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

Abstract

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 Ca2+-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.

Caspofungin Increases Fungal Chitin and Eosinophil and γδ T Cell–Dependent Pathology in Invasive Aspergillosis

“…Previous studies have reported increased A. fumigatus chitin expression when β-1,3-glucan synthesis is inhibited by caspofungin (36, 37). To confirm if caspofungin increases chitin exposure in germinating conidia, we cultured and germinated the clinical isolate Af293 (low/normal chitin expressing (43)) in the presence or absence of caspofungin and subsequently stained with the chitin-binding wheat germ agglutinin (WGA) for flow cytometric analysis.…”

“…Not surprisingly, antifungal drugs induce a significant amount of stress by directly inhibiting cell wall or membrane synthesis. For example, direct inhibition of the β-1,3-glucan synthesis pathway by in vitro growth in the presence of caspofungin resulted in a cell wall architecture characterized by increased levels of chitin in A. fumigatus (36, 37). However, a connection between increased caspofungin-mediated chitin exposure and increased eosinophil activation and pathology has not been described.…”

Medical mycology and fungal immunology: new research perspectives addressing a major world health challenge

“…Chitin particles also have been shown to induce IL-10 in the colon and offset the pathology associated with inflammatory gut disorders [16]. Moreover, echinocandin-treated cells of Candida and Aspergillus upregulate chitin production in their walls to offset damage inflicted on cell wall β-1,3-glucan [17,18]. Such chitin-rich cells may be less inflammatory in vivo , thus limiting the immune response required for their elimination by the immune system [17].…”

A complex game of hide and seek: the search for new antifungals

“…47 Treatment with echinocandins, normally reserved for human, can sometimes stimulate chitin synthesis, which in turn causes the fungi to be more resistant to antifungal treatment. 48 As a result, this pan-usage of echinocandins could increase the rate of resistance development.…”