Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus

Abstract

Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn’t localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don’t play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn’t show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated “paradoxical growth effect” at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.

In VitroandIn VivoAssessment of FK506 Analogs as Novel Antifungal Drug Candidates

“…In C. albicans, which causes both superficial and systemic candidiasis (29), calcineurin is required for survival in the presence of serum (30)(31)(32)(33). In A. fumigatus, which causes life-threatening systemic and invasive aspergillosis (34), the calcineurin pathway regulates conidial germination and hyphal growth (35). In human fungal pathogens, deletion of calcineurin genes considerably attenuates their infectivity and virulence (36).…”

Target Of Rapamycin pathway in the white-rot fungus Phanerochaete chrysosporium

“…One of them is PcFKBP12 which is an unusual location for this protein. In A. fumigatus, change in localization of AfFKBP12 has been observed in response to the immunosuppressant FK506 but not its secretion [34]. Due to amino acid conservation, the FRB domain of TOR and FKBP12 from P. chrysosporium can interact with rapamycin.…”

Leveraging Fungal Calcineurin-Inhibitor Structures, Biophysics and Dynamics to Design Selective and Non-Immunosuppressive FK506 Analogs

“…The deletion of A. fumigatus AfFKBP12 encoding gene leads to FK506 resistance, establishing its central role for calcineurin inhibition 25 Fig. 1).…”