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BMC Genomics Nov 2023Aspergillus fumigatus is a major fungal pathogen that causes severe problems due to its increasing resistance to many therapeutic agents. Fludioxonil is a compound that...
Distinct transcriptional responses to fludioxonil in Aspergillus fumigatus and its ΔtcsC and Δskn7 mutants reveal a crucial role for Skn7 in the cell wall reorganizations triggered by this antifungal.
BACKGROUND
Aspergillus fumigatus is a major fungal pathogen that causes severe problems due to its increasing resistance to many therapeutic agents. Fludioxonil is a compound that triggers a lethal activation of the fungal-specific High Osmolarity Glycerol pathway. Its pronounced antifungal activity against A. fumigatus and other pathogenic molds renders this agent an attractive lead substance for the development of new therapeutics. The group III hydride histidine kinase TcsC and its downstream target Skn7 are key elements of the multistep phosphorelay that represents the initial section of the High Osmolarity Glycerol pathway. Loss of tcsC results in resistance to fludioxonil, whereas a Δskn7 mutant is partially, but not completely resistant.
RESULTS
In this study, we compared the fludioxonil-induced transcriptional responses in the ΔtcsC and Δskn7 mutant and their parental A. fumigatus strain. The number of differentially expressed genes correlates well with the susceptibility level of the individual strains. The wild type and, to a lesser extend also the Δskn7 mutant, showed a multi-faceted stress response involving genes linked to ribosomal and peroxisomal function, iron homeostasis and oxidative stress. A marked difference between the sensitive wild type and the largely resistant Δskn7 mutant was evident for many cell wall-related genes and in particular those involved in the biosynthesis of chitin. Biochemical data corroborate this differential gene expression that does not occur in response to hyperosmotic stress.
CONCLUSIONS
Our data reveal that fludioxonil induces a strong and TcsC-dependent stress that affects many aspects of the cellular machinery. The data also demonstrate a link between Skn7 and the cell wall reorganizations that foster the characteristic ballooning and the subsequent lysis of fludioxonil-treated cells.
Topics: Aspergillus fumigatus; Antifungal Agents; Fungal Proteins; Glycerol; Cell Wall; Dioxoles; Pyrroles
PubMed: 37964194
DOI: 10.1186/s12864-023-09777-5 -
Journal of Clinical Microbiology Feb 2024Azole resistance in (AFM) is increasing and often associated with alterations. We evaluated the activity of isavuconazole and other mold-active azoles against 731 AFM...
Azole resistance in (AFM) is increasing and often associated with alterations. We evaluated the activity of isavuconazole and other mold-active azoles against 731 AFM isolates causing invasive aspergillosis collected in Europe (EU; = 449) and North America (NA; = 282). Isolates were submitted to CLSI susceptibility testing and epidemiological cutoff value (ECV) criteria. A posaconazole ECV of 0.5 mg/L was used as no CLSI ECV was determined. Azole non-wild-type (NWT) isolates were submitted for sequencing by whole genome sequencing. Overall, isavuconazole activity (92.7%/94.0% WT in EU/NA) was comparable to other azoles (WT rate range, 90.9%-96.4%/91.8%-98.6%, respectively), regardless of the region. A total of 79 (10.8%) azole NWT isolates were detected, and similar rates of these isolates were noted in EU (10.7%) and NA (11.0%). Although most AFM were WT to azoles, increasing azole NWT rates were observed in NA (from 6.0% in 2017 to 29.3% in 2021). Azole NWT rates varied from 4.9% (2019) to 20.6% (2018) in EU without an observed trend. alterations occurred in 56.3%/54.8% of azole NWT from EU/NA, respectively. The TR/L98H alteration was observed only in EU isolates (72.0% of EU isolates), while I242V occurred only in NA isolates (58.3%). Isavuconazole remained active (MIC, ≤1 mg/L) against 18.5/47.1% of azole NWT AFM exhibiting alterations in EU/NA, along with voriconazole (29.6/82.4%; MIC, ≤1 mg/L) and posaconazole (48.1/88.2%; MIC, ≤0.5 mg/L). Fourteen different alterations were detected in 44 of 79 NWT isolates. The activity of the azoles varied in AFM that displayed alterations. IMPORTANCE A few microbiology laboratories perform antifungal susceptibility testing locally for systemically active antifungal agents. The identification of emerging azole-resistant is worrisome. As such, there is a critical role for antifungal surveillance in tracking emerging resistance among both common and uncommon opportunistic fungi. Differences in the regional prevalence and antifungal resistance of these fungi render local epidemiological knowledge essential for the care of patients with a suspected invasive fungal infection.
Topics: Humans; Aspergillus fumigatus; Azoles; Antifungal Agents; Fungi; Europe; Invasive Fungal Infections; Drug Resistance, Fungal; Microbial Sensitivity Tests; Fungal Proteins; Nitriles; Pyridines; Triazoles
PubMed: 38193696
DOI: 10.1128/jcm.01141-23 -
International Journal of Molecular... Dec 2023Chronic rhinosinusitis (CRS) is characterized by inflammatory cell infiltration in the sinonasal mucosa. Eosinophil and neutrophil extracellular traps (EETs and NETs,...
Chronic rhinosinusitis (CRS) is characterized by inflammatory cell infiltration in the sinonasal mucosa. Eosinophil and neutrophil extracellular traps (EETs and NETs, respectively) are prominently found in CRS. This study aimed to investigate the effect of airborne fungi, and , on EET and NET formation. Nasal epithelial cells, eosinophils, and neutrophils were isolated from eosinophilic CRS (ECRS), non-ECRS (NECRS), and healthy control. We determined eosinophil and neutrophil transepithelial migration after fungal treatment. We then determined the release of EETs and NETs by fungi using Sytox Green staining and determined the role of reactive oxygen species (ROS) using ROS inhibitors. We identified more abundant EETs and NETs in ECRS than in NECRS. and enhanced eosinophil and neutrophil transepithelial migration. strongly induced EET and NET formation in CRS and, simultaneously, suppressed fungal metabolic activity. EET formation in CRS is associated with nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and NET formation with NADPH-oxidase and mitochondrial ROS. , but not , induced EET and NET formation, and peripheral blood eosinophils and neutrophils exhibited different immune responses against following the inflammatory status of the host. -induced EET and NET formation plays a crucial role in CRS pathogenesis.
Topics: Humans; Neutrophils; Extracellular Traps; Eosinophils; Reactive Oxygen Species; NADP; Rhinosinusitis; Chronic Disease; Sinusitis; Aspergillus; Aspergillus fumigatus; NADPH Oxidases
PubMed: 38139091
DOI: 10.3390/ijms242417264 -
BioRxiv : the Preprint Server For... Aug 2023, an important pulmonary fungal pathogen causing several diseases collectively called aspergillosis, relies on asexual spores or conidia for initiating host infection....
, an important pulmonary fungal pathogen causing several diseases collectively called aspergillosis, relies on asexual spores or conidia for initiating host infection. Here, we used a phylogenomic approach to compare proteins in the conidial surface of , two closely related non-pathogenic species, and , and the cryptic pathogen . After identifying 62 proteins uniquely expressed on the conidial surface, we deleted 42 genes encoding conidial proteins. We found deletion of 33 of these genes altered susceptibility to macrophage killing, penetration and damage to epithelial cells, and cytokine production. Notably, a gene that encodes glycosylasparaginase, which modulates levels of the host pro-inflammatory cytokine IL-1β, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that conidial surface proteins and effectors are important for evasion and modulation of the immune response at the onset of fungal infection.
PubMed: 37662192
DOI: 10.1101/2023.08.22.553365 -
Acta Biomaterialia Oct 2023Fungal keratitis (FK) is a severe infectious corneal disease. Since traditional eye drops exhibit poor dissolution and high corneal toxicity, the efficacy of current...
Fungal keratitis (FK) is a severe infectious corneal disease. Since traditional eye drops exhibit poor dissolution and high corneal toxicity, the efficacy of current treatments for FK remains limited. It is needed to develop new approaches to control the cornea damage from FK. In this study, a nanobody (Nb) specific to β-glucan in the fungal cell wall was prepared. The conjugate of the Nb with natamycin (NAT), a traditional antifungal drug, was synthesized. Firstly, we found the Nb specific to β-glucan inhibited fungal growth by disrupting cell wall and biofilm formation.. In addition, the content of β-glucan in the fungal cell wall decreased after Nb treatment. The Nb also reduced the adhesion ability of fungal conidia to human corneal epithelial cells (HCECs). Further, we examined the difference between NAT and Nb-NAT in antifungal growth. Nb-NAT showed better antifungal effects than NAT which was caused by the interaction between Nb and β-glucan. Moreover, Nb concentration below 0.5 mg/mL did not affect the viability of HCECs. Nb-NAT had less cytotoxicity and ocular surface irritation than NAT. Nb specific to β-glucan attenuated Aspergillus fumigatus (A. fumigatus) virulence and relieved inflammatory responses in FK. Nb-NAT treatment of the cornea improved therapeutic effects compared with NAT. It decreased clinical scores and expression level of inflammatory factors. To our knowledge, this study is the first to report a Nb specific to β-glucan and Nb-NAT for the treatment of FK. These unique functions of the Nb specific to β-glucan and Nb-NAT would render it as an alternative molecule to control fungal infections including FK. STATEMENT OF SIGNIFICANCE: Fungal keratitis is a corneal disease with a high rate of blindness. Due to the poor dissolution and high corneal toxicity exhibited by traditional eye drops, the efficacy of current therapeutic treatments for fungal keratitis (FK) remains limited. To enhance the therapeutic effect of natamycin in treating fungal keratitis, this study developed an innovative approach by preparing a β-glucan-specific nanobody and loading it with the antifungal drug natamycin. The β-glucan-specific nanobody has the ability to control both fungal pathogen invasion and inflammation, which can cause damage to the cornea in FK. The conjugation with the β-glucan-specific nanobody significantly increased the antifungal capacity of natamycin and reduced its toxicity. The further application of natamycin conjugated with the β-glucan-specific nanobody could be expanded to other diseases caused by fungal pathogen infections.
Topics: Humans; Antifungal Agents; Natamycin; Single-Domain Antibodies; Keratitis; Eye Infections, Fungal; Ophthalmic Solutions
PubMed: 37579912
DOI: 10.1016/j.actbio.2023.08.019 -
Microorganisms Aug 2023Transient and prolonged waterlogging stress (WS) stimulates ethylene (ET) generation in plants, but their reprogramming is critical in determining the plants' fate under...
Transient and prolonged waterlogging stress (WS) stimulates ethylene (ET) generation in plants, but their reprogramming is critical in determining the plants' fate under WS, which can be combated by the application of symbiotically associated beneficial microbes that induce resistance to WS. The present research was rationalized to explore the potential of the newly isolated 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing fungal endophytic consortium of (MA1) and (MA4) on maize growth promotion under WS. MA1 and MA4 were isolated from the seeds of L., which ably produced a sufficient amount of IAA, proline, phenols, and flavonoids. MA1 and MA4 proficiently colonized the root zone of maize ( L.). The symbiotic association of MA1 and MA4 promoted the growth response of maize compared with the non-inoculated plants under WS stress. Moreover, MA1- and MA4-inoculated maize plants enhanced the production of total soluble protein, sugar, lipids, phenolics, and flavonoids, with a reduction in proline content and HO production. MA1- and MA4-inoculated maize plants showed an increase in the DPPH activity and antioxidant enzyme activities of CAT and POD, along with an increased level of hormonal content (GA and IAA) and decreased ABA and ACC contents. Optimal stomatal activity in leaf tissue and adventitious root formation at the root/stem junction was increased in MA1- and MA4-inoculated maize plants, with reduced lysigenous aerenchyma formation, ratio of cortex-to-stele, water-filled cells, and cell gaps within roots; increased tight and round cells; and intact cortical cells without damage. MA1 and MA4 induced a reduction in deformed mesophyll cells, and deteriorated epidermal and vascular bundle cells, as well as swollen metaxylem, phloem, pith, and cortical area, in maize plants under WS compared with control. Moreover, the transcript abundance of ethylene-responsive gene , responsible for the induction of the WS tolerance in maize, showed optimally reduced expression sufficient for induction in WS tolerance, in MA1- and MA4-inoculated maize plants under WS compared with the non-inoculated control. The existing research supported the use of MA1 and MA4 isolates for establishing the bipartite mutualistic symbiosis in maize to assuage the adverse effects of WS by optimizing ethylene production.
PubMed: 37630585
DOI: 10.3390/microorganisms11082025 -
Medical Mycology Nov 2023Aspergillus fumigatus is a fungal species causing diverse diseases in humans. The use of azoles for treatments of A. fumigatus diseases has resulted in azole resistance....
Aspergillus fumigatus is a fungal species causing diverse diseases in humans. The use of azoles for treatments of A. fumigatus diseases has resulted in azole resistance. Azoles are also widely used in the environment for crop protection, which resulted in azole resistance. Resistance is primarily due to mutations in cyp51A, which encodes the target protein for azoles. Here we addressed the occurrence of azole resistance in soils from a vast part of Switzerland. We aimed to associate the use of azoles in the environment with the occurrence of azole resistance. We targeted sample sites from different agricultural environments as well as sites with no agricultural practice (natural sites and urban sites). Starting from 327 sites, 113 A. fumigatus isolates were recovered (2019-2021), among which 19 were azole-resistant (15 with TR34/L98H and four with TR46/Y121F/T289A resistance mutations in cyp51A). Our results show that azole resistance was not associated with a specific agricultural practice. Azoles could be chemically detected in investigated soils, however, their presence was not associated with the occurrence of azole-resistant isolates. Interestingly, genetic markers of resistance to other fungicides were detected but only in azole-resistant isolates, thus reinforcing the notion that A. fumigatus cross-resistance to fungicides has an environmental origin. In conclusion, this study reveals the spreading of azole resistance in A. fumigatus from the environment in Switzerland. The proximity of agricultural areas to urban centers may facilitate the transmission of resistant strains to at-risk populations. Thus, vigilant surveillance is required to maintain effective treatment options for aspergillosis.
Topics: Humans; Aspergillus fumigatus; Azoles; Fungicides, Industrial; Antifungal Agents; Soil; Switzerland; Fungal Proteins; Drug Resistance, Fungal; Microbial Sensitivity Tests
PubMed: 37930839
DOI: 10.1093/mmy/myad110 -
Cell Chemical Biology Jul 2023Rising drug resistance among pathogenic fungi, paired with a limited antifungal arsenal, poses an increasing threat to human health. To identify antifungal compounds, we...
Rising drug resistance among pathogenic fungi, paired with a limited antifungal arsenal, poses an increasing threat to human health. To identify antifungal compounds, we screened the RIKEN natural product depository against representative isolates of four major human fungal pathogens. This screen identified NPD6433, a triazenyl indole with broad-spectrum activity against all screening strains, as well as the filamentous mold Aspergillus fumigatus. Mechanistic studies indicated that NPD6433 targets the enoyl reductase domain of fatty acid synthase 1 (Fas1), covalently inhibiting its flavin mononucleotide-dependent NADPH-oxidation activity and arresting essential fatty acid biosynthesis. Robust Fas1 inhibition kills Candida albicans, while sublethal inhibition impairs diverse virulence traits. At well-tolerated exposures, NPD6433 extended the lifespan of nematodes infected with azole-resistant C. albicans. Overall, identification of NPD6433 provides a tool with which to explore lipid homeostasis as a therapeutic target in pathogenic fungi and reveals a mechanism by which Fas1 function can be inhibited.
Topics: Humans; Antifungal Agents; Candida albicans; Aspergillus fumigatus; Virulence; Microbial Sensitivity Tests
PubMed: 37369212
DOI: 10.1016/j.chembiol.2023.06.005 -
PLoS Pathogens Oct 2023The Aspergillus fumigatus unfolded protein response (UPR) is a two-component relay consisting of the ER-bound IreA protein, which splices and activates the mRNA of the...
The Aspergillus fumigatus unfolded protein response (UPR) is a two-component relay consisting of the ER-bound IreA protein, which splices and activates the mRNA of the transcription factor HacA. Spliced hacA accumulates under conditions of acute ER stress in vitro, and UPR null mutants are hypovirulent in a murine model of invasive pulmonary infection. In this report, we demonstrate that a hacA deletion mutant (ΔhacA) is furthermore avirulent in a model of fungal keratitis, a corneal infection, and an important cause of ocular morbidity and unilateral blindness worldwide. Interestingly, we demonstrate that A. fumigatus hacA is spliced in infected lung samples, but not in the cornea, suggesting the amount of ER stress experienced by the fungus varies upon the host niche. To better understand how the UPR contributes to fungal cell biology across a spectrum of ER-stress levels, we employed transcriptomics on the wild-type and ΔhacA strains in glucose minimal media (low stress), glucose minimal media with dithiothreitol (high stress), and gelatin minimal media as a proxy for the nutrient stress encountered in the cornea (mid-level stress). These data altogether reveal a unique HacA-dependent transcriptome under each condition, suggesting that HacA activity is finely-tuned and required for proper fungal adaptation in each environment. Taken together, our results indicate that the fungal UPR could serve as an important antifungal target in the setting of both invasive pulmonary and corneal infections.
Topics: Animals; Mice; Aspergillus fumigatus; Unfolded Protein Response; Keratitis; Nutrients; Glucose; Fungal Proteins
PubMed: 37906600
DOI: 10.1371/journal.ppat.1011435 -
BioRxiv : the Preprint Server For... Sep 2023Protein acetylation is a crucial post-translational modification that controls gene expression and a variety of biological processes. Sirtuins, a prominent class of NAD...
Protein acetylation is a crucial post-translational modification that controls gene expression and a variety of biological processes. Sirtuins, a prominent class of NAD -dependent lysine deacetylases, serve as key regulators of protein acetylation and gene expression in eukaryotes. In this study, six single knockout strains of fungal pathogen were constructed, in addition to a strain lacking all predicted sirtuins (SIRTKO). Phenotypic assays suggest that sirtuins are involved in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. deletion resulted in attenuation of virulence, as demonstrated in murine and infection models. The absence of AfSirE leads to altered acetylation status of proteins, including histones and non-histones, resulting in significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat infections or in combination therapy with available antifungals.
PubMed: 37808717
DOI: 10.1101/2023.09.25.558961