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

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.