Biomedical subjects

Geoffrey D Robson

Publications and source records attributed to Geoffrey D Robson.

15 recordsLinked to original sources

PubMed2006

The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress.

We have examined the contribution of metacaspases to the growth and stress response of the opportunistic human mould pathogen, Aspergillus fumigatus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic-like programmed cell death. Single metacaspase-deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase-deficient mutant, DeltacasA/DeltacasB, was constructed by disrupting both genes. Stationary phase cultures of wild-type A. fumigatus were associated with the appearance of typical markers of apoptosis, including elevated proteolytic activity against caspase substrates, phosphatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phosphatidylserine exposure was not observed in stationary phase cultures of the DeltacasA/DeltacasB mutant, although caspase activity and viability was indistinguishable from wild type. The mutant retained wild-type virulence and showed no difference in sensitivity to a range of pro-apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the DeltacasA/DeltacasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contributes to the apoptotic-like loss of membrane phospholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress.

Amino Acid Sequence↗

PubMed2005

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

Allergens↗

PubMed2005

Correlation between in vitro growth rate and in vivo virulence in Aspergillus fumigatus.

We describe a kinetic microbroth method of measuring the growth rate of Aspergillus fumigatus spectrophotometrically. Using this method, growth rates were determined for nine A. fumigatus isolates for which an LD90 value in immunosuppressed CD-1 mice had previously been obtained. Comparison of the growth rates and LD90 values of these isolates suggests that a correlation exists between the two parameters.

Animals↗

PubMed2005

Transcriptome analysis of recombinant protein secretion by Aspergillus nidulans and the unfolded-protein response in vivo.

Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.

Aspergillus nidulans↗

PubMed2004

Glutamic protease distribution is limited to filamentous fungi.

Glutamic proteases are a distinct, and recently re-classified, group of peptidases that are thought to be found only in fungi. We have identified and analysed the distribution of over 20 putative glutamic proteases from all fungal species whose genomes have been sequenced so far. Although absent from the Saccharomycetales class, glutamic proteases appear to be present in all other ascomycetes species examined. A large number of coding regions for glutamic proteases were also found clustered together in the Phanerochaete chrysosporium genome, despite apparently being absent from three other species of Basidiomycota.

Fungi↗

PubMed2004

The Aspergillus niger annexin, anxC3.1 is constitutively expressed and is not essential for protein secretion.

An annexin, anxC3.1, was isolated and characterised from the industrially important filamentous fungus Aspergillus niger. anxC3.1 is a single copy gene encoding a 506 amino acid predicted protein which contains four annexin repeats. Disruption of the anxC3.1 gene did not lead to any visible changes in phenotype, nor in the levels of secreted protein, nor specifically in glucoamylase production, suggesting no major role in secretion. anxC3.1 expression was found to be unaltered under a variety of conditions such as increased secretion, altered nitrogen source, heat shock, and decreased Ca2+ levels, indicating that anxC3.1 is constitutively expressed. This is the first reported functional characterisation of a fungal annexin.

Amino Acid Sequence↗

PubMed2004

Use of expressed sequence tag analysis and cDNA microarrays of the filamentous fungus Aspergillus nidulans.

The use of microarrays in the analysis of gene expression is becoming widespread for many organisms, including yeast. However, although the genomes of a number of filamentous fungi have been fully or partially sequenced, microarray analysis is still in its infancy in these organisms. Here, we describe the construction and validation of microarrays for the fungus Aspergillus nidulans using PCR products from a 4092 EST conidial germination library. An experiment was designed to validate these arrays by monitoring the expression profiles of known genes following the addition of 1% (w/v) glucose to wild-type A. nidulans cultures grown to mid-exponential phase in Vogel's minimal medium with ethanol as the sole carbon source. The profiles of genes showing statistically significant differential expression following the glucose up-shift are presented and an assessment of the quality and reproducibility of the A. nidulans arrays discussed.

Aspergillus nidulans↗

PubMed2004

Combining transcriptome data with genomic and cDNA sequence alignments to make confident functional assignments for Aspergillus nidulans genes.

Whole genome sequencing of several filamentous ascomycetes is complete or in progress; these species, such as Aspergillus nidulans, are relatives of Saccharomyces cerevisiae. However, their genomes are much larger and their gene structure more complex, with genes often containing multiple introns. Automated annotation programs can quickly identify open reading frames for hypothetical genes, many of which will be conserved across large evolutionary distances, but further information is required to confirm functional assignments. We describe a comparative and functional genomics approach using sequence alignments and gene expression data to predict the function of Aspergillus nidulans genes. By highlighting examples of discrepancies between the automated genome annotation and cDNA or EST sequencing, we demonstrate that the greater complexity of gene structure in filamentous fungi demands independent data on gene expression and the gene sequence be used to make confident functional assignments.

Aspergillus nidulans↗

PubMed2004

Comparison of extracellular phospholipase activities in clinical and environmental Aspergillus fumigatus isolates.

Extracellular phospholipase production by environmental and clinical isolates of Aspergillus fumigatus collected from several centres world-wide were compared. All isolates produced extracellular phospholipases which included phospholipase C and a phospholipid acyl hydrolase (phospholipase A and/or phospholipase B) activity. Clinical isolates of A. fumigatus produced the largest zone sizes in a diffusion assay and clinical isolates produced more extracellular phospholipase C than environmental isolates. However, environmental isolates of A. fumigatus showed increased acyl hydrolase activity compared to clinical isolates of A. fumigatus. This study suggests that extracellular phospholipase C activity, but not extracellular acyl hydrolase activity may be important in the pathogenicity of A. fumigatus.

Aspergillus fumigatus↗

PubMed2004

Oxidative and amphotericin B-mediated cell death in the opportunistic pathogen Aspergillus fumigatus is associated with an apoptotic-like phenotype.

When protoplasts of the opportunistic fungal pathogen Aspergillus fumigatus were treated with low but toxic levels of hydrogen peroxide (0.1 mM) or amphotericin B (0.5 microg ml(-1)), loss of cell viability and death were associated with a number of phenotypic changes characteristic of apoptosis. The percentage of protoplasts staining positive with annexin V-FITC, an indicator of the externalization of phosphatidylserine and an early marker of apoptosis, rose to approximately 55 % within 1 h. This was followed by a similar increase in apoptotic DNA fragmentation detected by the TUNEL assay, and led to a loss of cell permeability and death in approximately 90 % of protoplasts, as indicated by the uptake of propidium iodide. The development of an apoptotic phenotype was blocked when protoplasts were pre-treated with the protein synthesis inhibitor cycloheximide, indicating active participation of the cell in the process. However, no significant activity against synthetic caspase substrates was detected, and the inclusion of the cell-permeant broad-spectrum caspase inhibitor Z-VAD-fmk did not block the development of the apoptotic-like phenotype. Higher concentrations of H(2)O(2) (1.8 mM) and amphotericin B (1 microg ml(-1)) caused protoplasts to die without inducing an apoptotic phenotype. As predicted, the fungistatic antifungal agent itraconazole, which inhibits growth without causing immediate cell death, did not induce an apoptotic-like phenotype.

Amphotericin B↗

PubMed2003

Entry into the stationary phase is associated with a rapid loss of viability and an apoptotic-like phenotype in the opportunistic pathogen Aspergillus fumigatus.

When the opportunistic pathogen Aspergillus fumigatus entered the stationary phase, there was a rapid loss in cell viability which was associated with the appearance of markers characteristic of apoptosis, namely annexin V-FITC binding to the cytoplasmic membrane, demonstrating exposure of phosphatidylserine to the outer leaflet of the membrane; and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining of the nuclei, indicating DNA fragmentation. This was followed later by a loss of membrane integrity as revealed by propidium iodide staining. The development of the apoptotic phenotype was blocked when the protein synthesis inhibitor cycloheximide was added to the culture 1h prior to the onset of the stationary phase, demonstrating active participation of the cell. In addition, intracellular activity against substrates specific for caspase-1 and -8 also increased on stationary phase entry and the development of the apoptotic phenotype was blocked when the cell permeant caspase inhibitor Z-FAD-fmk was present in the medium. Cell death in A. fumigatus during the stationary phase therefore appears to share similarities to apoptotic cell death in higher eukaryotes and to be dependent on a caspase-like activity.

Amino Acid Chloromethyl Ketones↗

PubMed2003

Development and application of an assay for uranyl complexation by fungal metabolites, including siderophores.

An assay to detect UO(2)(2+) complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B. Schwyn and J. B. Neilands, Anal. Biochem. 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides. In this assay the discoloration of two dyed agars (one containing a CAS-Fe(3+) dye and the other containing a CAS-UO(2)(2+) dye) caused by ligands was quantified. The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added. The ratio of the discoloration on the CAS-UO(2)(2+) agar to the discoloration on the CAS-Fe(3+) agar was independent of the amount of siderophore added. A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park. The fungi were screened for the production of UO(2)(2+) chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph Aureobacterium flavescens JG-9. This organism is highly sensitive to the presence of hydroxamate siderophores. However, the CAS-based assay was found to be less sensitive than the A. flavescens JG-9 assay. No significant difference between the results for each site for the two tests was found. Three isolates were selected for further study and were identified as two Pencillium species and a Mucor species. Our results show that the new assay can be effectively used to screen fungi for the production of UO(2)(2+) chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.

Actinomycetales↗

PubMed2000

Glucoamylase::green fluorescent protein fusions to monitor protein secretion in Aspergillus niger.

A glucoamylase::green fluorescent protein fusion (GLA::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and GLA514). Expression of GLA::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa, and that fluorescence was most intense at hyphal apices. Extracellular GLA::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of the medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect GLA::sGFP by Western analysis. A strain containing the GLA::sGFP construct was subjected to UV mutagenesis and survivors screened for mutations in the general secretory pathway. Three mutants were isolated that were unable to form a halo on either starch or gelatin medium. All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, GLA::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation. These results indicate that the GLA::sGFP fusion constructs can be used as convenient fluorescent markers to study the dynamics of protein secretion in vivo and as a tool in the isolation of mutants in the general secretory pathway.

Aspergillus niger↗

PubMed1996

How do highly branched (colonial) mutants of Fusarium graminearum A3/5 arise during Quorn myco-protein fermentations?

Chlorate-resistant, highly branched (colonial) mutants and auxotrophic mutants were used to study the nuclear distribution, morphology and growth of heterokaryons of the Quorn myco-protein fungus, Fusarium graminearum A3/5. The results showed that for several complementary homokaryons, even a strong selective pressure was insufficient to maintain heterokaryons in a 'balanced' condition (i.e. exhibiting a wild-type or near wild-type phenotype). Furthermore, the margins of heterokaryotic colonies generally contained nuclei from only one of the parental homokaryons, indicating imperfect nuclear mixing within the mycelium. These observations suggest that recessive, colonial mutants may appear during Quorn myco-protein production following shear-induced separation of hyphal fragments which contain a sufficiently high ratio of colonial:wild-type nuclei for the colonial phenotype to be expressed.

Culture Media↗

PubMed1995

Choline- and acetylcholine-induced changes in the morphology of Fusarium graminearum: evidence for the involvement of the choline transport system and acetylcholinesterase.

The response of Fusarium graminearum to choline, acetylcholine and a number of related analogues was investigated and their ability to induce a morphological response quantified. A number of mutants resistant to the alkylating agent nitrogen mustard (nim strains) were generated and found to have lost the ability to transport choline. These mutants were found to be insensitive to choline and acetylcholine but not to betaine, ethanolamine and other analogues. In addition, the non-competitive inhibitor hemicholinium-3 was also found to reduce the morphological effect of choline, proving that transport of choline into the hypha is essential for the morphological response. Acetylcholinesterase inhibitors blocked the morphological response to acetylcholine but had no effect on the response to choline, suggesting the presence of a membrane- or wall-bound acetylcholinesterase that hydrolyses acetylcholine to choline which subsequently induces the morphological response. Studies on the in vivo chitin synthase activity revealed that addition of choline caused a transient 75% increase in chitin synthase activity within 30 s, the rate rapidly returning to that observed before the addition of choline. No such effect was observed with the nim mutants.

Acetylcholine↗