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

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.

Molecular Characterization of Gß-Like Protein CpcB Involved in Antifungal Drug Susceptibility and Virulence in A. fumigatus.

Aspergillus fumigatus is an airborne human fungal pathogen that can survive in a wide range of environmental condition. G protein complex transduces external signals from a variety of stimuli outside a cell to its interior effectors in all eukaryotes. Gß-like CpcB (cross pathway control B) belongs to a WD40 repeat protein family with the conserved G-H and W-D residues. Previous studies have demonstrated that Gß-like proteins cooperate with related signal transduction proteins to function during many important developmental processes in A. fumigatus. However, the molecular characteristics of Gß-like CpcB have not yet been identified. In this study, we demonstrated that the G-H residues in WD repeat 1, 2, 3, and the W-D residue in WD repeat 2 of CpcB are required not only to control normal hyphal growth and conidiation but also to affect antifungal drug susceptibility. The enhanced drug resistance might be due to reduced intracellular drug accumulation and altered ergosterol component. Moreover, we find that the first G-H residue of CpcB plays an important role in the virulence of A. fumigatus. To our knowledge, this is the first report for finding the importance of the conserved G-H and W-D residues for a Gß-like protein in understanding of G protein functions.