Treatment of Aspergillus fumigatus with
echinocandins such as
caspofungin inhibits the synthesis of cell wall β-1,3-glucan, which triggers a compensatory stimulation of
chitin synthesis. Activation of
chitin synthesis can occur in response to sub-MICs of
caspofungin and to CaCl2 and
calcofluor white (CFW), agonists of the
protein kinase C (PKC), and Ca(2+)-
calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding
chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the
chitin synthase enzymes that were required for the compensatory upregulation of
chitin synthesis. Only the ΔAfchsG mutant was hypersensitive to
caspofungin, and all other ΔAfchs mutants tested remained capable of increasing their
chitin content in response to treatment with CaCl2 and CFW and
caspofungin. The resulting increase in cell wall
chitin content correlated with reduced susceptibility to
caspofungin in the wild type and all ΔAfchs mutants tested, with the exception of the ΔAfchsG mutant, which remained sensitive to
caspofungin. In vitro exposure to the
chitin synthase inhibitor,
nikkomycin Z, along with
caspofungin demonstrated synergistic efficacy that was again AfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC
caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of
caspofungin. In addition, intrahyphal hyphae were formed in response to
echinocandin treatment. These in vitro data demonstrate that A. fumigatus has the potential to survive
echinocandin treatment in vivo by AfChsG-dependent upregulation of
chitin synthesis.
Chitin-rich cells may, therefore, persist in human tissues and act as the focus for
breakthrough infections.