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Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in severely immunocompromised patients. Although A. fumigatus is the most common cause of IBPA, infections with A. terreus are emerging and mortality rates are comparable to A. Fumigates infections. Although A. terreus is distributed ubiquitously, the incidence of A. Terreus mediated IBPA is comparably low.
To elucidate the pathogenesis of A. terreus aspergillosis in comparison to A. Fumigates mediated IBPA, an alternative infection model and two distinct pulmonary murine infection models were established and characterised in this study. To induce lethal infections by A. terreus an at least 100 times higher infectious dose was required than for A. fumigatus, but even with the highest infectious dose only 50% mortality was observed in corticosteroidtreated mice. However, surviving mice transiently displayed clinical symptoms and bioluminescence imaging revealed transient fungal growth, suggesting that the immune system of surviving animals was able to control the infection. In moribund animals, the disease pattern largely resembled A. fumigatus IBPA. However, persistent, ungerminated but viable conidia were frequently found in alveolar macrophages and pulmonary epithelial cells of surviving animals. In contrast to A. fumigatus infections, all mice infected with A. Terreus developed a fatty liver degeneration, possibly due to the production of toxic secondary metabolites. Thus, at least in mice, persistence and subclinical liver damage represent unique features of A. terreus mediated IBPA. Furthermore, these models provide the necessary tools to investigate the pathogenesis of A. terreus mediated aspergillosis in detail. The persistence and incomplete germination of conidia in macrophages and epithelial cells observed in vivo led to the hypothesis that the initial steps of disease establishment might be fundamentally different between A. terreus and A. fumigatus. Since alveolar macrophages represent the first immune cells facing inhaled conidia in the lung, the interaction of A. terreus and A. fumigatus conidia with these phagocytes was investigated. Interestingly, A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia. This was likely due to the higher exposure of β-1,3-glucan and galactomannan on the surface of A. Terreus resting and pre-swollen conidia. In agreement with the increased PAMP exposure observed on conidia, blocking of dectin-1 and the mannose receptor, the ligand-specific PRRs, significantly reduced phagocytosis of A. terreus to basal levels, but had only a moderate effect on phagocytosis of A. fumigatus. While A. fumigatus prevents phagolysosomal acidification which allows germination within this organelle, A. terreus conidia persisted in fully matured and acidified phagolysosomes after phagocytosis. The acidic pH of the phagolysosome prevented germination of A. terreus conidia, thus resulting in significantly reduced macrophage cytotoxicity. Blocking phagolysosome acidification by the specific v- ATPase inhibitor bafilomycin A increased A. terreus germination within phagolysosomes and consequently cytotoxicity. Thus, it appears that the two fungal species have evolved different interaction strategies with macrophages: While A. fumigatus interferes with phagosome maturation and escapes from phagocytes by germination, A. terreus remains viable but trapped within acidified phagolysosomes. However, possibly because germlings are more sensitive than conidia, A. fumigatus is inactivated to a higher extent by macrophages.
To determine factors involved in phagolysosome interaction, this work focused on pigments which are frequently used for morphological species discrimination in Aspergillus diagnostics. These pigments are part of the cell wall, provide defence against physiological stresses and can moreover interact with PRRs of immune cells. In A. fumigatus, the polyketide synthase PksP produces the pigment DHN-melanin, which prevents phagolysosome acidification, but is absent in A. terreus. However, recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, in A. terreus inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice.
In summary, this study demonstrates for the first time that A. terreus and A. Fumigates interact fundamentally different with the immune system during disease establishment. Although aspergillosis caused by A. terreus resembles A. fumigatus IBPA, significant differences in the interaction with macrophages suggest a modified pathogenicity strategy. While A. fumigatus hides from phagocytosis and prevents phagolysosome acidification which allows escape from macrophages by germination, A._terreus is rapidly phagocytosed, does not interfere with phagolysosomal maturation but persists within macrophages. Consequently, the pathogenicity strategy of A. fumigatus cannot be taken as a general model for all aspergilli but requires species-specific investigation.