active ingredient for controlling brown rot and, as a result, strains of M. fructicola resistant to this ingredient have emerged in many Brazilian states producing stone fruit. The aim of this study was to investigate the mechanisms associated with the resistance of M. fructicola to DMI tebuconazole. By sequencing the M. fructicola CYP51 (MfCYP51) gene, encoding the azole target sterol 14α-demethylase, a mutation was identified at the nucleotide position 1,492, causing the amino acid substitution from glycine to serine at the codon position 461, associated with reduced tebuconazole sensitivity. In addition, it was observed that MfCYP51 gene expression could play a secondary role in DMI fungicide resistance of M. fructicola strains in Brazil. However, for the specific isolate found to exhibit elevated expression levels of MfCYP51, no insertions that would trigger gene expression were found. Based on the point mutation associated with tebuconazole resistance, an allele-specific polymerase chain reaction method was developed to quickly identify resistant genotypes within the Brazilian population. This is the first report determining molecular mechanisms for DMI resistance identification for M. fructicola isolates from Brazil. This information provides an important advancement for risk assessment of DMI fungicides used to manage brown rot of stone fruit.

Evaluation of the combination mode of azoles antifungal inhibitors with CACYP51 and the influence of Site-directed mutation.

14α-demethylase (CYP51) is an essential metabolic enzyme for fungal survival and has been considered as an interesting target for the development of new antifungal inhibitors. Azoles antifungal inhibitors in the treatment of fungal diseases are good candidates via the interaction with the target enzyme CYP51 of fungus. In the study, we constructed the homology model for Candida albicans CYP51 (CACYP51) and analyzed the active site. In order to better understand the structural characteristics of azoles inhibitors and combination mode, the common feature pharmacophore model and the molecular docking were performed. The results suggest that the azoles inhibitors consist of three chemical features: the aromatic groups, phenyl groups and the azoles groups. The aromatic groups of inhibitors occupy the upper of active pocket, the phenyl groups and azoles groups occupy the bottom of active pocket. Further validation studies found these amino acid residues Tyr118, His310 and Ser378 play an important role in the substrate binding, and these amino acid residues with site-directed mutation will weaken the combining ability of the inhibitors.

Disrupting Hepatocyte Cyp51 from Cholesterol Synthesis Leads to Progressive Liver Injury in the Developing Mouse and Decreases RORC Signalling.

Development of mice with hepatocyte knockout of lanosterol 14α-demethylase (HCyp51-/-) from cholesterol synthesis is characterized by the progressive onset of liver injury with ductular reaction and fibrosis. These changes begin during puberty and are generally more aggravated in the knockout females. However, a subgroup of (pre)pubertal knockout mice (runts) exhibits a pronounced male prevalent liver dysfunction characterized by downregulated amino acid metabolism and elevated Casp12. RORC transcriptional activity is diminished in livers of all runt mice, in correlation with the depletion of potential RORC ligands subsequent to CYP51 disruption. Further evidence for this comes from the global analysis that identified a crucial overlap between hepatic Cyp51-/- and Rorc-/- expression profiles. Additionally, the reduction in RORA and RORC transcriptional activity was greater in adult HCyp51-/- females than males, which correlates well with their downregulated amino and fatty acid metabolism. Overall, we identify a global and sex-dependent transcriptional de-regulation due to the block in cholesterol synthesis during development of the Cyp51 knockout mice and provide in vivo evidence that sterol intermediates downstream of lanosterol may regulate the hepatic RORC activity.

Competitive Fitness of Phakopsora pachyrhizi Isolates with Mutations in the CYP51 and CYTB Genes.

Soybean rust (Phakopsora pachyrhizi) in Brazil is mainly controlled with applications of fungicides, including demethylation inhibitors (DMI) and quinone outside inhibitors (QoI). Isolates with less sensitivity to DMI and QoI have been reported, and these have been found to have mutations in the CYP51 and CYTB genes, respectively. There have been no reports of fitness costs in isolates with mutations in CYP51 and CYTB, and the aim of this work was to compare the competitive ability of isolates with lower DMI or QoI sensitivities with that of sensitive (wild-type) isolates. Urediniospores of sensitive wild-type isolates and isolates with different CYP51 or CYTB alleles were mixed and inoculated on detached soybean leaves. After 3 weeks, urediniospores were harvested and used as inoculum for the next disease cycle. Frequencies of relevant target site mutations were monitored using the pyrosequencing method over four disease cycles. Isolates with lower DMI sensitivity and different CYP51 alleles had competitive disadvantages compared with a DMI-sensitive, wild-type CYP51 isolate. In contrast, the isolate with the F129L mutation in the CYTB gene competed equally well with a QoI-sensitive, wild-type CYTB isolate under the conditions of this experiment. The CYP51 and CYTB alleles were stable in all isolates over four disease cycles when cultivated alone.