The filamentous fungus Aspergillus fumigatus is responsible for approximately 4% of all tertiary hospital deaths in Europe [1]. A. fumigatus has emerged as a significant human pulmonary pathogen capable of inducing disease in patients undergoing immunosuppressive therapy or those with pre-existing pulmonary malfunction [2,3]. Invasive aspergillosis is the most serious form of the disease, involving the invasion of viable tissue and resulting in a mortality rate of 80-95% [4,5]. Circumvention of the host immune response facilitates in vivo fungal dissemination, and recent work has demonstrated that the modified diketopiperazine, gliotoxin, secreted by A. fumigatus, is capable of specifically blocking the respiratory burst in humans by inhibiting assembly of the NADPH oxidase in isolated polymorphonuclear leukocytes [6]. In addition, the release of hydroxamate-type siderophores, to facilitate iron acquisition by the organism, is also essential for fungal virulence [7].Although classically referred to as secondary metabolites, gliotoxin and siderophores, in addition to a diverse range of other bioactive components, may Aspergillus fumigatus is an important human fungal pathogen. The Aspergillus fumigatus genome contains 14 nonribosomal peptide synthetase genes, potentially responsible for generating metabolites that contribute to organismal virulence. Differential expression of the nonribosomal peptide synthetase gene, pes1, in four strains of Aspergillus fumigatus was observed. The pattern of pes1 expression differed from that of a putative siderophore synthetase gene, sidD, and so is unlikely to be involved in iron acquisition. The Pes1 protein (expected molecular mass 698 kDa) was partially purified and identified by immunoreactivity, peptide mass fingerprinting (36% sequence coverage) and MALDI LIFT-TOF ⁄ TOF MS (four internal peptides sequenced). A pes1 disruption mutant (Dpes1) of Aspergillus fumigatus strain 293.1 was generated and confirmed by Southern and western analysis, in addition to RT-PCR. The Dpes1 mutant also showed significantly reduced virulence in the Galleria mellonella model system (P < 0.001) and increased sensitivity to oxidative stress (P ¼ 0.002) in culture and during neutrophil-mediated phagocytosis. In addition, the mutant exhibited altered conidial surface morphology and hydrophilicity, compared to Aspergillus fumigatus 293.1. It is concluded that pes1 contributes to improved fungal tolerance against oxidative stress, mediated by the conidial phenotype, during the infection process.Abbreviations CGD, chronic granulomatous disease; NRP synthetase, nonribosomal peptide synthetase; PNS, postnuclear supernatant; ROS, reactive oxygen species.

Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bc), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bc class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass = 20 kDa; pI = 5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean ± standard deviation, n = 3) of 3.13 ± 0.27 and 3.43 ± 1.0 lmol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.

Analysis of the genome of the human pathogen, Aspergillus fumigatus, revealed the presence of several putative glutathione transferase (GST) open reading frames. Three A. fumigatus GST genes, termed gstA, B, and C, were cloned and recombinant proteins expressed in Escherichia coli. Functional analysis of recombinant gstA-C conWrms that the enzymes exhibit GST activity and glutathione peroxidase activity. RT-PCR conWrmed low basal expression of gstA and gstC which was markedly up-regulated (at least 4£-10£) in the presence of either H 2 O 2 or 1-chloro-2,4-dinitrobenzene (CDNB). GstB expression was only observed in the presence of CDNB. These results demonstrate for the Wrst time the existence of three functional GSTs in A. fumigatus and strongly suggest a role for these enzymes in the response of the organism to both oxidative stress and xenobiotic presence. 

In fungi, nonribosomal peptide synthetases (NRP synthetases) are large multi-functional enzymes containing adenylation, thiolation (or peptidyl carrier protein, PCP) and condensation domains. These enzymes are often encoded within gene clusters. Multiple NRP synthetase ORFs have also been identified in fungi (14 in Aspergillus fumigatus). LeaA, a methyltransferase, is involved in secondary metabolite gene cluster regulation in Aspergillus spp. The NRP synthetases GliP and FtmA respectively direct the biosynthesis of the toxic metabolites gliotoxin and brevianamide F, a precursor of bioactive prenylated alkaloids. The NRP synthetase Pes1 has been shown to mediate resistance to oxidative stress, and in plant-pathogenic ascomycetes (e.g. Cochliobolus heterostrophus) an NRP synthetase, encoded by the NPS6 gene, significantly contributes to virulence and resistance to oxidative stress. Adenylation (A) domains within NRP synthetases govern the specificity of amino acid incorporation into nonribosomally synthesized peptides. To date there have only been limited demonstrations of A domain specificity (e.g. A. fumigatus GliP and in Beauveria bassiana) in fungi. Indeed, only in silico prediction data are available on A domain specificity of NRP synthetases from most fungi. NRP synthetases are activated by 49-phosphopantetheinylation of serine residues within PCP domains by 49-phosphopantetheinyl transferases (49-PPTases). Coenzyme A acts as the 49-phosphopantetheine donor, and labelled coenzyme A can be used to affinity-label apo-NRP synthetases. Emerging fungal gene disruption and gene cluster expression strategies, allied to proteomic strategies, are poised to facilitate a greater understanding of the coding potential of NRP synthetases in fungi.

Three non-ribosomal peptide synthetase genes, termed sidD, sidC and sidE, have been identified in Aspergillus fumigatus. Gene expression analysis by RT-PCR confirms that expression of both sidD and C was reduced by up to 90% under iron-replete conditions indicative of a likely role in siderophore biosynthesis. SidE expression was less sensitive to iron levels. In addition, two proteins purified from mycelia grown under iron-limiting conditions corresponded to SidD ( approximately 200 kDa) and SidC (496 kDa) as determined by MALDI ToF peptide mass fingerprinting and MALDI LIFT-ToF/ToF. Siderophore synthetases are unique in bacteria and fungi and represent an attractive target for antimicrobial chemotherapy.