Calcium signalling and homeostasis are essential for the growth, differentiation and virulence of filamentous fungi. During infection, A. fumigatusmust balance concomitant demands to: (1) withstand toxic levels of exogenous calcium (3-5 mM) in the host environment which can be >100,000x that of the fungal cytosolic free calcium ([Ca2+]c) concentration; (2) appropriately integrate homeostatic and stress-responsive adaptations; and (3) undergo normal calcium signalling. There is evidence for calcium signalling regulating numerous processes including spore germination and hyphal tip growth. The low resting level of [Ca2+]c (50-100 nM) is maintained by Ca2+ -pumps and -antiporters, and cytoplasmic Ca2+ -buffering. However, [Ca2+]c becomes an intracellular signal when its concentration is transiently increased. We have developed two methods for measuring and imaging [Ca2+]c: (1) 96-well plate luminometry using the genetically encoded, bioluminescent aequorin; and (2) fluorescence microscopy using the genetically encoded calcium-sensitive, fluorescent protein G-CaMP5. Aequorin is ideally suited for quantitative measurements of [Ca2+]c calcium signatures in cell populations whereas fluorescence imaging of the G-CaMP5 is good for single cell and subcellular measurements of [Ca2+]c. Using the aequorin methodology we have found that transient increases in [Ca2+]c with specific, reproducible calcium signatures in A. fumigatus arise from exposure to stresses such as high external calcium. In our analysis, [Ca2+]c spikes in actively growing hyphal tips have been imaged using G-CaMP5. Exposure of conidial germlings to high external calcium induces dramatic and very dynamic changes in [Ca2+]c with the generation of localized [Ca2+]c transients and waves. Furthermore, there is considerable heterogeneity in the [Ca2+]c responses of different germlings within the cell population. Calcium imaging and measurement using genetically encoded probes, particularly when combined with pharmacological and genetic analyses, will provide major new insights into calcium signalling in filamentous fungi.
Full conference title:
27th Fungal Genetics Conference
- Fungal Genetics Conference 27th (2013)