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Cerium oxide (CeO2) nanoparticles, as a metal oxide nanomaterial, are increasingly used for various industrial and biomedical applications. Although their cytotoxicity to bacteria and the associated mechanisms have attracted particular attention, the mechanisms behind their antifungal effects have remained unclear. This study investigated the antifungal properties of CeO2, focusing on Aspergillus oryzae. CeO2 inhibited fungal spore germination on solid substrates, and the effect was fungistatic rather than fungicidal. CeO2 inhibited fungal growth, especially under UV irradiation, and induced reactive oxygen species (ROS) production. Tocopherol reduced the intracellular ROS levels and the growth-inhibitory effects of CeO2, suggesting that ROS are involved in these growth-inhibitory effects. Transcriptomic analysis revealed upregulated expression of genes related to phospholipases and phosphate metabolism. CeO2 affected phosphate ion concentration in the medium, potentially influencing cellular responses. This research provided valuable insights into the antifungal effects of CeO2 application, which differ from those of conventional photocatalysts like TiO2., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Fungi are ubiquitously present in our living environment and are responsible for crop and infectious diseases. Developing new antifungal agents is constantly needed for their effective control. Here, we investigated fungal cellular responses to an array of antifungal compounds, including plant- and bacteria-derived antifungal compounds. The pathogenic fungus Aspergillus fumigatus generated reactive oxygen species in its hyphae after exposure to the antifungal compounds thymol, farnesol, citral, nerol, salicylic acid, phenazine-1-carbonic acid, and pyocyanin, as well as under oxidative and high-temperature stress conditions. The production of nitric oxide (NO) was determined using diaminofluorescein-FM diacetate (DAF-FM DA) and occurred in response to antifungal compounds and stress conditions. The application of reactive oxygen species or NO scavengers partly suppressed the inhibitory effects of farnesol on germination. However, NO production was not detected in the hyphae using the Greiss method. An LC/MS analysis also failed to detect DAF-FM-T, a theoretical product derived from DAF-FM DA and NO, in the hyphae after antifungal treatments. Thus, the cellular state after exposure to antifungal agents may be more complex than previously believed, and the role of NO in fungal cells needs to be investigated further., (© 2022. The Author(s).)

Aspergillosis associated with azole-resistant Aspergillus fumigatus has a mortality rate that can approach 90% in certain patient populations. The best-understood avenue for azole resistance involves changes in the cyp51A gene that encodes the target of azole drugs, lanosterol α-14 demethylase. The most common azole resistance allele currently described is a linked change corresponding to a change in the coding sequence of cyp51A and a duplication of a 34-bp region in the promoter leading to a tandem repeat (TR). Our previous studies identified a positively acting transcription factor called AtrR that binds to the promoter of cyp51A as well as that of an important membrane transporter protein gene called abcG1 In this work, we characterize two different mutant alleles of atrR , either an overproducing or an epitope-tagged form, causing constitutive activation of this factor. Using an epitope-tagged allele of atrR for chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), the genomic binding sites for AtrR were determined. Close to 900 genes were found to have an AtrR response element (ATRE) in their promoter regions. Transcriptome evaluation by RNA sequencing (RNA-seq) indicated that both alleles led to elevated transcription of a subset of target genes. An electrophoretic mobility shift assay and DNase I protection mapping localized the ATREs in both the abcG1 and cyp51A promoters. The ATRE in cyp51A was located within the 34-bp repeat element. Virulence in a murine model was compromised when AtrR was either deleted or overproduced, indicating that the proper dosage of this factor is key for pathogenesis. IMPORTANCE Aspergillus fumigatus are often treated with azole drugs, but resistance to these antifungal agents is increasing. Mortality from aspergillosis associated with azole-resistant fungi is extremely high. Previous work has identified transcriptional control of the azole drug target-encoding gene A. fumigatus are often treated with azole drugs, but resistance to these antifungal agents is increasing. Mortality from aspergillosis associated with azole-resistant fungi is extremely high. Previous work has identified transcriptional control of the azole drug target-encoding gene cyp51A as an important contributor to resistance in A. fumigatus Here, we demonstrate that the transcription factor AtrR binds to a region in the cyp51A promoter that is associated with alleles of this gene conferring clinically important azole resistance. Using high-throughput genomic technologies, we also uncover a large suite of target genes controlled by AtrR. These data indicate that AtrR coordinately regulates many different processes involved in drug resistance, metabolism, and virulence. Our new understanding of AtrR function provides important new insight into the pathogenesis of A. fumigatus ., (Copyright © 2019 Paul et al.)

We designed primers and cycling probes to detect the tandem repeat (TR) of cyp51A promoter in Aspergillus fumigatus. A control-probe was designed to anneal to the outside of the TR region, whereas a TR-probe was designed to anneal to the inside of the TR region. For amplification and probe-hydrolysis detection, the CycleavePCR system was used. Although the difference between Ct values of the wild-type genome for the control-probe and the TR-probe was around -0.1, the difference between Ct values of TR-harboring strains was around 0.7. These data indicate that this is a simple method to detect TR in azole-resistant A. fumigatus.

Aspergillus fumigatus is a saprophytic fungus that is a major causative pathogen for aspergillosis. Only a few classes of antifungals are used for treating this life-threatening fungal infection. Azoles are the first-line drugs and are widely used for the management and prophylaxis of aspergillosis. An emerging issue is the increasing incidence of resistant isolates worldwide. In particular, environmentally derived tandem-repeat-type azole-resistant mutations, such as Cyp51A TR34/L98H, and Cyp51A TR46/Y121F/T289A, have emerged over the last decade. In particular, azole-resistant isolates were prevalent in clinical settings in European countries; many of the reports are from the Netherlands, UK, and Germany. In contrast, reports on azole-resistant A. fumigatus isolates from East Asian countries are still few and have only recently begun to increase. Herein, all literature on East Asian azole-resistant A. fumigatus isolates were reviewed, and a complete list of resistant isolates from China, Japan, Taiwan, and Korea is provided. As of this report, the total numbers of tandem-repeat-type azole-resistant isolates are 26, 3, 32, and 1 in China, Japan, Taiwan, and Korea, respectively.

Recently, azole-resistant Aspergillus fumigatus containing a 34-bp or 46-bp tandem repeat in the promoter region of cyp51A combined with amino acid substitution(s) has appeared in the environment worldwide, including several Asian countries. In this study, we isolated the 34-bp tandem repeat-containing azole-resistant A. fumigatus strain OKH50 from a patient in Japan in May 2016. The patient had not been treated with medical azoles before the strain isolation, suggesting that the resistant property was acquired before infection. In addition, the patient had not traveled overseas. Our analysis of short tandem repeats of the strain indicates that the strain is strongly related to the 34-bp tandem repeat-containing isolates from European countries and Asia-Oceania countries but not to susceptible isolates from Japan, suggesting that the strain was introduced from overseas and might spread in Japan., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Aspergillus niger and its related species, known as Aspergillus section Nigri , are ubiquitously distributed across the globe and are often isolated from clinical specimens. In Japan, Aspergillus section Nigri is second most often isolated from clinical specimens following Aspergillus fumigatus We determined the species of Aspergillus section Nigri isolated in Japan by DNA sequencing of partial β-tubulin genes and investigated drug susceptibility by the CLSI M38-A2 method. The collection contained 20 Aspergillus niger , 59 Aspergillus welwitschiae , and 39 Aspergillus tubingensis strains. Drug susceptibility testing revealed 30 to 55% of A. niger , 6.8 to 18.6% of A. welwitschiae , and 79.5 to 89.7% of A. tubingensis isolates to be less susceptible (so-called resistant) to itraconazole (ITC) and/or voriconazole (VRC) according to the epidemiologic cutoff values (ECVs) proposed for A. niger previously. MIC distributions of ITC or VRC showed no remarkable differences between clinical and environmental isolates. When the cyp51A sequences were compared between susceptible and resistant strains, 18 amino acid mutations were specific for resistant isolates of A. niger and A. tubingensis ; however, none of them were confirmed to be associated with azole resistance. Three nonrelated A. welwitschiae isolates possessed a partial deletion in cyp51A , likely attributable to being more susceptible to azoles than other isolates. One of five ITC-resistant A. tubingensis isolates showed higher expression of cyp51A than did susceptible strains. Our results show that cyp51A point mutations may have no association with azole resistance but that in some cases the overexpression of cyp51A may lead to the azole resistance in these species., (Copyright © 2017 American Society for Microbiology.)

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B)., Competing Interests: The authors have declared that no competing interests exist.

Azole-resistant strains of Aspergillus fumigatus containing a tandem repeat in the cyp51A promoter and amino acid substitution(s) have been isolated in the environment worldwide; however, this type of resistant strain had never been isolated from the environment in Japan. Our previous study indicated that an azole-resistant A. fumigatus strain OKH50 containing a 34-bp tandem repeat in cyp51A promoter with L98H substitution in Cyp51A was isolated from a patient in Obihiro of Hokkaido, Japan. In this study, we collected azole-resistant Aspergillus spp. by air sampling from the environment in Japan. One Aspergillus-like colony was isolated from one of 10 sampling sites surveyed. The strain Env1 was confirmed as A. fumigatus by nucleotide sequencing and possessed a 34-bp tandem repeat in the promoter region of cyp51A with L98H substitution in Cyp51A. A. fumigatus Env1 has the identical short tandem repeat pattern with the OKH50 strain, indicating that these strains are closely related with each other. Additionally, the short tandem repeat pattern is closely related to Danish and Iranian environmental isolates, suggesting that azole-resistant strains have crossed transnational boundaries and are now present in Japan, and therefore, further analysis throughout Japan is required to determine the distribution of this type of azole-resistant A. fumigatus.

Azoles are widely used for controlling fungal growth in both agricultural and medical settings. The target protein of azoles is CYP51, a lanosterol 14-α-demethylase involved in the biosynthesis of ergosterol. Recently, a novel azole resistance mechanism has arisen in pathogenic fungal species Aspergillus fumigatus. Resistant strains contain a 34-bp or 46-bp tandem repeat (TR) in the promoter of cyp51A, and have disseminated globally in a short period of time. In this study, we investigated whether an azole-resistant strain with a 46-bp TR (TR46/Y121F/T289A) could be sensitised to azoles by deletion of srbA, encoding a direct regulator of cyp51A. The loss of SrbA did not affect colony growth or conidia production, but decreased expression of cyp51A. The srbA deletion strain showed hyper-susceptibility to medical azoles as well as azole fungicides, while its sensitivity to non-azole fungicides was unchanged. This is the first demonstration that deletion of a regulator of cyp51A can sensitise an azole-resistant A. fumigatus strain. This finding may assist in the development of new drugs to help combat life-threatening azole-resistant fungal pathogens.

Multi-azole resistant Aspergillus fumigatus carrying TR46/Y121F/T289A was isolated from a patient in Japan in Dec 2013. This strain grouped into the same clade of the ones which were clinically isolated in France and Germany. A. fumigatus harboring this mutation could be rapidly diffused outside the Eurasian continent., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.