Identification of immunogenic antigens from Aspergillus fumigatus by direct multiparameter characterization of specific conventional and regulatory CD4+ T cells.

CD4(+) T cells orchestrate immune responses against fungi, such as Aspergillus fumigatus, a major fungal pathogen in humans. The complexity of the fungal genome and lifestyle questions the existence of one or a few immune-dominant Ags and complicates systematic screening for immunogenic Ags useful for immunotherapy or diagnostics. In this study, we used a recently developed flow cytometric assay for the direct ex vivo characterization of A. fumigatus-specific CD4(+) T cells for rapid identification of physiological T cell targets in healthy donors. We show that the T cell response is primarily directed against metabolically active A. fumigatus morphotypes and is stronger against membrane protein fractions compared with cell wall or cytosolic proteins. Further analysis of 15 selected single A. fumigatus proteins revealed a highly diverse reactivity pattern that was donor and protein dependent. Importantly, the parallel assessment of T cell frequency, phenotype, and function allowed us to differentiate between proteins that elicit strong memory T cell responses in vivo versus Ags that induce T cell exhaustion or no reactivity in vivo. The regulatory T cell (Treg) response mirrors the conventional T cell response in terms of numbers and target specificity. Thus, our data reveal that the fungal T cell immunome is complex, but the ex vivo characterization of reactive T cells allows us to classify Ags and to predict potential immunogenic targets. A. fumigatus-specific conventional T cell responses are counterbalanced by a strong Treg response, suggesting that Treg-depletion strategies may be helpful in improving antifungal immunity.

The CCAAT-binding complex coordinates the oxidative stress response in eukaryotes.

The heterotrimeric CCAAT-binding complex is evolutionary conserved in eukaryotic organisms. The corresponding Aspergillus nidulans CCAAT-binding factor (AnCF) consists of the subunits HapB, HapC and HapE. All of the three subunits are necessary for DNA binding. Here, we demonstrate that AnCF senses the redox status of the cell via oxidative modification of thiol groups within the histone fold motif of HapC. Mutational and in vitro interaction analyses revealed that two of these cysteine residues are indispensable for stable HapC/HapE subcomplex formation and high-affinity DNA binding of AnCF. Oxidized HapC is unable to participate in AnCF assembly and localizes in the cytoplasm, but can be recycled by the thioredoxin system in vitro and in vivo. Furthermore, deletion of the hapC gene led to an impaired oxidative stress response. Therefore, the central transcription factor AnCF is regulated at the post-transcriptional level by the redox status of the cell serving for a coordinated activation and deactivation of antioxidative defense mechanisms including the specific transcriptional activator NapA, production of enzymes such as catalase, thioredoxin or peroxiredoxin, and maintenance of a distinct glutathione homeostasis. The underlying fine-tuned mechanism very likely represents a general feature of the CCAAT-binding complexes in eukaryotes.

Aspects on evolution of fungal beta-lactam biosynthesis gene clusters and recruitment of trans-acting factors.

Penicillins and cephalosporins are beta-lactam antibiotics. The formation of hydrophobic penicillins has been reported in fungi only, notably Penicillium chrysogenum and Aspergillus (Emericella) nidulans, whereas the hydrophilic cephalosporins are produced by both fungi, e.g., Acremonium chrysogenum (cephalosporin C), and bacteria. The producing bacteria include Gram-negatives and Gram-positives, e.g., Streptomyces clavuligerus (cephamycin C) and Lysobacter lactamgenus (cephabacins), respectively. The evolutionary origin of beta-lactam biosynthesis genes has been the subject of discussion for many years, and two main hypotheses have been proposed: (i) horizontal gene transfer (HGT) from bacteria to fungi or (ii) vertical decent. There are strong arguments in favour of HGT, e.g., unlike most other fungal genes, beta-lactam biosynthesis genes are clustered and some of these genes lack introns. In contrast to S. clavuligerus, all regulators of fungal beta-lactam biosynthesis genes represent wide-domain regulators that are not part of the gene cluster. If bacterial regulators were co-transferred with the gene cluster from bacteria to fungi, most likely they would have been non-functional in eukaryotes and lost during evolution. Recently, the penicillin biosynthesis gene aatB was discovered, which is not part of the penicillin biosynthesis gene cluster and is even located on a different chromosome. The aatB gene is regulated by the same regulators AnCF and AnBH1 as the penicillin biosynthesis gene aatA (penDE). Data suggest that aatA and aatB are paralogues derived by duplication of a common ancestor gene. This data supports a model in which part of the beta-lactam biosynthesis gene cluster was transferred to some fungi, i.e., the acvA and ipnA gene without a regulatory gene. We propose that during the assembly of aatA and acvA-ipnA into a single gene cluster, recruitment of transcriptional regulators occurred along with acquisition of the duplicated aatA ancestor gene and its cis-acting sites.

The thioredoxin system of the filamentous fungus Aspergillus nidulans: impact on development and oxidative stress response.

Redox regulation has been shown to be of increasing importance for many cellular processes. Here, redox homeostasis was addressed in Aspergillus nidulans, an important model organism for fundamental biological questions such as development, gene regulation or the regulation of the production of secondary metabolites. We describe the characterization of a thioredoxin system from the filamentous fungus A. nidulans. The A. nidulans thioredoxin A (AnTrxA) is an 11.6-kDa protein with a characteristic thioredoxin active site motif (WCGPC) encoded by the trxA gene. The corresponding thioredoxin reductase (AnTrxR), encoded by the trxR gene, represents a homodimeric flavoprotein with a native molecular mass of 72.2 kDa. When combined in vitro, the in Escherichia coli overproduced recombinant proteins AnTrxA and AnTrxR were able to reduce insulin and oxidized glutathione in an NADPH-dependent manner indicating that this in vitro redox system is functional. Moreover, we have created a thioredoxin A deletion strain that shows decreased growth, an increased catalase activity, and the inability to form reproductive structures like conidiophores or cleistothecia when cultivated under standard conditions. However, addition of GSH at low concentrations led to the development of sexual cleistothecia, whereas high GSH levels resulted in the formation of asexual conidiophores. Furthermore, by applying the principle of thioredoxin-affinity chromatography we identified several novel putative targets of thioredoxin A, including a hypothetical protein with peroxidase activity and an aldehyde dehydrogenase.

Interaction of HapX with the CCAAT-binding complex--a novel mechanism of gene regulation by iron.

Iron homeostasis requires subtle control systems, as iron is both essential and toxic. In Aspergillus nidulans, iron represses iron acquisition via the GATA factor SreA, and induces iron-dependent pathways at the transcriptional level, by a so far unknown mechanism. Here, we demonstrate that iron-dependent pathways (e.g., heme biosynthesis) are repressed during iron-depleted conditions by physical interaction of HapX with the CCAAT-binding core complex (CBC). Proteome analysis identified putative HapX targets. Mutual transcriptional control between hapX and sreA and synthetic lethality resulting from deletion of both regulatory genes indicate a tight interplay of these control systems. Expression of genes encoding CBC subunits was not influenced by iron availability, and their deletion was deleterious during iron-depleted and iron-replete conditions. Expression of hapX was repressed by iron and its deletion was deleterious during iron-depleted conditions only. These data indicate that the CBC has a general role and that HapX function is confined to iron-depleted conditions. Remarkably, CBC-mediated regulation has an inverse impact on the expression of the same gene set in A. nidulans, compared with Saccharomyces cerevisae.