cyp51A mutations, protein modeling, and efflux pump gene expression reveals multifactorial complexity towards understanding Aspergillus section Nigri azole resistance mechanism.

Black Aspergillus species are the most common etiological agents of otomycosis, and pulmonary aspergillosis. However, limited data is available on their antifungal susceptibility profiles and associated resistance mechanisms. Here, we determined the azole susceptibility profiles of black Aspergillus species isolated from the Indian environment and explored the potential resistance mechanisms through cyp51A gene sequencing, protein homology modeling, and expression analysis of selected genes cyp51A, cyp51B, mdr1, and mfs based on their role in imparting resistance against antifungal drugs. In this study, we have isolated a total of 161 black aspergilli isolates from 174 agricultural soil samples. Isolates had variable resistance towards medical azoles; approximately 11.80%, 3.10%, and 1.24% of isolates were resistant to itraconazole (ITC), posaconazole (POS), and voriconazole (VRC), respectively. Further, cyp51A sequence analysis showed that non-synonymous mutations were present in 20 azole-resistant Aspergillus section Nigri and 10 susceptible isolates. However, Cyp51A homology modeling indicated insignificant protein structural variations because of these mutations. Most of the isolates showed the overexpression of mdr1, and mfs genes. Hence, the study concluded that azole-resistance in section Nigri cannot be attributed exclusively to the cyp51A gene mutation or its overexpression. However, overexpression of mdr1 and mfs genes may have a potential role in drug resistance.

Correction: Design and synthesis of eugenol/isoeugenol glycoconjugates and other analogues as antifungal agents against Aspergillus fumigatus.

[This corrects the article DOI: 10.1039/D2MD00138A.].

Unveiling the Cell Wall-Targeting Mechanisms and Multifaceted Virulence Modulation by a Eugenol Glycoconjugate against Aspergillus fumigatus: Insights from in vitro and in ovo Studies.

AIM: The primary objective of this study was to elucidate the putative cell wall-associated targets of compound 6i, a glycoconjugate of eugenol, in Aspergillus fumigatus, while also evaluating its toxicity and assessing histopathologic alterations in the liver, heart, and kidney of compound 6i-treated embryos using an in ovo model. METHOD: To achieve this aim, compound 6i was synthesized, and a series of biochemical assays were performed to determine its impact on the fungal cell wall. Additionally, qRT-PCR and LC-MS/MS analyses were conducted to investigate changes in gene and protein expression profiles associated with melanin biosynthesis, conidiation, siderophore production, transcriptional regulation of ß-glucan biosynthesis, and calcineurin activity in A. fumigatus. RESULTS: The experimental findings revealed that compound 6i exhibited notable antifungal activity against A. fumigatus by perturbing cell wall integrity, hindering ergosterol, glucan, and chitin biosynthesis, and inhibiting catalase production. Moreover, relative gene expression and proteomic analyses demonstrated that compound 6i exerted both down-regulatory and up-regulatory effects on several crucial genes and proteins involved in the aforementioned fungal processes. Furthermore, increased expression of oxidative stress-related proteins was observed in the presence of compound 6i. Notably, the glycoconjugate of eugenol did not elicit cytotoxicity in the liver, heart, and kidney of chick embryos. CONCLUSION: The current investigation elucidated the multifaceted mechanisms by which compound 6i exerts its antifungal effects against A. fumigatus, primarily through targeting cell wall components and signaling pathways. These findings underscore the potential of the eugenol glycoconjugate as a promising antifungal candidate, warranting further exploration and development for combating A. fumigatus infections.

Liver Extracellular Matrix-Based Nanofiber Scaffolds for the Culture of Primary Hepatocytes and Drug Screening.

Immortalized liver cell lines and primary hepatocytes are currently used as in vitro models for hepatotoxic drug screening. However, a decline in the viability and functionality of hepatocytes with time is an important limitation of these culture models. Advancements in tissue engineering techniques have allowed us to overcome this challenge by designing suitable scaffolds for maintaining viable and functional primary hepatocytes for a longer period of time in culture. In the current study, we fabricated liver-specific nanofiber scaffolds with polylactic acid (PLA) along with a decellularized liver extracellular matrix (LEM) by the electrospinning technique. The fabricated hybrid PLA-LEM scaffolds were more hydrophilic and had better swelling properties than the PLA scaffolds. The hybrid scaffolds had a pore size of 38 ± 8 µm and supported primary rat hepatocyte cultures for 10 days. Increased viability (2-fold increase in the number of live cells) and functionality (5-fold increase in albumin secretion) were observed in primary hepatocytes cultured on the PLA-LEM scaffolds as compared to those on conventional collagen-coated plates on day 10 of culture. A significant increase in CYP1A2 enzyme activity was observed in hepatocytes cultured on PLA-LEM hybrid scaffolds in comparison to those on collagen upon induction with phenobarbital. Drugs like acetaminophen and rifampicin showed the highest toxicity in hepatocytes cultured on hybrid scaffolds. Also, the lethal dose of these drugs in rodents was accurately predicted as 1.6 g/kg and 594 mg/kg, respectively, from the corresponding IC50 values obtained from drug-treated hepatocytes on hybrid scaffolds. Thus, the fabricated liver-specific electrospun scaffolds maintained primary hepatocyte viability and functionality for an extended period in culture and served as an effective ex vivo drug screening platform to predict an accurate in vivo drug-induced hepatotoxicity.

Systems-wide analysis of A. fumigatus using kinetic modeling of metabolic pathways to identify putative drug targets.

Aspergillosis is a major causative factor for morbidity in those with impaired immune systems, often caused by Aspergillus fumigatus. The diagnosis and treatment are difficult due to the diversity of individuals and risk factors and still pose a challenge for medical professionals. To understand the pathogenicity of any organism, it is critical to identify the significant metabolic pathways that are involved. Our work focused on developing kinetic models of critical pathways crucial for the survival of A. fumigatus using COPASI. While focusing on the folate biosynthesis, ergosterol biosynthesis and glycolytic pathway; sensitivity, time-course and steady-state analysis were performed to find the proteins/enzymes that are essential in the pathway and can be considered as potential drug targets. For further analysis of the interaction of drug targets identified, a protein-protein interaction (PPI) network was built, and hub nodes were identified using the Cytohubba package from Cytoscape. Based on the findings, dihydropteroate-synthase, dihydrofolate-reductase, 4-amino-4-deoxychorismate synthase, HMG-CoA-reductase, PG-isomerase and hexokinase could act as potential drug targets. Further, molecular docking and MM-GBSA analysis were performed with ligands chosen from DrugBank, and PubChem, and validated by experimental evidence and existing literature based on results from kinetic modeling and PPI network analysis. Based on docking scores and MM-GBSA results, molecular simulations were carried out for 1AJ2-dapsone, 1DIS-sulfamethazine, 1T02-lovastatin and 70YL-3-bromopyruvic acid complexes, which validated our findings. Our study provides a deeper insight into the mechanisms of A. fumigatus's metabolism to reveal dapsone, sulfamethazine, lovastatin and 3-bromopyruvic acid as potential drugs for the treatment of Aspergillosis.Communicated by Ramaswamy H. Sarma.

Design and Synthesis of 1,3-Diynes as Potent Antifungal Agents against Aspergillus fumigatus.

Eugenol and isoeugenol, secondary metabolites isolated from the plant Myristica fragrans have displayed antifungal activities against Aspergillus fumigatus (IC50 1900 µM). Compounds having conjugated unsaturation have been of great use as antifungals i. e. amphotericin B, nystatin and terbinafine etc. Hence, in the present study, we have designed and synthesised 1,3-diynes by utilizing Glaser-Hay and Cadiot-Chodkiewicz coupling reactions to furnish possible antifungal agents. Synthesis of 1,6-diphenoxyhexa-2,4-diyne derivatives was achieved by Cu(I) catalysed coupling of propargylated eugenol, isoeugenol, guaiacol, vanillin and dihydrogenated eugenol or eugenol in good to excellent yields. All the synthesized compounds were evaluated against pathogenic fungus A. fumigatus. Among all the synthesized compounds, one of the compounds was found to be exhibiting promising antifungal activity with IC50 value of 7.75 µM thereby suggesting that this type of scaffold could pave the way for developing new antifungal agents. The most active compound was found to be low cytotoxic when assayed against L-132 cancer cell line. Effect of the most active compound on ergosterol biosynthesis has also been studied. Also, the most active compound exhibited significant anti-biofilm activity although the concentration was found to be higher than its anti-fungal activity. Morphological changes in the biofilm were remarkable under confocal laser scanning microscopy.

4-Allyl-2-methoxyphenol modulates the expression of genes involved in efflux pump, biofilm formation and sterol biosynthesis in azole resistant Aspergillus fumigatus.

Introduction: Antifungal therapy for aspergillosis is becoming problematic because of the toxicity of currently available drugs, biofilm formation on host surface, and increasing prevalence of azole resistance in Aspergillus fumigatus. Plants are rich source of bioactive molecules and antimicrobial activity of aromatic bioactive compounds draws attention because of its promising biological properties. The present study elucidated the antibiofilm activity of 4-allyl-2-methoxyphenol (eugenol) against azole-resistant environmental A. fumigatus isolates. Methods: Soil samples were collected from agricultural fields across India; azole-resistant A. fumigatus (ARAF) were isolated followed by their molecular identification. Antibiofilm activity of eugenol was calculated via tetrazolium based-MTT assay. The expression of the multidrug efflux pumps genes MDR1, MDR4, transporters of the MFS gene, erg11A gene encoding 14α demethylase, and transcription regulatory genes, MedA, SomA and SrbA, involved in biofilm formation of A. fumigatus were calculated by quantitative real time PCR. Results: Out of 89 A. fumigatus isolates, 10 were identified as azole resistant. Eugenol exhibited antibiofilm activity against ARAF isolates, ranging from 312 to 500 µg/mL. Confocal laser scanning microscopy analysis revealed absence of extracellular matrix of ARAF biofilm after eugenol treatment. The gene expression indicated significantly low expression of efflux pumps genes MDR1, MDR4, erg11A and MedA in eugenol treated ARAF isolates when compared with untreated isolates. Conclusions: Our results demonstrate that eugenol effects the expression of efflux pump and biofilm associated genes as well as inhibits biofilm formation in azole resistant isolates of A. fumigatus.

Design and synthesis of eugenol/isoeugenol glycoconjugates and other analogues as antifungal agents against Aspergillus fumigatus.

Glycoconjugates are biologically significant molecules as they tend to serve a wide range of intra- and extra-cellular processes depending on their size and complexity. The secondary metabolites of the plant Myristica fragrans, eugenol and isoeugenol, have shown antifungal activities (IC50 1900 µM). Therefore, we envisioned that glycoconjugates based on these two scaffolds could prove to be potent antifungal agents. Triazole-containing compounds have shown prominent activities as antifungal agents. Based on this, we opined that a Cu(i) catalyzed click reaction could serve as the bridging tool between a eugenol/isoeugenol moiety and sugars to synthesize eugenol/isoeugenol based glycoconjugates. In our present work, we have coupled propargylated eugenol/isoeugenol and azido sugar to furnish eugenol/isoeugenol based glycoconjugates. In another approach, we have carried out hydroxylation of the double bond of eugenol and subsequent azidation of a primary alcohol followed by intramolecular coupling reactions leading to various other analogues. All the synthesized compounds were assayed against an opportunistic pathogenic fungus, Aspergillus fumigatus. Among the synthesized compounds, two analogues have exhibited significant antifungal activities with IC50 values of 5.42 and 9.39 µM, respectively. The study suggested that these two analogues inhibit cell wall-associated melanin hydrophobicity along with the number of conidia. The synthesized compounds were found to be non-cytotoxic to an untransformed cell line.

Isoeugenol affects expression pattern of conidial hydrophobin gene RodA and transcriptional regulators MedA and SomA responsible for adherence and biofilm formation in Aspergillus fumigatus.

Aspergillus fumigatus is one of the major pathogenic fungal species, causing life-threatening infections. Due to a limited spectrum of available antifungals, exploration of new drug targets as well as potential antifungal molecules has become pertinent. Rodlet layer plays an important role in adherence of fungal conidia to hydrophobic cell surfaces in host, which also leads to A. fumigatus biofilm formation, contributing factor to fungal pathogenicity. From decades, natural sources have been known for the development of new active molecules. The present study investigates effect of isoeugenol on genes responsible for hydrophobins (RodA), adhesion as well as biofilm formation (MedA and SomA) of A. fumigatus. Minimum inhibitory concentrations (MIC and IC50) of isoeugenol against A. fumigatus were determined using broth microdilution assay. The IC50 results showed reduced hydrophobicity and biofilm formation as well as eradication after treatment with the compound and electron micrograph data corroborated these findings. The qRT-PCR showed a significant downregulation of genes RodA, MedA, SomA and pksP involved in hydrophobicity and biofilm formation. SwissADME studies potentiated drug-like propensity for isoeugenol which formed four hydrogen bonds with low binding energy (- 4.54 kcal/mol) at the catalytic site of RodA protein studied via AutoDock4. Hence, the findings conclude that isoeugenol inhibits conidial hydrophobicity and biofilm formation of A. fumigatus and further investigations are warranted in this direction.

Gene expression, molecular docking, and molecular dynamics studies to identify potential antifungal compounds targeting virulence proteins/genes VelB and THR as possible drug targets against Curvularia lunata.

Curvuluria lunata is a melanized fungus pathogenic to both plants and animals including humans, causing from mild, febrile to life-threatening illness if not well treated. In humans, it is an etiological agent of keratomycosis, sinusitis, and onychomycosis in immunocompromised and immunocompetent patients. The development of multiple-drug-resistant strains poses a critical treatment issue as well as public health problem. Natural products are attractive prototypes for drug discovery due to their broad-spectrum efficacy and lower side effects. The present study explores possible targets of natural antifungal compounds (α-pinene, eugenol, berberine, and curcumin) against C. lunata via gene expression analysis, molecular docking interaction, and molecular dynamics (MD) studies. Curcumin, berberine, eugenol, and α-pinene exhibited in vitro antifungal activity at 78 µg/ml, 156 µg/ml, 156 µg/ml, and 1250 µg/ml, respectively. In addition, treatment by these compounds led to the complete inhibition of conidial germination and hindered the adherence when observed on onion epidermis. Several pathogenic factors of fungi are crucial for their survival inside the host including those involved in melanin biosynthesis, hyphal growth, sporulation, and mitogen-activated protein kinase (MAPK) signalling. Relative gene expression of velB, brn1, clm1, and pks18 responsible for conidiation, melanin, and cell wall integrity was down-regulated significantly. Results of molecular docking possessed good binding affinity of compounds and have confirmed their potential targets as THR and VelB proteins. The docked structures, having good binding affinity among all, were further refined, and rescored from their docked poses through 100-ns long MD simulations. The MDS study revealed that curcumin formed a stable and energetically stabilized complex with the target protein. Therefore, the study concludes that the antifungal compounds possess significant efficacy to inhibit C. lunata growth targeting virulence proteins/genes involved in spore formation and melanin biosynthesis.

Molecular virulence determinants of Magnaporthe oryzae: disease pathogenesis and recent interventions for disease management in rice plant.

Magnaporthe oryzae, causative agent of the rice blast disease, is a major concern for the loss in yield of rice crop across the globe. It is known for its characteristic melanised dome-shaped appressorium containing a dense melanin layer. The melanised layer is of considerable importance as it is required to generate turgor pressure for initiating peg formation, consequently rupturing the plant cuticle. Various virulence factors play an important role in the disease progression as well as pathogenesis of the fungus. Some of the proteins encoded by virulence genes are associated with signalling, secondary metabolism, protein deprivation, defence responses and conidiation. The purpose of this review is to describe various fungal virulence determinants and provide insights into the molecular mechanisms that are involved in progression of the disease. Besides, the recent molecular approaches being employed to combat the rice blast have also been elaborated.

SEM and qRT-PCR revealed quercetin inhibits morphogenesis of Aspergillus flavus conidia via modulating calcineurin-Crz1 signalling pathway.

ASPERGILLUS FLAVUS: exploits diverse mechanisms to survive during exposure to antifungal agents including morphogenesis. Germination of dormant conidia involves cascades of reactions integrated into the signalling pathway. This study documents the effect of phytochemical-quercetin on A. flavus during germination of conidia using scanning electron microscopy (SEM). Significant inhibition of conidial swelling of A. flavus in comparison to control was observed at 4 and 7 h Quantitative real-time PCR for genes from calcium signalling pathway and heat-shock proteins family showed up-regulation of heat shock (Hsp70 and Hsp90) and calcium signalling pathway genes (calcium-transporting ATPase and calmodulin) in response to quercetin at initial 4 h in comparison to control sample whereas up-regulation of Hsp70, calcineurin and transcription factor Crz1, were observed in both the treated samples. Gene encoding for calcium-kinase, cAMP, Rho-gdp, Plc and Pkc showed a constitutively higher level of expression in quercetin-treated sample in comparison to control at both time points. These data showed a clear response from genes encoding calcineurin-Crz1 signalling pathways and may find its application in the screening of antifungal agents. ABBREVIATIONS: Hsp: Hear shock protein; MIC: Minimum Inhibitory Concentration; SEM: Scanning Electron Microscopy; qRT-PCR: Quantitative Real-Time Polymerase Chain Reaction.

cis-9-Hexadecenal, a Natural Compound Targeting Cell Wall Organization, Critical Growth Factor, and Virulence of Aspergillus fumigatus.

Aspergillus fumigatus causes several nosocomial pulmonary infections and accounts for high morbidity and mortality rate globally. Among various virulence factors, 1,8-dihydroxynaphthalene-melanin plays an important role in the survival during unfavorable conditions both in vivo and in vitro, masks various molecular patterns associated with A. fumigatus, and protects it from the host immune system. In the present study, we aim to understand the potential of cis-9-hexadecenal as an antimelanogenic compound and its role in modulating other associated virulence factors in A. fumigatus. cis-9-Hexadecenal is a bioactive compound that belongs to C16 mono-unsaturated fatty-aldehyde groups. Minimum effective concentration of cis-9-hexadecenal affecting A. fumigatus melanin biosynthesis was determined using broth microdilution method. The spectrophotometric analysis revealed reduced melanin content (91%) and hydrophobicity (59%) at 0.293 mM of cis-9-hexadecenal. Cell surface organizational changes using electron microscopy showed altered demelanized smooth A. fumigatus conidial surface without any protrusions after cis-9-hexadecenal treatment. The transcript analysis of polyketide synthase (PKS) pksP/alb1 gene was quantified through qRT-PCR which revealed an upregulated expression. Total proteome profiling conducted through LC-MS-MS showed upregulated PKS enzyme but other downstream proteins involved in the 1,8-dihydroxynaphthalene-melanin biosynthesis pathway were absent. The homology modeling of PKS using Expasy's web server predicted that PKS is stable at varied conditions and is hydrophilic in nature. The Ramachandran plot by PROCHECK confirmed the 3-D structure of PKS to be reliable. Docking analysis using AutoDock-4.2.6 predicted the binding of cis-9-hexadecenal and PKS at Thr-264 and Ser-171 residue via hydrogen bonding at a low binding energy of -4.95 kcal/mol.

Anti-melanogenic activity of Myristica fragrans extract against Aspergillus fumigatus using phenotypic based screening.

BACKGROUND: Aspergillus fumigatus, an opportunistic fungal pathogen is associated with a wide array of diseases. It produces 1, 8-dihydroxy naphthalene (DHN) melanin that imparts greenish grey color to conidia and is an important virulence factor. It masks various molecular patterns associated with A. fumigatus and protects the fungus from host immune system. Myristica fragrans, enriched with secondary metabolites has been traditionally used for the treatment of infectious and inflammatory diseases. The present study was aimed to explore the anti-melanogenic effect of M. fragrans extracts on A. fumigatus. METHODS: M. fragrans extracts (hexane, chloroform, methanol and ethanol) were prepared through polarity guided extraction. Phytochemical analysis was performed to detect the chemical constituents of the extracts. The minimum effective concentration (MEC) of the extracts against A. fumigatus melanin was determined by broth micro-dilution assay. Various virulence factors were assayed by spectrophotometric methods. Electron microscopic studies were performed to evaluate the effect of the hexane extract of M. fragrans on A. fumigatus cell surface morphology. The major active compounds of the extract were detected by gas chromatography-mass spectrometry (GC-MS). Docking was performed to study the interaction between the major identified compounds and the ketosynthase domain of polyketide synthase protein. RESULTS: The results indicated that the hexane extract of M. fragrans inhibited melanin production (76.09%), reduced ergosterol content (83.63%) and hydrophobicity of the cell (72.2%) at the MEC of 0.078 mg/mL. Altered conidial surface, disappearance of protrusions and absence of melanin layer on outer cell surface was observed in electron microscopy. Forty-two compounds were identified by GC-MS. The main constituents were identified as sabinene (12.2%), linoleic acid (11.7%), hexadecanoic acid (10.5%), safrole (8.1%) and elemicin (7.8%). Docking studies revealed that hexadecanoic acid, its derivative compound cis-9-hexadecenal and isoeugenol have lower binding energy forming proper hydrogen bond with ketosynthase domain of polyketide synthase protein. CONCLUSION: The study concludes that the extract of M. fragrans has potential antifungal properties that can be explored in combination with available antifungals. This combination approach may be helpful for large number of patients suffering with A. fumigatus infections.

Molecular Insights Into Development and Virulence Determinants of Aspergilli: A Proteomic Perspective.

Aspergillus species are the major cause of health concern worldwide in immunocompromised individuals. Opportunistic Aspergilli cause invasive to allergic aspergillosis, whereas non-infectious Aspergilli have contributed to understand the biology of eukaryotic organisms and serve as a model organism. Morphotypes of Aspergilli such as conidia or mycelia/hyphae helped them to survive in favorable or unfavorable environmental conditions. These morphotypes contribute to virulence, pathogenicity and invasion into hosts by excreting proteins, enzymes or toxins. Morphological transition of Aspergillus species has been a critical step to infect host or to colonize on food products. Thus, we reviewed proteins from Aspergilli to understand the biological processes, biochemical, and cellular pathways that are involved in transition and morphogenesis. We majorly analyzed proteomic studies on A. fumigatus, A. flavus, A. terreus, and A. niger to gain insight into mechanisms involved in the transition from conidia to mycelia along with the role of secondary metabolites. Proteome analysis of morphotypes of Aspergilli provided information on key biological pathways required to exit conidial dormancy, consortia of virulent factors and mycotoxins during the transition. The application of proteomic approaches has uncovered the biological processes during development as well as intermediates of secondary metabolite biosynthesis pathway. We listed key proteins/ enzymes or toxins at different morphological types of Aspergillus that could be applicable in discovery of novel therapeutic targets or metabolite based diagnostic markers.