The cyclase-associated protein contributes to antifungal susceptibility and virulence in Aspergillus fumigatus

Abstract

Aspergillus fumigatus is the most prevalent pathogenic mould that contributes to high morbidity and mortality in immunocompromised patients. Here, we characterized the functions of the cyclase-associated protein (CAP) in A. fumigatus. To study the role of CAP in virulence and antifungal susceptibility of A. fumigatus, CAP gene knockout strain (ΔCAP) and complemented strain (R-ΔCAP) were constructed. ΔCAP showed a reduced growth rate, abnormal hyphal development, and increased susceptibility to cell wall-perturbing agents (Congo red, calcofluor white, and SDS), oxidative stress-inducing agents (H2O2 and menadione), calcineurin inhibitors (FK506 and CsA), and voriconazole (VRC) and itraconazole. Transcriptome analysis revealed that differentially expressed genes responsible for regulating growth, hyphal development, cell wall synthesis, stress responses and antifungal susceptibility were identified in ΔCAP. To identify CAP-interacting proteins, an A. fumigatus strain expressing the CAP protein fused with a C-terminus 6×his tag was constructed and designated Afcap6his. After extracting Afcap6his and Af293 proteins, actin and adenylate cyclase were identified by coimmunoprecipitation (co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, ΔCAP showed downregulated actin expression, AC-cAMP-PKA pathway activity and efflux pump genes (AfuMDR1, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1B) expression as well as increased calcineurin activity. By using an invasive pulmonary aspergillosis (IPA) murine model, ΔCAP exhibited attenuated virulence and increased VRC therapeutic efficiency. Thus, CAP plays an important role in regulating antifungal susceptibility and virulence of A. fumigatus.

Keywords

• Aspergillus fumigatus
• cyclase-associated protein
• antifungal susceptibility
• virulence
• VRC efficacy