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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 -
MicrobiologyOpen Jan 2020Chitin is one of the key components of fungal cell wall, and chitin deacetylases (CDAs) have been found in fungi; however, their functions remain unknown. Aspergillus...
Chitin is one of the key components of fungal cell wall, and chitin deacetylases (CDAs) have been found in fungi; however, their functions remain unknown. Aspergillus fumigatus is known to cause fatal invasive aspergillosis (IA) among immunocompromised patients with a high mortality rate. Although the A. fumigatus cell wall has long been taken as a unique target for drug development, its dynamic remodeling is complicated and not well understood. Seven putative CDAs are annotated in the A. fumigatus genome. In this study, we analyzed the function of the putative CDAs, Cod4 and Cod7, in A. fumigatus. Biochemical analysis of recombinant proteins showed that Cod4 preferentially deacetylated (GlcNAc) and was less active on chitooligosaccharides with DP > 5, whereas Cod7 was unable to catalyze deacetylation. Simulation of three-dimensional structure revealed that both Cod4 and Cod7 shared a similar folding pattern with HyPgdA from Helicobacter pylori and, similar to HyPgdA, a substitution of Thr8 by Ala8 in Cod7 abolished its CDA activity. Deletion of the cod4, cod7, or both in A. fumigatus led to polarity abnormality and increased conidiation. Furthermore, the expression level of the genes related to polarity was upregulated in the mutants. Our results demonstrated that Cod4 and Cod7 were involved in polarity, though Cod4 was inactive.
Topics: Acetylglucosamine; Amidohydrolases; Aspergillus fumigatus; Cell Wall; Chitin; Gene Expression Regulation, Fungal; Mutation
PubMed: 31602821
DOI: 10.1002/mbo3.943 -
PLoS Genetics Dec 2019Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated...
Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options.
Topics: Animals; Aspergillus fumigatus; Calcium; Caspofungin; Cell Wall; Disease Models, Animal; Drug Resistance, Fungal; Fungal Proteins; Gene Expression Regulation, Fungal; Gene Regulatory Networks; Mice; Mutation; Pulmonary Aspergillosis; Stress, Physiological; Transcription Factors; Virulence
PubMed: 31887136
DOI: 10.1371/journal.pgen.1008551 -
Chemistry & Biodiversity Apr 2023The endophyte Nemania primolutea, inhibited the growth of Penicillium chrysogenum in the coculture system. Four new compounds, nemmolutines A-B (1-2), and penigenumin...
The endophyte Nemania primolutea, inhibited the growth of Penicillium chrysogenum in the coculture system. Four new compounds, nemmolutines A-B (1-2), and penigenumin (3) from N. primolutea, penemin (4) from P. chrysogenum were isolated from the coculture. On the other hand, P. chrysogenum inhibited the Aspergillus fumigatus in the coculture. Induced metabolites (13-16) with monasone naphthoquinone scaffolds including a new one from P. chrysogenum were produced by the coculture of P. chrysogenum, and A. fumigatus. Interesting, cryptic metabolites penicichrins A-B isolated from wild P. chrysogenum induced by host Ziziphus jujuba medium were also found in induced P. chrysogenum cultured in PDB ordinary medium. So the induction of penicichrin production by supplementing with host extract occurred in the fungus P. chrysogenum not the host medium. The productions of penicichrins were the spontaneous metabolism, and the metabolites (13-16) were the culture driven. Compounds 4, 6, 8, 10, 11, 14, and 15 showed significant antifungal activities against the phytopathogen Alternaria alternata with MIC of 1-8 μg/mL, and compounds 7, 9, and 12 indicated significant antifeedant activities against silkworms with feeding deterrence indexes (FDIs) of 92 %, 66 %, and 64 %. The carboxy group in 4-(2-hydroxybutynoxy)benzoic acid derivatives, and xylabisboeins; the hydroxy group in mellein derivatives; and the quinoid in monasone naphthoquinone increased the antifungal activities.
Topics: Antifungal Agents; Aspergillus fumigatus; Penicillium; Penicillium chrysogenum; Ascomycota; Culture Techniques
PubMed: 36859575
DOI: 10.1002/cbdv.202300004 -
MBio Nov 2020Antibiotic resistance is an increasing threat to human health. In the case of , which is both an environmental saprobe and an opportunistic human fungal pathogen,...
Antibiotic resistance is an increasing threat to human health. In the case of , which is both an environmental saprobe and an opportunistic human fungal pathogen, resistance is suggested to arise from fungicide use in agriculture, as the azoles used for plant protection share the same molecular target as the frontline antifungals used clinically. However, limiting azole fungicide use on crop fields to preserve their activity for clinical use could threaten the global food supply via a reduction in yield. In this study, we clarify the link between azole fungicide use on crop fields and resistance in a prototypical human pathogen through systematic soil sampling on farms in Germany and surveying fields before and after fungicide application. We observed a reduction in the abundance of on fields following fungicide treatment in 2017, a finding that was not observed on an organic control field with only natural plant protection agents applied. However, this finding was less pronounced during our 2018 sampling, indicating that the impact of fungicides on population size is variable and influenced by additional factors. The overall resistance frequency among agricultural isolates is low, with only 1 to 3% of isolates from 2016 to 2018 displaying resistance to medical azoles. Isolates collected after the growing season and azole exposure show a subtle but consistent decrease in susceptibility to medical and agricultural azoles. Whole-genome sequencing indicates that, despite the alterations in antifungal susceptibility, fungicide application does not significantly affect the population structure and genetic diversity of in fields. Given the low observed resistance rate among agricultural isolates as well the lack of genomic impact following azole application, we do not find evidence that azole use on crops is significantly driving resistance in in this context. Antibiotic resistance is an increasing threat to human health. In the case of , which is an environmental fungus that also causes life-threatening infections in humans, antimicrobial resistance is suggested to arise from fungicide use in agriculture, as the chemicals used for plant protection are almost identical to the antifungals used clinically. However, removing azole fungicides from crop fields threatens the global food supply via a reduction in yield. In this study, we survey crop fields before and after fungicide application. We find a low overall azole resistance rate among agricultural isolates, as well as a lack of genomic and population impact following fungicide application, leading us to conclude azole use on crops does not significantly contribute to resistance in .
Topics: Agriculture; Aspergillus fumigatus; Azoles; Dose-Response Relationship, Drug; Drug Resistance, Fungal; Fungicides, Industrial; Genetics, Population; Genotype; Humans; Microbial Sensitivity Tests; Phylogeny; Population Dynamics; Soil Microbiology
PubMed: 33234685
DOI: 10.1128/mBio.02213-20 -
Current Protocols in Microbiology Sep 2019Aspergillus fumigatus is an opportunistic human pathogenic mold. DNA extraction from this fungus is usually performed by mechanical perturbation of cells, as it...
Aspergillus fumigatus is an opportunistic human pathogenic mold. DNA extraction from this fungus is usually performed by mechanical perturbation of cells, as it possesses a rigid and complex cell wall. While this is not problematic for single isolates, it can be time consuming for large numbers of strains if using traditional DNA extraction procedures. Therefore, in this article we describe a fast and efficient thermal-shock method to release DNA from spores of A. fumigatus and other filamentous fungi without the need for complex extraction methods. This is especially important for high-throughput PCR analyses of mutants in 96- or 384-well formats in a very short period of time without any concern about sample cross-contamination. This method is currently being used to validate the protein-coding gene and non-coding RNA knockout libraries in A. fumigatus generated in our laboratory, and could be used in the future for diagnostics purposes. © 2019 The Authors.
Topics: Aspergillosis; Aspergillus fumigatus; Containment of Biohazards; DNA, Fungal; Humans; Polymerase Chain Reaction; Spores, Fungal
PubMed: 31518062
DOI: 10.1002/cpmc.89 -
MBio Aug 2021Azole resistance in pathogenic Aspergillus fumigatus has become a global public health issue threatening the use of medical azoles. The environmentally occurring...
Azole resistance in pathogenic Aspergillus fumigatus has become a global public health issue threatening the use of medical azoles. The environmentally occurring resistance mutations, TR/L98H (TR) and TR/Y121F/T289A (TR), are widespread across multiple continents and emerging in the United States. We used whole-genome single nucleotide polymorphism (SNP) analysis on 179 nationally represented clinical and environmental A. fumigatus genomes from the United States along with 18 non-U.S. genomes to evaluate the genetic diversity and foundation of the emergence of azole resistance in the United States. We demonstrated the presence of clades of A. fumigatus isolates: clade A (17%) comprised a global collection of clinical and environmental azole-resistant strains, including all strains with the TR/L98H allele from India, The Netherlands, the United Kingdom, and the United States, and clade B (83%) consisted of isolates without this marker mainly from the United States. The TR/L98H polymorphism was shared among azole-resistant A. fumigatus strains from India, The Netherlands, the United Kingdom, and the United States, suggesting the common origin of this resistance mechanism. Six percent of azole-resistant A. fumigatus isolates from the United States with the TR resistance marker had a mixture of clade A and clade B alleles, suggestive of recombination. Additionally, the presence of equal proportions of both mating types further suggests the ongoing presence of recombination. This study demonstrates the genetic background for the emergence of azole resistance in the United States, supporting a single introduction and subsequent propagation, possibly through recombination of environmentally driven resistance mutations. Aspergillus fumigatus is one of the most common causes of invasive mold infections in patients with immune deficiencies and has also been reported in patients with severe influenza and severe acute respiratory syndrome coronavirus 2 (SARs-CoV-2). Triazole drugs are the first line of therapy for this infection; however, their efficacy has been compromised by the emergence of azole resistance in A. fumigatus, which was proposed to be selected for by exposure to azole fungicides in the environment [P. E. Verweij, E. Snelders, G. H. J. Kema, E. Mellado, et al., Lancet Infect Dis 9:789-795, 2009, https://doi.org/10.1016/S1473-3099(09)70265-8]. Isolates with environmentally driven resistance mutations, TR/L98H (TR) and TR/Y121F/T289A (TR), have been reported worldwide. Here, we used genomic analysis of a large sample of resistant and susceptible A. fumigatus isolates to demonstrate a single introduction of TR in the United States and suggest its ability to spread into the susceptible population is through recombination between resistant and susceptible isolates.
Topics: Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Cytochrome P-450 Enzyme System; Drug Resistance, Fungal; Fungal Proteins; Genome, Fungal; Humans; Microbial Sensitivity Tests; Polymorphism, Single Nucleotide; Triazoles; United States; Whole Genome Sequencing
PubMed: 34372699
DOI: 10.1128/mBio.01803-21 -
Microbiology Spectrum Feb 2022Aspergillus fumigatus is an important opportunistic pathogenic fungus that causes invasive aspergillosis in immunocompromised humans. Regulated fungal growth is...
Aspergillus fumigatus is an important opportunistic pathogenic fungus that causes invasive aspergillosis in immunocompromised humans. Regulated fungal growth is essential for disease development and progression. Thus, screening for genes that regulate fungal growth may lead to the identification of potential therapeutic targets for invasive aspergillosis (IA). Screening of the transfer DNA (T-DNA) random-insertion A. fumigatus mutants identified a severe growth deficiency mutant AFM2954 and featured as the mutated gene described as a putative intracellular protein transporter of unknown function. The deletion of exhibited severe growth defects and significantly increased the nematode and mouse survival rates and decreased the fungal loads and histopathological damages in mouse lungs. Transcriptomic analyses revealed expression changes associated with the cell wall synthesis, the tricarboxylic acid cycle (TCA cycle), and oxidative phosphorylation genes in the mutant. Deletion of the gene resulted in resistance to cell wall-perturbing agents and thickened cell wall as well as reduced ATP contents and mitochondrial membrane potential, suggested that affected the cell wall synthesis and mitochondrial function of A. fumigatus. All together, our study uncovered novel functions of in growth and virulence of A. fumigatus and provided a theoretical basis for the development of new therapeutic target for treating IA patients. Aspergillus fumigatus is the main causative agent of invasive aspergillosis in immunocompromised hosts, with up to 90% lethality. Nevertheless, the fungal factors that regulate the pathogenesis of A. fumigatus remain largely unknown. Better understanding of the mechanisms controlling growth of A. fumigatus may provide novel therapeutic targets. In the present study, we characterized in the opportunistic pathogen A. fumigatus. The function of remains unknown. We proved its important role in growth and virulence, likely because of its effects on cell wall synthesis and mitochondrial functions.
Topics: Amino Acid Sequence; Animals; Aspergillosis; Aspergillus fumigatus; Fungal Proteins; Gene Expression Regulation, Fungal; Humans; Male; Mice; Mice, Inbred BALB C; Sequence Alignment; Sequence Deletion; Virulence
PubMed: 35107385
DOI: 10.1128/spectrum.01558-21 -
Medical Mycology Jul 2021Azole resistance in Aspergillus fumigatus is increasing worldwide and can affect prognosis. It is mostly mediated by cytochrome P51 (CYP51) mutations. In lung transplant...
UNLABELLED
Azole resistance in Aspergillus fumigatus is increasing worldwide and can affect prognosis. It is mostly mediated by cytochrome P51 (CYP51) mutations. In lung transplant recipients (LTR), little is known regarding the prevalence and clinical impact of CYP51 mutations. One hundred thirty-one consecutive A. fumigatus isolates from 103 patients were subjected to CYP51A genotyping through PCR and sequencing. Antifungal susceptibility testing was performed using the Sensititre YeastOne YO-9© broth microdilution technique. Correlations between genotype, phenotype, clinical manifestations of Aspergillus infection, and clinical outcomes were made. Thirty-four (26%) isolates harbored mutations of CYP51A; N248K (n = 14) and A9T (n = 12) were the most frequent. Three isolates displayed multiple point mutations. No significant influences of mutational status were identified regarding azole MICs, the clinical presentation of Aspergillus disease, 1-year all-cause mortality, and clinical outcomes of invasive forms. In the specific context of lung transplant recipients, non-hotspot CYP51A-mutated isolates are regularly encountered; this does not result in major clinical consequences or therapeutic challenges.
LAY SUMMARY
In 131 isolates of Aspergillus fumigatus isolates originating from 103 lung transplant recipients, the CYP51A polymorphism rate was 26%, mostly represented by N248K and A9T mutations. These mutations, however, did not significantly impact azoles minimal inhibitory concentrations or clinical outcomes.
Topics: Antifungal Agents; Aspergillus fumigatus; Cytochrome P-450 Enzyme System; Fungal Proteins; Humans; Lung Transplantation; Microbial Sensitivity Tests; Phenotype; Point Mutation; Polymorphism, Genetic; Prevalence; Transplant Recipients
PubMed: 33418565
DOI: 10.1093/mmy/myaa110 -
MBio Aug 2021The prevalence of Aspergillus fumigatus colonization in individuals with cystic fibrosis (CF) and subsequent fungal persistence in the lung is increasingly recognized....
The prevalence of Aspergillus fumigatus colonization in individuals with cystic fibrosis (CF) and subsequent fungal persistence in the lung is increasingly recognized. However, there is no consensus for clinical management of A. fumigatus in CF individuals, due largely to uncertainty surrounding A. fumigatus CF pathogenesis and virulence mechanisms. To address this gap in knowledge, a longitudinal series of A. fumigatus isolates from an individual with CF were collected over 4.5 years. Isolate genotypes were defined with whole-genome sequencing that revealed both transitory and persistent A. fumigatus in the lung. Persistent lineage isolates grew most readily in a low-oxygen culture environment, and conidia were more sensitive to oxidative stress-inducing conditions than those from nonpersistent isolates. Closely related persistent isolates harbored a unique allele of the high-osmolarity glycerol (HOG) pathway mitogen-activated protein kinase kinase, Pbs2 (). Data suggest this novel allele arose and is necessary for the fungal response to osmotic stress in a low-oxygen environment through hyperactivation of the HOG (SakA) signaling pathway. Hyperactivation of the HOG pathway through comes at the cost of decreased conidial stress resistance in the presence of atmospheric oxygen levels. These novel findings shed light on pathoadaptive mechanisms of A. fumigatus in CF, lay the foundation for identifying persistent A. fumigatus isolates that may require antifungal therapy, and highlight considerations for successful culture of persistent Aspergillus CF isolates. Aspergillus fumigatus infection causes a spectrum of clinical manifestations. For individuals with cystic fibrosis (CF), allergic bronchopulmonary aspergillosis (ABPA) is an established complication, but there is a growing appreciation for A. fumigatus airway persistence in CF disease progression. There currently is little consensus for clinical management of A. fumigatus long-term culture positivity in CF. A better understanding of A. fumigatus pathogenesis mechanisms in CF is expected to yield insights into when antifungal therapies are warranted. Here, a 4.5-year longitudinal collection of A. fumigatus isolates from a patient with CF identified a persistent lineage that harbors a unique allele of the Pbs2 mitogen-activated protein kinase kinase (MAPKK) necessary for unique CF-relevant stress phenotypes. Importantly for A. fumigatus CF patient diagnostics, this allele provides increased fitness under CF lung-like conditions at a cost of reduced growth under standard laboratory conditions. These data illustrate a molecular mechanism for A. fumigatus CF lung persistence with implications for diagnostics and antifungal therapy.
Topics: Aspergillosis, Allergic Bronchopulmonary; Aspergillus fumigatus; Cystic Fibrosis; Genomics; Genotype; Glycerol; Host-Pathogen Interactions; Humans; Longitudinal Studies; Lung; Metabolic Networks and Pathways; Mutation; Osmolar Concentration; Signal Transduction
PubMed: 34465017
DOI: 10.1128/mBio.02153-21