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Journal of Microbiology, Immunology,... Aug 2022Rapid and reliable diagnostic methods for Aspergillus fumigatus infection are urgently needed. Clustered regularly interspaced short palindromic repeat...
BACKGROUND
Rapid and reliable diagnostic methods for Aspergillus fumigatus infection are urgently needed. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 13a (Cas13a) has high sensitivity and specificity in the diagnosis of viral infection. However, its potential use in detecting A. fumigatus remains unexplored. A highly sensitive and specific method using the CRISPR/Cas13a system was developed for the reliable and rapid detection of A. fumigatus.
METHODS
The conserved internal transcribed spacer (ITS) region of A. fumigatus was used to design CRISPR-derived RNA (crRNA) and the corresponding recombinase polymerase amplification (RPA) primer sequence with the T7 promoter for the CRISPR assay. Twenty-five clinical isolates and 43 bronchoalveolar lavage fluid (BALF) remaining from routine examinations of patients with confirmed pulmonary aspergillosis were collected to further validate the CRISPR assay.
RESULTS
No amplification signal was observed when genomic DNA from closely clinically related Aspergillus species, such as Aspergillus flavus, Aspergillus niger, and Aspergillus terreus, as well as Monascus purpureus Went and Escherichia coli, was tested by this assay, and the detection limit for A. fumigatus was 3 copies in a single reaction system. Validation experiments using the 25 clinical isolates demonstrated 91.7% specificity for the A. fumigatus section, and the sensitivity was 100% when first-generation sequencing was used as the standard. There was no significant difference between the PCR and CRISPR methods (P = 1.0), and the diagnosis results of the two methods were consistent (Kappa = 0.459, P = 0.003).
CONCLUSION
The study offers a new validated CRISPR/Cas13a technique for A. fumigatus detection, providing a simple, rapid and affordable test that is ready for application in the diagnosis of A. fumigatus infection.
Topics: Aspergillosis; Aspergillus fumigatus; Diagnostic Tests, Routine; Humans; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 34969623
DOI: 10.1016/j.jmii.2021.11.008 -
Future Microbiology Dec 2023(shortened to ) is a fungus (plural: fungi) that can cause a serious infection in some people. can become resistant to medicines known as azoles (isavuconazole,... (Review)
Review
WHAT IS THIS SUMMARY ABOUT?
(shortened to ) is a fungus (plural: fungi) that can cause a serious infection in some people. can become resistant to medicines known as azoles (isavuconazole, itraconazole, posaconazole, and voriconazole). This means they stop working and are not able to kill the fungus. Fungi can become resistant through changes in their genes, which are called mutations. Scientists looked at previously collected samples from people infected with and found that 36 of the samples showed resistance to an azole. In 35 of these samples, scientists looked for mutations in 50 genes. These 50 genes are known to play a role in azole resistance and/or are important for fungal survival.
WHAT WERE THE RESULTS?
In total, 18 out of 36 samples (50%) showed resistance to isavuconazole only. Of these, 12 had mutations in 4 genes important for fungal survival (called , , and ). Mutations were found in 2 genes that are the most common causes of azole resistance (called and ). The most common mutation, called TR34/L98H, was found in 9 samples. Of these, 8 samples showed resistance to all 4 of the azoles tested.
WHAT DO THE RESULTS OF THE STUDY MEAN?
Studying mutations that make fungi resistant to medicines helps to make sure that people with fungal infections get treated with medicines that will work for them.
Topics: Humans; Antifungal Agents; Fungal Proteins; Aspergillus fumigatus; Azoles; Drug Resistance, Fungal; Microbial Sensitivity Tests
PubMed: 37920995
DOI: 10.2217/fmb-2023-0023 -
Analytical Methods : Advancing Methods... Feb 2023has the potential to degrade lignocellulosic biomass, but the degradation mechanism is not clear. The purpose of this study is to analyze the differential proteins and...
has the potential to degrade lignocellulosic biomass, but the degradation mechanism is not clear. The purpose of this study is to analyze the differential proteins and metabolites produced by G-13 in the degradation of different lignin model compounds. Ferulic acid, sinapic acid, and -coumaric acid were used as carbon sources. By controlling the culture conditions, and adding a cellulose co-substrate and an auxiliary carbon source, the enzymatic production law of three lignin model compounds degraded by G-13 was investigated. Proteomics and metabolomics analysis were conducted for the two groups with the largest difference in enzyme activity expression. The results showed that a total of 1447 peptides were identified by proteomics analysis. Among them, 134 proteins were significantly changed, 73 proteins were up-regulated, and 61 proteins were down-regulated. The key proteins that degrade lignin model compounds are catechol dioxygenase, glutathione reductase, dextranase, isoamyl alcohol oxidase, glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase. Enrichment analysis of differential metabolite functions revealed that G-13 is associated with several pathways related to the degradation of lignin. Among them, starch and sucrose metabolism, pentose phosphate pathway, glutathione metabolism, and the -cleavage pathway of dihydroxylated aromatic rings are closely related to lignin degradation. The information presented in this paper will be helpful for future research on the degradation or depolymerization of natural lignocellulosic substrates.
Topics: Aspergillus fumigatus; Lignin; Proteomics; Fungal Proteins; Fungi; Carbon
PubMed: 36723181
DOI: 10.1039/d2ay01446g -
PLoS Pathogens Nov 2022The eukaryotic multisubunit Elongator complex has been shown to perform multiple functions in transcriptional elongation, histone acetylation and tRNA modification....
The eukaryotic multisubunit Elongator complex has been shown to perform multiple functions in transcriptional elongation, histone acetylation and tRNA modification. However, the Elongator complex plays different roles in different organisms, and the underlying mechanisms remain unexplored. Moreover, the biological functions of the Elongator complex in human fungal pathogens remain unknown. In this study, we verified that the Elongator complex of the opportunistic fungal pathogen Aspergillus fumigatus consists of six subunits (Elp1-6), and the loss of any subunit results in similarly defective colony phenotypes with impaired hyphal growth and reduced conidiation. The catalytic subunit-Elp3 of the Elongator complex includes a S-adenosyl methionine binding (rSAM) domain and a lysine acetyltransferase (KAT) domain, and it plays key roles in the hyphal growth, biofilm-associated exopolysaccharide galactosaminogalactan (GAG) production, adhesion and virulence of A. fumigatus; however, Elp3 does not affect H3K14 acetylation levels in vivo. LC-MS/MS chromatograms revealed that loss of Elp3 abolished the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification of tRNA wobble uridine (U34), and the overexpression of tRNAGlnUUG and tRNAGluUUC, which normally harbor mcm5s2U modifications, mainly rescues the defects of the Δelp3 mutant, suggesting that tRNA modification rather than lysine acetyltransferase is responsible for the primary function of Elp3 in A. fumigatus. Strikingly, global proteomic comparison analyses showed significantly upregulated expression of genes related to amino acid metabolism in the Δelp3 mutant strain compared to the wild-type strain. Western blotting showed that deletion of elp3 resulted in overexpression of the amino acid starvation-responsive transcription factor CpcA, and deletion of CpcA markedly reversed the defective phenotypes of the Δelp3 mutant, including attenuated virulence. Therefore, the findings of this study demonstrate that A. fumigatus Elp3 functions as a tRNA-modifying enzyme in the regulation of growth, GAG production, adhesion and virulence by maintaining intracellular amino acid homeostasis. More broadly, our study highlights the importance of U34 tRNA modification in regulating cellular metabolic states and virulence traits of fungal pathogens.
Topics: Humans; Uridine; Aspergillus fumigatus; Histone Acetyltransferases; Virulence; Proteomics; Chromatography, Liquid; Tandem Mass Spectrometry; RNA, Transfer; Amino Acids
PubMed: 36374932
DOI: 10.1371/journal.ppat.1010976 -
Medecine Et Maladies Infectieuses Aug 2020Aspergillus fumigatus is the predominant etiological agent of invasive aspergillosis (IA), a difficult-to-manage fungal disease associated with a high case fatality... (Review)
Review
Aspergillus fumigatus is the predominant etiological agent of invasive aspergillosis (IA), a difficult-to-manage fungal disease associated with a high case fatality rate. Azole antifungals, particularly voriconazole, have significantly improved the survival rate of patients with IA. However, the clinical advances made possible through the use of medical azoles could be threatened by the emergence of azole-resistant strains which has been reported in an ever-increasing number of countries over the last 10 years. The major resistance mechanism, that combines point mutation(s) in the coding sequence of cyp51A gene and an insertion of a tandem repeat in the promoter region of this gene which leads to its overexpression (TR/L98H and TR/Y121F/T289A), is presumed to be of environmental origin. However, the emergence of clinical and environmental azole-resistant strains without the cyp51A gene mutation suggests that other mechanisms could also be responsible for azole resistance (for example, overexpression of efflux pumps). The development of resistance may be linked to either long-term use of azole antifungals in patients with chronic aspergillosis (patient-acquired route) or selection pressure of the fungicides in the environment (environmental route). The fungicide-driven route could be responsible for resistance in azole-naive patients with IA. This literature review aims to summarize recent findings, focusing on the current situation of azole-resistance in A. fumigatus, and provides better understanding of the importance of the environmental route in resistance acquisition.
Topics: Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Azoles; Drug Resistance, Fungal; Fungal Proteins; Genotype; Humans; Microbial Sensitivity Tests; Voriconazole
PubMed: 31472992
DOI: 10.1016/j.medmal.2019.07.014 -
PLoS Pathogens Jul 2020Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute...
Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.
Topics: Animals; Aspergillosis; Aspergillus fumigatus; Fungal Proteins; Gene Expression Regulation, Fungal; Gliotoxin; Mice; Oxidative Stress; Transcription Factors; Virulence
PubMed: 32667960
DOI: 10.1371/journal.ppat.1008645 -
Frontiers in Cellular and Infection... 2019There are only few drugs available to treat fungal infections, and the lack of new antifungals, along with the emergence of drug-resistant strains, results in millions...
There are only few drugs available to treat fungal infections, and the lack of new antifungals, along with the emergence of drug-resistant strains, results in millions of deaths/year. An unconventional approach to fight microbial infection is to exploit nutritional vulnerabilities of microorganism metabolism. The metal gallium can disrupt iron metabolism in bacteria and cancer cells, but it has not been tested against fungal pathogens such as and . Here, we investigate activity of gallium nitrate III [Ga(NO)] against these human pathogens, to reveal the gallium mechanism of action and understand the interaction between gallium and clinical antifungal drugs. Ga(NO) presented a fungistatic effect against azole-sensitive and -resistant strains (MIC = 32.0 mg/L) and also had a synergistic effect with caspofungin, but not with azoles and amphotericin B. Its antifungal activity seems to be reliant on iron-limiting conditions, as the presence of iron increases its MIC value and because we observed a synergistic interaction between gallium and iron chelators against . We also show that an mutant (Δ) unable to grow in the absence of iron is more susceptible to gallium, reinforcing that gallium could act by disrupting iron homeostasis. Furthermore, we demonstrate that gallium has a fungistatic effect against different species of ranging from 16.0 to 256.0 mg/L, including multidrug-resistant , and . Our findings indicate that gallium can inhibit fungal pathogens under iron-limiting conditions, showing that Ga(NO) could be a potential therapy not only against bacteria but also as an antifungal drug.
Topics: Antifungal Agents; Aspergillus fumigatus; Azoles; Dose-Response Relationship, Drug; Drug Resistance, Fungal; Gallium; Kinetics; Microbial Sensitivity Tests
PubMed: 31921699
DOI: 10.3389/fcimb.2019.00414 -
Mycopathologia Aug 2023Aspergillus fumigatus is a genetically diverse fungal species, which is near ubiquitous in its global distribution and is the major cause of the life-threatening disease...
Aspergillus fumigatus is a genetically diverse fungal species, which is near ubiquitous in its global distribution and is the major cause of the life-threatening disease invasive aspergillosis. We present 3 de novo genome assemblies that were selected to be representative of the genetic diversity of clinical and environmental A. fumigatus. Sequencing using long-read Oxford Nanopore and subsequent assembly of the genomes yielded 10-23 contigs with an N50 of 4.05 Mbp to 4.93 Mbp.
Topics: Aspergillus fumigatus; Genome; Aspergillosis; Sequence Analysis, DNA
PubMed: 37227556
DOI: 10.1007/s11046-023-00740-2 -
Microbial Cell Factories Jan 2023Purification of L-methionine γ-lyase (MGL) from A. fumigatus was sequentially conducted using heat treatment and gel filtration, resulting in 3.04 of purification fold...
Purification of L-methionine γ-lyase (MGL) from A. fumigatus was sequentially conducted using heat treatment and gel filtration, resulting in 3.04 of purification fold and 73.9% of enzymatic recovery. The molecular mass of the purified MGL was approximately apparent at 46 KDa based on SDS-PAGE analysis. The enzymatic biochemical properties showed a maximum activity at pH 7 and exhibited plausible stability within pH range 5.0-7.5; meanwhile the highest catalytic activity of MGL was observed at 30-40 °C and the enzymatic stability was noted up to 40 °C. The enzyme molecule was significantly inhibited in the presence of Cu, Cd, Li, Mn, Hg, sodium azide, iodoacetate, and mercaptoethanol. Moreover, MGL displayed a maximum activity toward the following substrates, L-methionine < DL-methionine < Ethionine < Cysteine. Kinetic studies of MGL for L-methioninase showed catalytic activity at 20.608 mM and 12.34568 µM.min. Furthermore, MGL exhibited anticancer activity against cancerous cell lines, where IC were 243 ± 4.87 µg/ml (0.486 U/ml), and 726 ± 29.31 µg/ml (1.452 U/ml) against Hep-G2, and HCT116 respectively. In conclusion, A. fumigatus MGL had good catalytic properties along with significantly anticancer activity at low concentration which makes it a probably candidate to apply in the enzymotherapy field.
Topics: Aspergillus fumigatus; Kinetics; Carbon-Sulfur Lyases; Methionine
PubMed: 36635695
DOI: 10.1186/s12934-023-02019-z -
Journal de Mycologie Medicale Apr 2020Aspergillus infections are increasingly recognized as a global health problem because of limited antifungal drugs and occurrence of azole resistance worldwide. More... (Review)
Review
Aspergillus infections are increasingly recognized as a global health problem because of limited antifungal drugs and occurrence of azole resistance worldwide. More cyp51-mediated and non-cyp51-mediated mechanisms of azole resistance have been identified in clinical and laboratory studies in recent years with applications of molecular biotechnology including next-generation sequencing, reverse genetics and so on. In this review, current research on the molecular mechanisms of azole resistance in A. fumigatus were presented and summarized and meanwhile the putative clinical relevance of these findings from bench work were discussed. Important aims are to gain more insight to mechanism of azole resistance and provide some efficient lead for prevention strategy.
Topics: Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Azoles; Drug Resistance, Fungal; Fungal Proteins; Gene Expression Regulation, Fungal; High-Throughput Nucleotide Sequencing; Humans; Microbial Sensitivity Tests; Mutation; Sterol 14-Demethylase
PubMed: 32008963
DOI: 10.1016/j.mycmed.2019.100915