64 research outputs found
Siderophore Biosynthesis But Not Reductive Iron Assimilation Is Essential for Aspergillus fumigatus Virulence
The ability to acquire iron in vivo is essential for most microbial pathogens. Here we show that Aspergillus fumigatus does not have specific mechanisms for the utilization of host iron sources. However, it does have functional siderophore-assisted iron mobilization and reductive iron assimilation systems, both of which are induced upon iron deprivation. Abrogation of reductive iron assimilation, by inactivation of the high affinity iron permease (FtrA), has no effect on virulence in a murine model of invasive aspergillosis. In striking contrast, A. fumigatus l-ornithine-N 5-monooxygenase (SidA), which catalyses the first committed step of hydroxamate-type siderophore biosynthesis, is absolutely essential for virulence. Thus, A. fumigatus SidA is an essential virulence attribute. Combined with the absence of a sidA ortholog—and the fungal siderophore system in general—in mammals, these data demonstrate that the siderophore biosynthetic pathway represents a promising new target for the development of antifungal therapies
Small ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for regulation of protein synthesis
Small non-protein-coding RNAs (ncRNAs) have systematically been studied in various model organisms from Escherichia coli to Homo sapiens. Here, we analyse the small ncRNA transcriptome from the pathogenic filamentous fungus Aspergillus fumigatus. To that aim, we experimentally screened for ncRNAs, expressed under various growth conditions or during specific developmental stages, by generating a specialized cDNA library from size-selected small RNA species. Our screen revealed 30 novel ncRNA candidates from known ncRNA classes such as small nuclear RNAs (snRNAs) and C/D box-type small nucleolar RNAs (C/D box snoRNAs). Additionally, several candidates for H/ACA box snoRNAs could be predicted by a bioinformatical screen. We also identified 15 candidates for ncRNAs, which could not be assigned to any known ncRNA class. Some of these ncRNA species are developmentally regulated implying a possible novel function in A. fumigatus development. Surprisingly, in addition to full-length tRNAs, we also identified 5′- or 3′-halves of tRNAs, only, which are likely generated by tRNA cleavage within the anti-codon loop. We show that conidiation induces tRNA cleavage resulting in tRNA depletion within conidia. Since conidia represent the resting state of A. fumigatus we propose that conidial tRNA depletion might be a novel mechanism to down-regulate protein synthesis in a filamentous fungus
The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis
The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we
demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore,
exogenous H2O2 (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P 0.0101). While both mutants
exhibited significant sensitivity to both exogenous gliotoxin (P<0.001) and H2O2 (P<0.01), unexpectedly, exogenous
gliotoxin relieved H2O2-induced growth inhibition in a dose-dependent manner (0 to 10 g/ml). Gliotoxin-containing organic
extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P<0.05) the growth of the gliK26933 deletion mutant.
The A. fumigatus gliK26933 mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-
phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396.
These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus
gliK26933 mutant than in those of the wild type (P0.0024 [fold difference, 24] and P0.0003 [fold difference, 9.6], respectively)
and were absent from A. fumigatus gliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial
extracts of the A. fumigatus gliK26933 mutant compared to the wild type (P<0.001). Determination of the gliotoxin uptake rate
revealed a significant difference (P0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the
gliK46645 mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine
levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new
insights into gliotoxin functionality in A. fumigatus
The Role of Glutathione S-Transferase GliG in Gliotoxin Biosynthesis in Aspergillus fumigatus
Gliotoxin, a redox-active metabolite, is produced
by the opportunistic fungal pathogen Aspergillus
fumigatus, and its biosynthesis is directed by the
gli gene cluster. Knowledge of the biosynthetic
pathway to gliotoxin, which contains a disulfide
bridge of unknown origin, is limited, although L-Phe
and L-Ser are known biosynthetic precursors. Deletion
of gliG from the gli cluster, herein functionally
confirmed as a glutathione S-transferase, results in
abrogation of gliotoxin biosynthesis and accumulation
of 6-benzyl-6-hydroxy-1-methoxy-3-methylenepiperazine-
2,5-dione. This putative shunt metabolite
from the gliotoxin biosynthetic pathway contains an
intriguing hydroxyl group at C-6, consistent with
a gliotoxin biosynthetic pathway involving thiolation
via addition of the glutathione thiol group to a reactive
acyl imine intermediate. Complementation of gliG
restored gliotoxin production and, unlike gliT, gliG
was found not to be involved in fungal self-protection
against gliotoxin
The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Raw data have been deposited in the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE54810. Funding: This work was supported in part by grants to EMB from the MRC (G0501164) and BBSRC (BB/G009619/1), to EMB and NDR from the Wellcome Trust (WT093596MA), to MB from Imperial College London (Division of Investigative Sciences PhD Studentship), to HH from the ERA-NET PathoGenoMics project TRANSPAT, Austrian Science Foundation (FWF I282-B09), to SGF from the National Institutes of Health, USA (R01AI073829). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD
Preclinical evaluation of two 68Ga-siderophores as potential radiopharmaceuticals for Aspergillus fumigatus infection imaging
PURPOSE: Invasive pulmonary aspergillosis is mainly caused by Aspergillus fumigatus, and is one of the major causes of morbidity and mortality in immunocompromised patients. The mortality associated with invasive pulmonary aspergillosis remains high, mainly due to the difficulties and limitations in diagnosis. We have shown that siderophores can be labelled with (68)Ga and can be used for PET imaging of A. fumigatus infection in rats. Here we report on the further evaluation of the most promising (68)Ga-siderophore candidates, triacetylfusarinine (TAFC) and ferrioxamine E (FOXE). METHODS: Siderophores were labelled with (68)Ga using acetate buffer. Log P, protein binding and stability values were determined. Uptake by A. fumigatus was studied in vitro in cultures with high and low iron loads. In vivo biodistribution was determined in normal mice and an infection model was established using neutropenic rats inoculated with A. fumigatus. Static and dynamic muPET imaging was performed and correlated with CT images, and lung infection was evaluated ex vivo. RESULTS: (68)Ga-siderophores were labelled with high radiochemical purity and specific activity. (68)Ga-TAFC and (68)Ga-FOXE showed high uptake by A. fumigatus in iron-deficient cultures. In normal mice, (68)Ga-TAFC and (68)Ga-FOXE showed rapid renal excretion with high metabolic stability. In the rat infection model focal lung uptake was detected by muPET with both compounds and increased with severity of the infection, correlating with abnormal CT images. CONCLUSION: (68)Ga-TAFC and (68)Ga-FOXE displayed excellent in vitro stability and high uptake by A. fumigatus. Both compounds showed excellent pharmacokinetics, highly selective accumulation in infected lung tissue and good correlation with severity of disease in a rat infection model, which makes them promising agents for A. fumigatus infection imaging
Sub-Telomere Directed Gene Expression during Initiation of Invasive Aspergillosis
Aspergillus fumigatus is a common mould whose spores are a
component of the normal airborne flora. Immune dysfunction permits developmental
growth of inhaled spores in the human lung causing aspergillosis, a significant
threat to human health in the form of allergic, and life-threatening invasive
infections. The success of A. fumigatus as a pathogen is unique
among close phylogenetic relatives and is poorly characterised at the molecular
level. Recent genome sequencing of several Aspergillus species
provides an exceptional opportunity to analyse fungal virulence attributes
within a genomic and evolutionary context. To identify genes preferentially
expressed during adaptation to the mammalian host niche, we generated multiple
gene expression profiles from minute samplings of A. fumigatus
germlings during initiation of murine infection. They reveal a highly
co-ordinated A. fumigatus gene expression programme, governing
metabolic and physiological adaptation, which allows the organism to prosper
within the mammalian niche. As functions of phylogenetic conservation and
genetic locus, 28% and 30%, respectively, of the
A. fumigatus subtelomeric and lineage-specific gene
repertoires are induced relative to laboratory culture, and physically clustered
genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses
are a prominent feature. Locationally biased A. fumigatus gene
expression is not prompted by in vitro iron limitation, acid,
alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression
is favoured following ex vivo neutrophil exposure and in
comparative analyses of richly and poorly nourished laboratory cultured
germlings. We found remarkable concordance between the A.
fumigatus host-adaptation transcriptome and those resulting from
in vitro iron depletion, alkaline shift, nitrogen
starvation and loss of the methyltransferase LaeA. This first transcriptional
snapshot of a fungal genome during initiation of mammalian infection provides
the global perspective required to direct much-needed diagnostic and therapeutic
strategies and reveals genome organisation and subtelomeric diversity as
potential driving forces in the evolution of pathogenicity in the genus
Aspergillus
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