Antifungal and Antivirulence Activity of Vanillin and Tannic Acid Against Aspergillus fumigatus and Fusarium solani.

Aspergillus fumigatus and Fusarium solani infections have become severe health threat; both pathogens are considered a priority due to the increasing emergence of antifungal-resistant strains and high mortality rates. Therefore, the discovery of new therapeutic strategies has become crucial. In this study, we evaluated the antifungal and antivirulence effects of vanillin and tannic acid against Aspergillus fumigatus and Fusarium solani. The minimum inhibitory concentrations of the compounds were determined by the microdilution method in RPMI broth in 96-well microplates according to CLSI. Conidial germination, protease production, biofilm formation, and in vivo therapeutic efficacy assays were performed. The results demonstrated that vanillin and tannic acid had antifungal activity against Aspergillus fumigatus, while tannic acid only exhibited antifungal activity against Fusarium solani. We found that vanillin and tannic acid inhibited conidial germination and secreted protease production and biofilm formation of the fungal pathogens using sub-inhibitory concentrations. Besides, vanillin and tannic acid altered the fungal membrane permeability, and both compounds showed therapeutic effect against aspergillosis and fusariosis in an infection model in Galleria mellonella larvae. Our results highlight the antivirulence effect of vanillin and tannic acid against priority pathogenic fungi as a possible therapeutic alternative for human fungal infections.

Discovery of a Novel Potent Tetrazole Antifungal Candidate with High Selectivity and Broad Spectrum.

Thirty-one novel albaconazole derivatives were designed and synthesized based on our previous work. All compounds exhibited potent in vitro antifungal activities against seven pathogenic fungi. Among them, tetrazole compound D2 was the most potent antifungal with MIC values of <0.008, <0.008, and 2 µg/mL against Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, respectively, the three most common and critical priority pathogenic fungi. In addition, compound D2 also exhibited potent activity against fluconazole-resistant C. auris isolates. Notably, compound D2 showed a lower inhibitory activity in vitro against human CYP450 enzymes as well as a lower inhibitory effect on the hERG K+ channel, indicating a low risk of drug-drug interactions and QT prolongation. Moreover, with improved pharmacokinetic profiles, compound D2 showed better in vivo efficacy than albaconazole at reducing fungal burden and extending the survival of C. albicans-infected mice. Taken together, compound D2 will be further investigated as a promising candidate.

The C2H2-Type Transcription Factor ZfpA, Coordinately with CrzA, Affects Azole Susceptibility by Regulating the Multidrug Transporter Gene atrF in Aspergillus fumigatus.

The incidence of invasive aspergillosis caused by Aspergillus fumigatus has risen steadily over the past few decades due to the limited effective treatment options and the emergence of antifungal-resistant isolates. In clinic-isolated A. fumigatus, the azole resistance mechanism is primarily caused by mutations of the drug target and/or overexpression of drug efflux pumps. However, knowledge about how drug efflux pumps are transcriptionally regulated is limited. In this study, we found that loss of a C2H2 transcription factor ZfpA (zinc finger protein) results in the marked upregulation of a series of drug efflux pump-encoding genes, especially atrF, which contributes to azole drug resistance in A. fumigatus. CrzA is a previously identified positive transcription factor for genes of drug efflux pumps, and ZfpA transcriptionally inhibits expressions of drug efflux pumps in a CrzA-dependent way. Under the treatment of azoles, both ZfpA and CrzA transfer to nuclei and coregulate the expression of multidrug transporters and then keep normal drug susceptibility in fungal cells. Findings in this study demonstrated that ZfpA is not only involved in fungal growth and virulence potential but also negatively regulates antifungal drug susceptibility. IMPORTANCE Conserved across all kingdoms of life, ABC transporters comprise one of the largest protein families. They are associated with multidrug resistance, affecting aspects such as resistance to antimicrobials or anticancer drugs. Despite the importance of ABC transporters in multidrug resistance, the understanding of their regulatory network is still limited in A. fumigatus. Here, we found that the loss of the transcription factor ZfpA induces the expression of the ABC transporter gene atrF, altering azole susceptibility in A. fumigatus. ZfpA, coordinately with CrzA, affects the azole susceptibility by regulating the expression of the ABC transporter gene atrF. These findings reveal the regulatory mechanism of the ABC transporter gene atrF in A. fumigatus.

Clinical epidemiology of pulmonary aspergillosis in hospitalized patients and contribution of Cyp51A, Yap1, and Cdr1B mutations to voriconazole resistance in etiologic Aspergillus species.

Pulmonary aspergillosis is a life-threatening fungal infection with worldwide distribution. In the present study, clinical epidemiology of pulmonary aspergillosis and antifungal susceptibility of etiologic Aspergillus species were evaluated in one-hundred fifty patients with special focus on the frequency of voriconazole resistance. All the cases were confirmed by the clinical pictures, laboratory findings, and isolation of etiologic Aspergillus species which belonged to two major species, i.e., A. flavus and A. fumigatus. Seventeen isolates displayed voriconazole MIC greater than or equal to the epidemiological cutoff value. Expression of cyp51A, Cdr1B, and Yap1 genes was analyzed in voriconazole-intermediate/resistant isolates. In A. flavus, Cyp51A protein sequencing showed the substitutions T335A and D282E. In the Yap1 gene, A78C replacement led to Q26H amino acid substitution that was not reported previously in A. flavus resistant to voriconazole. No mutations associated with voriconazole resistance were found in the three genes of A. fumigatus. The expression of Yap1 was higher than that of two other genes in both A. flavus and A. fumigatus. Overall, voriconazole-resistant strains of both A. fumigatus and A. flavus demonstrated overexpression of Cdr1B, Cyp51A, and Yap1 genes compared to voriconazole-susceptible strains. Although there are still ambiguous points about the mechanisms of azole resistance, our results showed that mutations were not present in majority of resistant and intermediate isolates, while all of these isolates showed overexpression in all three genes studied. As a conclusion, it seems that the main reason of the emergence of mutation in voriconazole-resistant isolates of A. flavus and A. fumigatus is previous or prolonged exposure to azoles.

Virus Infection Impairs Fungal Response to Stress: Effect of Salt.

Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) weakens the resistance of biofilms of common A. fumigatus reference strain Af293 in intermicrobial competition with Pseudomonas aeruginosa, and sensitizes A. fumigatus for antifungal effects of nikkomycin Z. We compared the sensitivity of two virus-infected (VI) and one virus-free (VF) Af293 strains to hypertonic salt. Salt stress impairs the growth of VI and VF at all times; VF control growth always exceeds VI, and VF growth in salt always exceeds VI. Since VF growth exceeds VI in the presence and absence of salt, we also examined growth in salt as a percentage of control growth. Initially, as a percentage of control, VI exceeded VF, but at 120 h VF began to exceed VI consistently even by this measure; thus, at that time the growth of VF in salt surges in relation to control growth, or, alternatively, its growth in salt persists compared to the relative inhibition of VI. In summary, virus infection impairs the response of A. fumigatus to several different stresses, including hypertonic salt.

Triazole resistance in Aspergillus fumigatus exposed to new chiral fungicide mefentrifluconazole.

BACKGROUND: Triazole resistance in the human fungal pathogen Aspergillus fumigatus has been a growing challenge in clinic treatment with triazole drugs such as itraconazole. The fast evolvement of triazole resistance in A. fumigatus in the ecosystem has drawn great attention, and there has been a possible link between the application of triazole fungicides in agriculture and triazole resistance in A. fumigatus. The change in susceptibility of A. fumigatus exposed to the new chiral triazole fungicide mefentrifluconazole was investigated in this study. RESULTS: The results indicated that triazole resistance in A. fumigatus was acquired with exposure to mefentrifluconazole at a level of greater than or equal to 2 mg L-1 in liquid medium and soil (not at 0.4 nor 1 mg L-1 ). Interestingly, stereoselectivity was found in the acquisition of triazole resistance in A. fumigatus when exposed to mefentrifluconazole. R-mefentrifluconazole, which is very active on plant pathogens, exhibited stronger possibility in the development of the resistance in A. fumigatus than its antipode. Overexpression of cyp51A, AtrF, AfuMDR1 and AfuMDR4 were associated with the acquired resistance in A. fumigatus with hereditary stability. CONCLUSION: The results suggest that triazole resistance in A. fumigatus could be resulted from the selection of mefentrifluconazole at concentrations larger than 2 mg L-1 . Mefentrifluconazole should be applied within the dosage recommended by good agricultural practice to avoid the resistance in A. fumigatus in soil. This also may be applicable to other triazole fungicides. © 2022 Society of Chemical Industry.

Acidic/Alkaline Stress Mediates Responses to Azole Drugs and Oxidative Stress in Aspergillus fumigatus.

A human host exploits stresses such as acidic/alkaline pH, antifungal drugs, and reactive oxygen species to kill microbial pathogens such as the fungus Aspergillus fumigatus. However, A. fumigatus is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of the human host? In this observation, we show that simultaneous exposure to acidic pH and oxidative stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills A. fumigatus synergistically in vitro. Interestingly, A. fumigatus is resistant to the combination of alkaline pH and oxidative stress. Quantitative real-time PCR analyses showed that acidic/alkaline pH stress can mediate oxidative stress responses in A. fumigatus by regulating the expression of catalase-encoding genes. We further show that A. fumigatus is sensitive to the combination of acidic/alkaline stress and azole drug stress. Transcriptome analysis revealed that the sensitivity of A. fumigatus to azole drugs under acidic/alkaline conditions may be related to changes in genetic stability, sphingolipid metabolism, lipid metabolism, and amino acid metabolism. Collectively, our findings suggest that combinatorial stress represents a powerful fungicidal mechanism employed by hosts against pathogens, which suggests novel approaches to potentiate antifungal therapy. IMPORTANCE The human host combats fungal infections via phagocytic cells that recognize and kill fungal pathogens. Immune cells combat Aspergillus fumigatus infections with a potent mixture of chemicals, including reactive oxygen species, acidic/alkaline stress, and antifungal drugs. However, A. fumigatus is relatively resistant to these stresses in vitro. In this observation, we show that it is the combination of acidic/alkaline pH and oxidative or azole stress that kills A. fumigatus so effectively, and we define the molecular mechanisms that underlie this potency. Our findings suggest that combinatorial stress is a powerful fungicidal mechanism employed by hosts, which suggests novel approaches to potentiate antifungal therapy. This study provides a platform for future studies that will address the combinatorial impacts of various environmental stresses on A. fumigatus and other pathogenic microbes.

In Vitro Evaluation of Antimicrobial Peptides from the Black Soldier Fly (Hermetia Illucens) against a Selection of Human Pathogens.

Antimicrobial peptides (AMPs) are being explored as alternatives to traditional antibiotics to combat the rising antimicrobial resistance. Insects have proven to be a valuable source of new, potent AMPs with large structural diversity. For example, the black soldier fly has one of the largest AMP repertoires ever recorded in insects. Currently, however, this AMP collection has not yet undergone antimicrobial evaluation or in-depth in vitro characterization. This study evaluated the activity of a library of 36 black soldier fly AMPs against a panel of human pathogens (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus fumigatus) and a human cell line (MRC5-SV2). The activity profile of two cecropins (Hill-Cec1 and Hill-Cec10) with potent Gram-negative activity, was further explored by characterizing their hemolysis, time-to-kill kinetics, membrane-permeabilization properties, and anti-biofilm activity. Hill-Cec1 and Hill-Cec10 also showed high activity against other bacterial species, including Klebsiella pneumoniae and multi-drug resistant P. aeruginosa. Both AMPs are bactericidal and have a rapid onset of action with membrane-permeabilizing effects. Hill-Cec1 and Hill-Cec10 were also able to prevent P. aeruginosa biofilm formation, but no relevant effect was seen on biofilm eradication. Overall, Hill-Cec1 and Hill-Cec10 are promising leads for new antimicrobial development to treat critical infections caused by Gram-negative pathogens such as P. aeruginosa. IMPORTANCE With the ever growing antimicrobial resistance, finding new candidates for antimicrobial drug development is indispensable. Antimicrobial peptides have steadily gained attention as alternatives for conventional antibiotics, due to some highly desirable characteristics, such as their low propensity for resistance development. With this article, we aim to upgrade the knowledge on the activity of black soldier fly antimicrobial peptides and their potential as future therapeutics. To achieve this, we have evaluated for the first time a library of 36 synthetically produced peptides from the black soldier fly against a range of human pathogens and a human cell line. Two selected peptides have undergone additional testing to characterize their antimicrobial profile against P. aeruginosa, a clinically important Gram-negative pathogen with a high established resistance. Overall, this research has contributed to the search for new peptide drug leads to combat the rising antimicrobial resistance.

Quantitative Monitoring of Mycelial Growth of Aspergillus fumigatus in Liquid Culture by Optical Density.

Filamentous fungi form multicellular hyphae, which generally form pellets in liquid shake cultures, during the vegetative growth stage. Because of these characteristics, growth-monitoring methods commonly used in bacteria and yeast have not been applied to filamentous fungi. We have recently revealed that the cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) contribute to hyphal aggregation in Aspergillus oryzae. Here, we tested whether Aspergillus fumigatus shows dispersed growth in liquid media that can be quantitatively monitored, similar to that of yeasts. We constructed a double disruptant mutant of both the primary α-1,3-glucan synthase gene ags1 and the putative GAG synthase gene gtb3 in A. fumigatus AfS35 and found that the hyphae of this mutant were fully dispersed. Although the mutant lost α-1,3-glucan and GAG, its growth and susceptibility to antifungal agents were not different from those of the parental strain. Mycelial weight of the mutant in shake-flask cultures was proportional to optical density for at least 18 h. We were also able to quantify the dose response of hyphal growth to antifungal agents by measuring optical density. Overall, we established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly used for screening for novel antifungals against Aspergillus species. IMPORTANCE Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi so that we may apply the methods used for unicellular organisms such as yeast and bacteria. In the present study, by using the fungal pathogen Aspergillus fumigatus strain with modified hyphal surface polysaccharides, we succeeded in monitoring the hyphal growth quantitatively by optical density. The principle of this easy measurement by optical density could lead to a novel standard of hyphal quantification such as those that have been used for yeasts and bacteria. Dose response of hyphal growth by antifungal agents could also be monitored. This method could be useful for screening for novel antifungal reagents against Aspergillus species.

Kaempferol ameliorate the prognosis of Aspergillus fumigatus keratitis by reducing fungal load and inhibiting the Dectin-1 and p38 MAPK pathway.

Fungal keratitis is one of leading reasons for blindness in the world, which causes corneal blindness mainly due to excessive inflammatory responses. Kaempferol (KAE) is a natural flavonoid which has potent anti-inflammatory effects. However, whether KAE plays protective roles in fungal keratitis and the potentially protective mechanisms are unrevealed. Here we first investigated the anti-inflammatory and antifungal effects of KAE on Aspergillus fumigatus (A. fumigatus) keratitis in C57BL/6 mice. We found that treatment of KAE ameliorated the severity of keratitis, inhibited macrophages and neutrophils recruitment, depressed corneal fungal load, and declined the expression of TLR4 and Dectin-1 in A. fumigatus infected mice corneas. And in activated hyphae or Curdlan stimulated macrophages, pretreatment of KAE also significantly decreased the mRNA and protein expression of IL-1ß, TNF-α, MIP-2 and the phosphorylated-p38 (p-p38)/p38 MAPK ratio. In summary, KAE ameliorated the prognosis of fungal keratitis in C57BL/6 mice by reducing corneal fungal load, depressing the inflammatory cells recruitment, and downregulating the expression of inflammatory factors, and those effects depended on the inhibition of Dectin-1 and p38 MAPK pathway.

Regulation of gliotoxin biosynthesis and protection in Aspergillus species.

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

Discovery of a novel Aurora B inhibitor GSK650394 with potent anticancer and anti-aspergillus fumigatus dual efficacies in vitro.

Invasive fungal infections including Candidiasis and Aspergillosis are associated with considerable morbidity and mortality in immunocompromised individuals, such as cancer patients. Aurora B is a key mitotic kinase required for the cell division of eukaryotes from fungus to man. Here, we identified a novel Aurora B inhibitor GSK650394 that can inhibit the recombinant Aurora B from human and Aspergillus fumigatus, with IC50 values of 5.68 and 1.29 µM, respectively. In HeLa and HepG2 cells, GSK650394 diminishes the endogenous Aurora B activity and causes cell cycle arrest in G2/M phase. Further cell-based assays demonstrate that GSK650394 efficiently suppresses the proliferation of both cancer cells and Aspergillus fumigatus. Finally, the molecular docking calculation and site-directed mutagenesis analyses reveal the molecular mechanism of Aurora B inhibition by GSK650394. Our work is expected to provide new insight into the combinational therapy of cancer and Aspergillus fumigatus infection.

The role of Glabridin in antifungal and anti-inflammation effects in Aspergillus fumigatus keratitis.

PURPOSE: To investigate the effect of Glabridin (GLD) in Aspergillus fumigatus keratitis and its associated mechanisms. METHODS: Aspergillus fumigatus (A. fumigatus) conidia was inoculated in 96-well plate, and minimal inhibitory concentration (MIC) and biofilm formation ability were evaluated after GLD treatment. Spore adhesion ability was evaluated in conidia infected human corneal epithelial cells (HCECs). Keratitis mouse model was created by corneal intrastromal injection with A. fumigatus conidia, and GLD treatment started at the day after infection. The number of fungal colonies was calculated by plate count, and degree of corneal inflammation was assessed by clinical score. Flow cytometry, myeloperoxidase (MPO), and immunofluorescence staining (IFS) experiments were used to assess neutrophil infiltrations. PCR, ELISA and Western blot were conducted to determine levels of TLR4, Dectin-1 as well as downstream inflammatory factors. RESULTS: GLD treatment suppressed the proliferation, biofilm formation abilities and adhesive capability of A. fumigatus. In mice upon A. fumigatus infection, treatment of GLD showed significantly decreased severity of corneal inflammation, reduced number of A. fumigatus in cornea, and suppressed neutrophil infiltration in cornea. GLD treatment obviously inhibited mRNA and protein levels of Dectin-1, TLR4 and proinflammatory mediators such as IL-1ß, HMGB1, and TNF-α in mice corneas compared to the control group. CONCLUSION: GLD has antifungal and anti-inflammatory effects in fungal keratitis through suppressing A. fumigatus proliferation and alleviating neutrophil infiltration, and repressing the expression of TLR4, Dectin-1 and proinflammatory mediators.

Disulfiram inhibits IL-1ß secretion and inflammatory cells recruitment in Aspergillus fumigatus keratitis.

PURPOSE: Disulfiram, an inhibitor of gasdermin D-induced pore formation, is known to suppress interleukin (IL)-1ß secretion and pyroptosis. However, its effects on fungal keratitis remain unknown. Therefore, we investigated the role of disulfiram in Aspergillus fumigatus keratitis. METHODS: In vitro, Cell Count Kit-8 (CCK8) assay and cell scratch test were performed to determine optimal concentration. In vivo and in vitro experiments were conducted in a mouse model, human neutrophils, and mouse peritoneal macrophages. We pre-treated the mice or cells with disulfiram and infected them with A. fumigatus at specific times. We subsequently evaluated the development of fungal keratitis lesions, the recruitment of inflammatory cells, and the production of inflammatory cytokines using slit lamp microscopy, clinical evaluation, quantitative reverse transcription polymerase chain reaction, immunofluorescence staining, enzyme-linked immunosorbent assay, and western blotting. We also used slit lamp microscopy and clinical evaluation to assess the effect of natamycin with or without disulfiram. RESULTS: Disulfiram at 20 µM has no significant cytotoxic effect and does not affect cell migration. In the mouse model, disulfiram significantly suppressed inflammatory responses, reduced neutrophil and macrophage recruitment, and down-regulated myeloperoxidase and nitric oxide synthase levels at earlier stages of infection. Disulfiram had no effect on IL-1ß production and maturation, but it inhibited IL-1ß secretion in macrophages. Disulfiram combined with natamycin significantly increased corneal transparency in the mice model. CONCLUSION: Overall, disulfiram reduced the host immune response in fungal keratitis by attenuating neutrophil and macrophage recruitment and inhibiting IL-1ß secretion in macrophages. Disulfiram in combination with antifungal agents may serve as a novel therapeutic method for reducing corneal opacity in fungal keratitis.

Construction and activity evaluation of novel dual-target (SE/CYP51) anti-fungal agents containing amide naphthyl structure.

With the increase of fungal infection and drug resistance, it is becoming an urgent task to discover the highly effective antifungal drugs. In the study, we selected the key ergosterol bio-synthetic enzymes (Squalene epoxidase, SE; 14 α-demethylase, CYP51) as dual-target receptors to guide the construction of novel antifungal compounds, which could achieve the purpose of improving drug efficacy and reducing drug-resistance. Three different series of amide naphthyl compounds were generated through the method of skeleton growth, and their corresponding target products were synthesized. Most of compounds displayed the obvious biological activity against different Candida spp. and Aspergillus fumigatus. Among of them, target compounds 14a-2 and 20b-2 not only possessed the excellent broad-spectrum anti-fungal activity (MIC50, 0.125-2 µg/mL), but also maintained the anti-drug-resistant fungal activity (MIC50, 1-4 µg/mL). Preliminary mechanism study revealed the compounds (14a-2, 20b-2) could block the bio-synthetic pathway of ergosterol by inhibiting the dual-target (SE/CYP51) activity, and this finally caused the cleavage and death of fungal cells. In addition, we also discovered that compounds 14a-2 and 20b-2 with low toxic and side effects could exert the excellent therapeutic effect in mice model of fungal infection, which was worthy for further in-depth study.

Dual activity of Meloidogyne incognita-regulated Musa acuminata Pathogenesis-related-10 (MaPR-10) gene.

Plant immunity to pathogen infections is a dynamic response that involves multiple organelles and defence signalling systems such as hypersensitive response (HR) and systemic acquired resistance (SAR). The latter requires the function of Pathogenesis-related (PR) proteins, a common plant protein family with diverse roles in plant innate immunity. Our previous proteomics study showed that a PR gene (ITC1587_Bchr9_P26466_MUSBA) was differentially regulated during a compatible banana-M. incognita interaction, substantiating the isolation of this gene in the current study. Here, we successfully isolated and characterised Pathogenesis-related-10 (PR10) gene with ß-1,3-glucanase and ribonuclease (RNase) activities from two Musa acuminata cultivars (denoted as MaPR10) namely Berangan and Grand Naine (ITC1256). We found that MaPR10 cloned sequences possess glycine-rich loop domain and shared conserved motifs specific to PR10 gene group, confirming its identity as a member of this group. Interestingly, we also found a catalytic domain sequence for glycoside hydrolase family 16 (EXDXXE), unique only to MaPR10 cloned sequences. Two peptide variants closely related to the reference sequence ITC1587_Bchr9_P26466_MUSBA namely MaPR10-BeB5 and MaPR10-GNA5 were overexpressed and purified to test for their functionality. Here, we confirmed that both protein variants possess ß-1,3-glucanase and ribonuclease (RNase) activities, and inhibit the growth of Aspergillus fumigatus, a human opportunistic pathogen. To our knowledge, this is the first PR10 plant proteins with such properties to be reported thus far.

An expanded agar-based screening method for azole-resistant Aspergillus fumigatus.

Antifungal susceptibility testing is an essential tool for guiding antifungal therapy. Reference methods are complex and usually only available in specialised laboratories. We have designed an expanded agar-based screening method for the detection of azole-resistant Aspergillus fumigatus isolates. Normally, identification of resistance mechanisms is obtained only after sequencing the cyp51A gene and promoter. However, our screening method provides azole resistance detection and presumptive resistance mechanisms identification. A previous agar-based method consisting of four wells containing voriconazole, itraconazole, posaconazole and a growth control, detected azole resistance to clinical azoles. Here, we have modified the concentrations of voriconazole and posaconazole to adapt to the updated EUCAST breakpoints against A. fumigatus. We have also expanded the method to include environmental azoles to assess azole resistance and the azole resistance mechanism involved. We used a collection of A. fumigatus including 54 azole-resistant isolates with Cyp51A modifications (G54, M220, G448S, TR53 , TR34 /L98H, TR46 /Y121F/T289A, TR34 /L98H/S297T/F495I), and 50 azole susceptible isolates with wild-type Cyp51A. The screening method detects azole-resistant A. fumigatus isolates when there is growth in any of the azole-containing wells after 48h. The growth pattern in the seven azoles tested helps determine the underlying azole resistance mechanism. This approach is designed for surveillance screening of A. fumigatus azole-resistant isolates and can be useful for the clinical management of patients prior to antifungal susceptibility testing confirmation.

Design, synthesis, and biological activity evaluation of 2-(benzo[b]thiophen-2-yl)-4-phenyl-4,5-dihydrooxazole derivatives as broad-spectrum antifungal agents.

To discover antifungal compounds with broad-spectrum and stable metabolism, a series of 2-(benzo[b]thiophen-2-yl)-4-phenyl-4,5-dihydrooxazole derivatives was designed and synthesized. Compounds A30-A34 exhibited excellent broad-spectrum antifungal activity against Candida albicans with MIC values in the range of 0.03-0.5 µg/mL, and against Cryptococcus neoformans and Aspergillus fumigatus with MIC values in the range of 0.25-2 µg/mL. In addition, compounds A31 and A33 showed high metabolic stability in human liver microsomes in vitro, with the half-life of 80.5 min and 69.4 min, respectively. Moreover, compounds A31 and A33 showed weak or almost no inhibitory effect on the CYP3A4 and CYP2D6. The pharmacokinetic evaluation in SD rats showed that compound A31 had suitable pharmacokinetic properties and was worthy of further study.

Study on inhibitory activity and mechanism of chitosan oligosaccharides on Aspergillus Flavus and Aspergillus Fumigatus.

Chitosan oligosaccharides (COS) are a derivative of low molecular weight chitosan and are potent natural antimicrobial agents. The antimicrobial activity of COS against Aspergillus flavus and Aspergillus fumigatus was evaluated by minimum inhibitory concentration (MIC) and inhibition of mycelial growth. The MICs of COS against these two fungi were 31.2 and 15.6 mg/mL, respectively. COS treatment rendered fungal mycelia wrinkled and deformed with a fractured appearance. COS also increased cellular permeability leading to a significant leakage of cellular components indicating membrane damage. This compound also dose-dependently reduced chitin production and enhanced chitinase activity while enhancing the accumulation of reactive oxygen species (ROS). These characteristics suggested that COS has inhibitory effects against food spoilage fungi and acts on the cell wall and membrane and alters cellular metabolism. COS shows promise for food industry applications since it is non-toxic to higher organisms.