Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies

Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall beta-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 Ca2+-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (Delta Afchs) 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 Delta AfchsG mutant was hypersensitive to caspofungin, and all other Delta Afchs 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 Delta Afchs mutants tested, with the exception of the Delta AfchsG 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.