Despite concerted efforts, diagnosis of aspergillosis is still a great challenge to clinical microbiology laboratories. Along with the requirement for high sensitivity and specificity, species-specific identification is important. We developed rapid, sensitive and species-specific qPCR assays using the TaqMan technology for the detection and identification of Aspergillus fumigatus and Aspergillus terreus. The assays were designed to target orthologs of the Streptomyces factor C gene that are only found in a few species of filamentous fungi. Fungi acquired this gene through horizontal gene transfer and divergence of the gene allows identification of species. The assays have potential as a molecular diagnosis tool for the early detection of fungal infection caused by Aspergillus fumigatus and Aspergillus terreus, which merits future diagnostic studies. The assays were sensitive enough to detect a few genomic equivalents in blood samples.
New approaches for treatment of invasive fungal infections are necessary to cope with emerging resistant fungal pathogens of humans. In this paper, three different strategies are presented and evaluated to find new-type antifungal drugs and their targets. While experimental data obtained with potent chitinase inhibitors, e.g. allosamidin, and small-size antifungal proteins of fungal origin are encouraging more efforts are needed to verify and exploit the possible involvement of intracellular thiols, e.g. glutathione, and their metabolic anzymes in the pathogenesis of mycoses caused by dimorphic fingi. Chitinase inhibitors seem to hinder the cell separation of yeasts and the fragmentation of filamentous fungi quite effectively and, hence, they may be implicated in future therapies of systemic mycoses. In addition, small-size antifungal proteins possessing a broad inhibition spectrum may also provide us with promising new agents for the treatment of different kinds of (e.g. cutaneous) fungal infections.
Pairwise interactions between four antifungal compounds were studied. The β-1,3-glucan synthase inhibitor echinocandin B (ECB) showed synergistic effect with the cell wall hydrolase ChiB chitinase and EngA β-1,3-glucanase on Saccharomyces cerevisiae, Candida albicans, Aspergillus rugulosus and A. fumigatus. The antifungal protein of Penicillium chrysogenum (PAF) did not influence the antifungal activity of ChiB or EngA, but showed antagonistic effect with ECB on A. nidulans, A. rugulosus and A. fumigatus. PAF had no significant effect on the growth of the tested yeasts as it was expected and did not influence significantly the antifungal activity of ECB, ChiB or EngA against yeasts.
Stress sensitivity of three related phytopathogenic Fusarium species (Fusarium graminearum, Fusarium oxysporum and Fusarium verticillioides) to different oxidative, osmotic, cell wall, membrane, fungicide stressors and an antifungal protein (PAF) were studied in vitro. The most prominent and significant differences were found in oxidative stress tolerance: all the three F. graminearum strains showed much higher sensitivity to hydrogen peroxide and, to a lesser extent, to menadione than the other two species. High sensitivity of F. verticillioides strains was also detectable to an azole drug, Ketoconazole. Surprisingly, no or limited differences were observed in response to other oxidative, osmotic and cell wall stressors. These results indicate that fungal oxidative stress response and especially the response to hydrogen peroxide (this compound is involved in a wide range of plant-fungus interactions) might be modified on niche-specific manner in these phylogenetically related Fusarium species depending on their pathogenic strategy. Supporting the increased hydrogen peroxide sensitivity of F. graminearum, genome-wide analysis of stress signal transduction pathways revealed the absence one CatC-type catalase gene in F. graminearum in comparison to the other two species.
Yeast protein sequence-based homology search for glutathione (GSH) metabolic enzymes and GSH transporters demonstrated that Aspergillus nidulans has a robust GSH uptake and metabolic system with several paralogous genes. In wet laboratory experiments, two key genes of GSH metabolism, gcsA, and glrA, encoding γ-l-glutamyl-l-cysteine synthetase and glutathione reductase, respectively, were deleted. The gene gcsA was essential, and the ΔgcsA mutant required GSH supplementation at considerably higher concentration than the Saccharomyces cerevisiae gsh1 mutant (8–10 mmol l−1 vs. 0.5 μmol l−1). In addition to some functions known previously, both genes were important in the germination of conidiospores, and both gene deletion strains required the addition of extra GSH to reach wild-type germination rates in liquid cultures. Nevertheless, the supplementation of cultures with 10 mmol l−1 GSH was toxic for the control and ΔglrA strains especially during vegetative growth, which should be considered in future development of high GSH-producer fungal strains. Importantly, the ΔglrA strain was characterized by increased sensitivity toward a wide spectrum of osmotic, cell wall integrity and antimycotic stress conditions in addition to previously reported temperature and oxidative stress sensitivities. These novel phenotypes underline the distinguished functions of GSH and GSH metabolic enzymes in the stress responses of fungi.
PAF, which is produced by the filamentous fungus Pencicillium chrysogenum, is a small antifungal protein, triggering ROS-mediated apoptotic cell death in Aspergillus nidulans. In this work, we provide information on the function of PAF in the host P. chrysogenum considering that carbon-starving cultures of the Δpaf mutant strain showed significantly reduced apoptosis rates in comparison to the wild-type (wt) strain. Moreover, the addition of PAF to the Δpaf strain resulted in a twofold increase in the apoptosis rate. PAF was also involved in the regulation of the autophagy machinery of this fungus, since several Saccharomyces cerevisiae autophagy-related ortholog genes, e.g. those of atg7, atg22 and tipA, were repressed in the deletion strain. This phenomenon was accompanied by the absence of autophagosomes in the Δpaf strain, even in old hyphae.