Studies on the release of neutrophil extracellular traps and IFN-γ as part of the innate immune response to Aspergillus fumigatus and on the fungal stress response via the hybrid sensor kinase TcsC

Abstract

Aspergillus fumigatus is a saprophytic mold that naturally inhabits the soil. Asexual reproduction yields hardy conidia that circulate in the air and are inhaled daily by humans. The fungus seems not to have evolved distinct mechanisms of pathogenicity, but is capable of responding to many stressful environmental cues present in its naturally harsh niche. The robust conidia present no problem to a fully functioning immune system, but if the innate immune system is compromised, the conidia can become activated and differentiate within the lung tissue to form invasive and disseminating hyphae. The resulting disease is called aspergillosis and is difficult to detect and to treat. To date, scientists have yet to find the factor(s) missing during immunosuppression that allow a healthy patient to easily dispose of A. fumigatus. We explored two possibilities: the production of neutrophil extracellular traps (NETs) and the release of IFN-γ by natural killer (NK) cells. We report here that NETs alone cannot kill the fungus, but do inhibit polar growth. Elongation of hyphal tips is abrogated due to zinc starvation, likely a consequence of the zinc-chelating, NETs-associated protein calprotectin. NK cells alone are also incapable of fungicidal activity, but their release of IFN-γ upon contact with A. fumigatus abrogates hyphal growth by a yet unknown mechanism. In vitro studies of the innate immune response, though helpful, are far from representative of the in vivo response. Neither NETs nor IFN-γ alone can manage Aspergillus infection, but in combination, these and other immune assaults certainly can. The difficulty lies in identifying the precise combination of immune cells and cytokine milieu that in a healthy individual prevent infection. Additionally, we explored mechanisms by which the fungus responds to stress, namely the HOG MAPK pathway, historically involved in osmotic stress response. In filamentous fungi, certain stress signals are sensed by a cytoplasmic hybrid histidine kinase sensor and then passed through the HOG system via phosphorylation. We identified the putative hybrid sensor kinase in A. fumigatus, and generated a corresponding knockout mutant. The ΔtcsC mutant was indeed sensitive to osmotic stress, and resistant to the phenolpyrrole fungicide fludioxonil. In the wild type the addition of either osmotic stress or fludioxonil resulted in SakA phosphorylation and translocation to the nucleus. SakA, the Hog1 homolog in A. fumigatus, is located at the end of the HOG pathway, confirming the role of TcsC as the cytoplasmic sensor upstream of SakA. In hypoxia, on farnesol, and in high concentrations of divalent cations the ΔtcsC mutant exhibited a striking “fluffy” phenotype characterized by the production of tremendous aerial hyphae and little or no differentiation, i.e., no conidiation. Though the ΔtcsC mutant showed no change in virulence compared to wild type, components of the TcsC signalling pathway remain promising targets for antifungal agents