[Show abstract][Hide abstract] ABSTRACT: Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds.
[Show abstract][Hide abstract] ABSTRACT: Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.
[Show abstract][Hide abstract] ABSTRACT: Azoles are currently the mainstay of antifungal treatment both in agricultural and in clinical settings. Although the target site of azole action is well studied, the basis of azole resistance and the ultimate mode of action of the drug in fungi are poorly understood. To gain a deeper insight into these aspects of azole action, restriction-mediated plasmid integration (REMI) was used to create azole sensitive and resistant strains of the clinically important fungus Aspergillus fumigatus. Four azole sensitive insertions and four azole-resistant insertions were characterized. Three phenotypes could be re-created in wild-type AF210 by reintegration of rescued plasmid and a further four could be confirmed by complementation of the mutant phenotype with a copy of the wild-type gene predicted to be disrupted by the original insertional event. Six insertions were in genes not previously associated with azole sensitivity or resistance. Two insertions occur in transporter genes that may affect drug efflux, whereas others may affect transcriptional regulation of sterol biosynthesis genes and NADH metabolism in the mitochondrion. Two insertions are in genes of unknown function.
[Show abstract][Hide abstract] ABSTRACT: The mitochondrial phosphopantetheinyl transferase gene pptB of the opportunistic pathogen Aspergillus fumigatus has been identified and characterised. Unlike pptA, which is required for lysine biosynthesis, secondary metabolism, and iron assimilation, pptB is essential for viability. PptB is located in the mitochondria. In vitro expression of pptA and pptB has shown that PptB is specific for the mitochondrial acyl carrier protein AcpA.
Full-text · Article · Dec 2010 · Fungal Genetics and Biology
[Show abstract][Hide abstract] ABSTRACT: Genes that are essential for viability represent potential targets for the development of anti-infective agents. However,
relatively few have been determined in the filamentous fungal pathogen Aspergillus fumigatus. A novel solution employing parasexual genetics coupled with transposon mutagenesis using the Fusarium oxysporum transposon impala had previously enabled the identification of 20 essential genes from A. fumigatus; however, further use of this system required a better understanding of the mode of action of the transposon itself. Examination
of a range of conditions indicated that impala is activated by prolonged exposure to low temperatures. This newly identified property was then harnessed to identify 96
loci that are critical for viability in A. fumigatus, including genes required for RNA metabolism, organelle organization, protein transport, ribosome biogenesis, and transcription,
as well as a number of noncoding RNAs. A number of these genes represent potential targets for much-needed novel antifungal
[Show abstract][Hide abstract] ABSTRACT: In recent years the filamentous fungus Aspergillus fumigatus has become a significant cause of infection in man and as such has become the focus of much study. It is thought to be the leading mould pathogen in leukaemia and transplant patients and is responsible for mortality in a large number of individuals with immunological disorders. In an attempt to develop molecular mutagenesis tools for assessment of this organism, the genome of A. fumigatus was analysed to identify possible functional transposable elements. An apparently intact Fot1/Pogo type transposon with 65% identity to the active Tan1 element of Aspergillus niger was identified and designated Aft1. Aft1 is a 1.9kb element present in multiple (>20) highly conserved copies. It encodes a 332 amino acid transposase which contains all the functional motifs required for transposition. In addition, the transposase was expressed in cultures grown at 37 degrees C in all three strains assessed and excision analysis suggests Aft1 may be active and of use in transposon tagging experiments. Southern hybridisation patterns indicate that Aft1 is widely distributed amongst clinical isolates of A. fumigatus with considerable variation in genomic localisation. A comprehensive analysis of the genomic localisation of Aft1 in the sequenced strain AF293 show that one insertion is 30 bases upstream of a predicted gene encoding a G-protein coupled receptor. Expression analysis indicates that this gene has been inactivated by the insertion.
Full-text · Article · Feb 2008 · Fungal Genetics and Biology
[Show abstract][Hide abstract] ABSTRACT: Fungi secrete extracellular enzymes to enable them to harvest nutrients from the environment. In the case of pathogenic fungi these enzymes can also be pathogenesis factors. Here we report the identification in fungi of a complex family of extracellular phospholipase C (PLC) enzymes, homologous to the Pseudomonas aeruginosa PLCH_PSEAE. Database searches and phylogenetic analysis showed that the PLCs clustered into two groups with different evolutionary histories. One group, subdivided into PLC-A, -B, -C and -D, was found only in aspergilli and Neosartorya fischeri. Each species only ever showed three of the four PLCs except N. fischeri which had all four PLCs plus duplicate PLC-A, -B and -C genes. Modelling studies indicated that these PLCs had mechanistic similarities to phosphoesterases and aryl sulphatases, but that they probably did not differ in substrate specificity. The second group, PLC-E, was seen in a wider range of fungi including some species of aspergilli and was always found in a head-to-head arrangement with a copper oxidase, similar to the laccases. The PLC genes appear to have arisen from separate gene transfer events from bacteria or lower eukaryotes. Thus, aspergilli have acquired PLCs twice in the course of evolution.
Preview · Article · Nov 2006 · Mycological Research
[Show abstract][Hide abstract] ABSTRACT: The ADAMs are a family of integral membrane proteases involved in shedding and fusion events in animal tissues. Here, we report the identification of two ADAMs, ADM-A and ADM-B, in the pathogenic fungus Aspergillus fumigatus. The domain structure of metazoan ADAMs was seen in ADM-A and -B, although with some differences. ADAMs were identified in other filamentous fungi and phylogenetic analysis indicated that the fungal ADAMs were monophyletic and most closely related to metazoan ADAM 10 and 17. Recombinant ADM-B protease specifically cleaved casein and albumin while recombinant propeptide+protease was inactive. A sheddase function is therefore proposed for fungal ADAMs.
Preview · Article · Aug 2005 · FEMS Microbiology Letters
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Little is known of the phospholipid composition of Aspergillus species. The aim of this study was to determine individual phospholipid analogues in Aspergillus. Twenty-nine clinical and environmental isolates from five Aspergillus species were analysed. Fast atom bombardment mass spectrometric data were considered in two ranges, m/z 190-500 and m/z 500-1000, to facilitate the recognition of major fatty acyl groups and phospholipids. Quantitative comparison of major anions in both m/z ranges was undertaken. Confirmation of major phospholipid anions from eight representative isolates was achieved by tandem mass spectrometry. The major phospholipid families were phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS). Anions were detected consistent with the presence of specific phospholipid moieties, such as palmitoyl-linolenoyl phosphatidic acid, palmitoyl-oleoylphosphatidylethenolamine, oleoyl-linoleoyl-phophatidylserine and palmitoyl-linoleoyl-phosphatidylinositol. It appears that there is some commonality of sn1 or sn2 fatty acyl substituents, frequently C18:2 at sn1 accompanied by C16:0 at sn2, with differing molecular weights being attributable to analogues with differing head groups. Differences in certain phospholipids (e.g. minor peak with m/z 933) were detected between A. fumigatus, A. nidulans and other species examined which could have diagnostic value.
[Show abstract][Hide abstract] ABSTRACT: Azole-resistant oropharyngeal and oesophageal candidiasis is a recent phenomenon observed in patients with AIDS usually previously treated with fluconazole. Some variation has been observed in antifungal susceptibility testing among separate colonies of Candida albicans from the same patient. This raises the question of whether there are multiple clones present or simply phenotypic variation in expression of azole resistance. To address this question we took 18 isolates grown from multiple swabs taken before and after experimental azole therapy from a single HIV-positive individual with fluconazole-resistant oral candidiasis and compared morphotype, karyotype, PCR-based DNA typing and azole susceptibility. Ten of the isolates were from a single 2-day period. Amongst these 10 there were seven morphotypes, five karyotypes and four polymerase chain reaction (PCR) types. Three further morphotypes, one karyotype and two PCR types were found amongst the eight isolates obtained during the subsequent 4 months. Limited variation in susceptibility to two azoles--fluconazole and D0870--was also seen. This work emphasizes both the large genotype and phenotypic variability of C. albicans isolates in the mouth of AIDS patients with fluconazole resistance, and the difficulties in interpretation of present typing methods.
No preview · Article · Jan 1998 · Journal of Infection
[Show abstract][Hide abstract] ABSTRACT: Little is known of the phospholipid composition of Aspergillus species. The aim of this study was to determine individual phospholipid analogues in Aspergillus. Twentynine clinical and environmental isolates from five Aspergillus species were analysed. Fast atom bombardment mass spectrometric data were considered in two ranges, m/z 190-500 and m/z 500-1000, to facilitate the recognition of major fatty acyl groups and phospholipids. Quantitative comparison of major anions in both m/z ranges was undertaken. Confirmation of major phospholipid anions from eight representative isolates was achieved by tandem mass spectrometry. The major phospholipid families were phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS). Anions were detected consistent with the presence of specific phospholipid moieties, such as palmitoyl-linolenoyl phosphatidic acid, palmitoyl-oleoylphosphatidylethenolamine, oleoyl-linoleoyl-phophatidylserine and palmitoyl-linoleoyl-phosphatidylinositol. It appears that there is some commonality of snl or sn2 fatty acyl substituents, frequently C18:2 at snl accompanied by C,6:0 at sn2, with differing molecular weights being attributable to analogues with differing head groups. Differences in certain phospholipids (e.g. minor peak with m/z 933) were detected between A. fumigatus, A. nidulans and other species examined which could have diagnostic value.
[Show abstract][Hide abstract] ABSTRACT: We examined fast atom bombardment mass spectra (FAB-MS) of 29 clinical isolates of Aspergillus, from five pathogenic species, for the presence of phthioic acid anions (m/z 395·6) when grown at 37°C. Phthioic acid was
detected in only one of 12 A. fumigatus, three of nine A. terreus and one of four A. niger isolates. Phthioic acid is unlikely to be a major pathogenicity determinant of Aspergillus.
Preview · Article · Mar 1997 · Journal of medical and veterinary mycology: bi-monthly publication of the International Society for Human and Animal Mycology
[Show abstract][Hide abstract] ABSTRACT: Wound infections with Aspergillus are surprisingly rare given the quantity of spores in hospital air. We report the first case of infection of abdominal viscera due to Aspergillus in a patient with a laparostomy for Crohn's disease. Amphotericin B effected a cure. Sampling of air from the patient's environment yielded one isolated of Aspergillus fumigatus that matched the patient's isolates. Other examples of surgical wounds being contaminated by Aspergillus are reviewed.
No preview · Article · Oct 1996 · Journal of Infection
[Show abstract][Hide abstract] ABSTRACT: Although several typing methods have been described for Shiga-like toxin-producing Escherichia coli O157, the methods are somewhat cumbersome. Using 22 isolates of Escherichia coli O157 and five other Escherichia coli isolates, two primers (M13 core sequence and 970-11) were found to give excellent differentiation between isolates using random amplified polymorphic DNA (RAPD). Using only the presence or absence of variable bands, a matrix of 20 variable characters was identified. From these characters, similarity coefficients were calculated and a phenogram constructed. All of the Escherichia coli O157 isolates were easily distinguished from the non-O157 Escherichia coli isolates. Using a 95% similarity cutoff, we found 13 RAPD types among the 22 Escherichia coli O157 isolates. Isolates thought to be identical by toxin and phage typing as well as by epidemiological association were distinguished, and others thought to be distinct by lack of epidemiological association were identical. RAPD using M13 and 970-11 primers is a potentially useful typing tool for Escherichia coli isolates of serotype O157 and possibly other Escherichia coli isolates.
No preview · Article · May 1996 · European Journal of Clinical Microbiology
[Show abstract][Hide abstract] ABSTRACT: Extracellular phospholipase activity has been implicated in the pathogenesis of several bacterial infections. Recently, extracellular phospholipase activity has been proposed as a virulence factor in the opportunistic yeast Candida albicans. Aspergillus fumigatus is the most pathogenic member of its genus, responsible for > 90% of infections. Previously, no specific virulence factors have been determined. We investigated the ability of A. fumigatus to produce extracellular phospholipases at 37 degrees C. Fast atom bombardment was used to compare lipid-containing media before and at 5-h intervals during shaking culture of A. fumigatus. Lipids were extracted and analyzed. Many anions corresponding to phospholipid breakdown products were identified. Specific anion species identified indicated phospholipase A, B, C (PLC), and D activities. PLC activity was further investigated by using the synthetic substrate p-nitrophenylphosphorylcholine. PLC activity was initially observed after 30 h of growth and accumulated in broth cultures up to 50 h. At 55 h, there was a sharp increase in PLC activity which coincided with cultures reaching the stationary phase. Activity of the PLC was measured at different temperatures, with greater activity occurring at 37 degrees C than at lower temperatures. Phospholipases could represent a virulence determinant in A. fumigatus.
Full-text · Article · Apr 1996 · Infection and Immunity
[Show abstract][Hide abstract] ABSTRACT: In the last four years, several molecular typing methods have been applied to various Aspergillus species, primarily A. fumigatus and A. niger. Many ‘clones’ or DNA types exist in the environment and in patients. The most discriminatory methods appear to be restriction fragment length polymorphism analysis with or without specific probes and random amplification of polymorphic DNA analysis. Insufficient work on interlaboratory reproducibility has been done. Most patients are infected by single DNA types, although occasionally multiple isolates are detected. Little work has been done with regard to detailed hospital epidemiology using molecular typing methods.
No preview · Article · Jun 1995 · Journal of Hospital Infection
[Show abstract][Hide abstract] ABSTRACT: Invasive aspergillosis is often nosocomially acquired and carries a high mortality. Molecular typing methods to discriminate isolates have now been developed. Using simple restriction endonuclease (Sal1 and Xho1) digestion of total genomic DNA, we have typed 25 epidemiologically-related isolates of A. fumigatus from six hospital episodes of invasive aspergillosis. Eight DNA types were found and in each case the DNA type matched precisely the epidemiological data. Thus DNA typing of A. fumigatus can provide the means to match isolates from linked sources and distinguish isolates from diverse origins.
Full-text · Article · Mar 1995 · Epidemiology and Infection