Synergistic effects of putative Ca²⁺-binding sites of calmodulin in fungal development, temperature stress and virulence of Aspergillus fumigatus

ABSTRACT

In pathogenic fungi, calcium-calmodulin-dependent serine-threonine-specific phosphatase calcineurin is involved in morphogenesis and virulence. Therefore, calcineurin and its tightly related protein complexes are attractive antifungal drug targets. However, there is limited knowledge available on the relationship between in vivo Ca²⁺-binding sites of calmodulin (CaM) and its functions in regulating stress responses, morphogenesis, and pathogenesis. In the current study, we demonstrated that calmodulin is required for hyphal growth, conidiation, and virulence in the human fungal pathogen, Aspergillus fumigatus. Site-directed mutations of calmodulin revealed that a single Ca²⁺-binding site mutation had no significant effect on A. fumigatus hyphal development, but multiple Ca²⁺-binding site mutations exhibited synergistic effects, especially when cultured at 42 °C, indicating that calmodulin function in response to temperature stress depends on its Ca²⁺-binding sites. Western blotting implied that mutations in Ca²⁺-binding sites caused highly degraded calmodulin fragments, suggesting that the loss of Ca²⁺-binding sites results in reduced protein stability. Moreover, normal intracellular calcium homeostasis and the nuclear translocation of the transcriptional factor CrzA are dependent on Ca²⁺-binding sites of AfCaM, demonstrating that Ca²⁺-binding sites of calmodulin are required for calcium signalling and its major transcription factor CrzA. Importantly, in situ mutations for four Ca²⁺-binding sites of calmodulin resulted in an almost complete loss of virulence in the Galleria mellonella wax moth model. This study shed more light on the functional characterization of putative calcium-binding sites of calmodulin in the morphogenesis and virulence of A. fumigatus, which enhances our understanding of calmodulin biological functions in cells of opportunistic fungal pathogens.

KEYWORDS:

• Calcium homeostasis
• calmodulin
• EF-hand
• virulence
• A. fumigatus

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (NSFC) (grants 82172292 and 31861133014 to L.L.), and the National Key R&D Program of China (2019YFA0904900), and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Conceptualization, Y.Z. and L.L.; methodology, X.L. and R.F.; validation, X.L.; formal analysis, X.L. and R.F.; investigation, X.L., R.F., and P.L.; resources, L.L.; data curation, X.L., R.F., and P.L.; writing – original draft preparation, X.L. and R.F.; writing – review and editing, Y.Z. and L.L.; visualization, X.L. and R.F.; supervision, L.L.; project administration, L.L.; funding acquisition, L.L. All authors have read and agreed to the published version of the manuscript.

Data Availability statement

The authors confirm that data supporting the findings of this study are available within the article.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2023.2290757

Additional information

Funding

The work was supported by the National Natural Science Foundation of China [82172292]; National Natural Science Foundation of China [31861133014]; National Key R&D Program of China [2019YFA0904900].