71 research outputs found
Genes Differentially Expressed in Conidia and Hyphae of Aspergillus fumigatus upon Exposure to Human Neutrophils
Aspergillus fumigatus is the most common etiologic agent of invasive aspergillosis in immunocompromised patients. Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells. To our knowledge, this is the first study that investigates the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD) which are defective in the production of reactive oxygen species.Transcriptional profiles of conidia and hyphae exposed to neutrophils, either from normal donors or from CGD patients, were obtained by using the genome-wide microarray. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack.This work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans
Dual Organism Transcriptomics of Airway Epithelial Cells Interacting with Conidia of Aspergillus fumigatus
Background
Given the complex nature of the responses that can occur in host-pathogen interactions, dual transcriptomics offers a powerful method of elucidating these interactions during infection. The gene expression patterns of Aspergillus fumigatus conidia or host cells have been reported in a number of previous studies, but each focused on only one of the interacting organisms. In the present study, we profiled simultaneously the transcriptional response of both A. fumigatus and human airway epithelial cells (AECs).
Methodology
16HBE14o- transformed bronchial epithelial cells were incubated with A. fumigatus conidia at 37Β°C for 6 hours, followed by genome-wide transcriptome analysis using human and fungal microarrays. Differentially expressed gene lists were generated from the microarrays, from which biologically relevant themes were identified. Human and fungal candidate genes were selected for validation, using RT-qPCR, in both 16HBE14o- cells and primary AECs co-cultured with conidia.
Principal Findings
We report that ontologies related to the innate immune response are activated by co-incubation with A. fumigatus condia, and interleukin-6 (IL-6) was confirmed to be up-regulated in primary AECs via RT-qPCR. Concomitantly, A. fumigatus was found to up-regulate fungal pathways involved in iron acquisition, vacuolar acidification, and formate dehydrogenase activity
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
Conidiation Color Mutants of Aspergillus fumigatus Are Highly Pathogenic to the Heterologous Insect Host Galleria mellonella
The greater wax moth Galleria mellonella has been widely used as
a heterologous host for a number of fungal pathogens including Candida
albicans and Cryptococcus neoformans. A positive
correlation in pathogenicity of these yeasts in this insect model and animal
models has been observed. However, very few studies have evaluated the
possibility of applying this heterologous insect model to investigate virulence
traits of the filamentous fungal pathogen Aspergillus
fumigatus, the leading cause of invasive aspergillosis. Here, we have
examined the impact of mutations in genes involved in melanin biosynthesis on
the pathogenicity of A. fumigatus in the G.
mellonella model. Melanization in A. fumigatus confers
bluish-grey color to conidia and is a known virulence factor in mammal models.
Surprisingly, conidial color mutants in B5233 background that have deletions in
the defined six-gene cluster required for DHN-melanin biosynthesis caused
enhanced insect mortality compared to the parent strain. To further examine and
confirm the relationship between melanization defects and enhanced virulence in
the wax moth model, we performed random insertional mutagenesis in the Af293
genetic background to isolate mutants producing altered conidia colors. Strains
producing conidia of previously identified colors and of novel colors were
isolated. Interestingly, these color mutants displayed a higher level of
pathogenicity in the insect model compared to the wild type. Although some of
the more virulent color mutants showed increased resistance to hydrogen
peroxide, overall phenotypic characterizations including secondary metabolite
production, metalloproteinase activity, and germination rate did not reveal a
general mechanism accountable for the enhanced virulence of these color mutants
observed in the insect model. Our observations indicate instead, that
exacerbated immune response of the wax moth induced by increased exposure of
PAMPs (pathogen-associated molecular patterns) may cause self-damage that
results in increased mortality of larvae infected with the color mutants. The
current study underscores the limitations of using this insect model for
inferring the pathogenic potential of A. fumigatus strains in
mammals, but also points to the importance of understanding the innate immunity
of the insect host in providing insights into the pathogenicity level of
different fungal strains in this model. Additionally, our observations that
melanization defective color mutants demonstrate increased virulence in the
insect wax moth, suggest the potential of using melanization defective mutants
of native insect fungal pathogens in the biological control of insect
populations
Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis of Aspergillus fumigatus Keratitis
Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1Ξ² and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that Ξ²-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1β/β corneas have impaired IL-1Ξ² and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high Ξ²-glucan. In contrast to Dectin 1β/β mice, cellular infiltration into infected TLR2β/β, TLR4β/β, and MD-2β/β mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4β/β mice, but not TLR2β/β or MD-2β/β mice. We also found that TRIFβ/β and TIRAPβ/β mice exhibited no fungal-killing defects, but that MyD88β/β and IL-1R1β/β mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which Ξ²-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1Ξ², and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing
Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis of Aspergillus fumigatus Keratitis
Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1Ξ² and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that Ξ²-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1β/β corneas have impaired IL-1Ξ² and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high Ξ²-glucan. In contrast to Dectin 1β/β mice, cellular infiltration into infected TLR2β/β, TLR4β/β, and MD-2β/β mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4β/β mice, but not TLR2β/β or MD-2β/β mice. We also found that TRIFβ/β and TIRAPβ/β mice exhibited no fungal-killing defects, but that MyD88β/β and IL-1R1β/β mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which Ξ²-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1Ξ², and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing
A Role for the Unfolded Protein Response (UPR) in Virulence and Antifungal Susceptibility in Aspergillus fumigatus
Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ΞhacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37Β°C, but cell wall integrity was disrupted at 45Β°C, resulting in a dramatic loss in viability. The ΞhacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ΞhacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy
MALDI-TOF analysis of mixtures of 3-deoxyanthocyanidins and anthocyanins
The mass spectrometric analysis of 3-deoxyanthocyanidins and anthocyanins present in crude extracts from sorghum plant tissue was carried out by matrix-assisted laser desorption ionization mass spectrometry (MALDI). Sensitivities to as low as 5 pmol/ΞΌl were easily attained for pure samples of the anthocyanidin, pelargonidin, and the anthocyanin, malvin. Sensitivities to the level of 15 pmol/ΞΌl were attained for 3- deoxyanthocyanidins present in crude extracts from sorghum plant tissue. Through its capability for analyzing very small quantities of these compounds in unpurified samples, MALDI provided a sensitive means for the detection of these flavonoid pigments in plant tissues.link_to_subscribed_fulltex
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