Live-cell imaging of pathogenic fungal hyphae reveal dynamic cellular responses to clinical antifungals

Antifungal susceptibility testing quantifies end-point fungal biomass in liquid cultures initiated from non-invasive yeast or spore morphologies. However, end-point analyses obscure informative spatio-temporal responses to drug exposures. In the major fungal pathogens Aspergillus fumigatus and Candida albicans we used microfluidic-coupled, fluorescence-mediated live-cell imaging to capture the real-time responses of fungal hyphae to clinical concentrations of AmBisome or Caspofungin. In both fungi, AmBisome exposure caused rapid growth arrest, extensive hyphal vacuolation and membrane blebbing. Responses to Caspofungin exposure were slower with initial lytic effects occurring after 1.5 or 4 hours in A.fumigatus and C.albicans, respectively. Whilst C.albicans hyphae undergo unsalvageable hyphal lysis in response to Caspofungin, A.fumigatus exhibit several compensatory growth behaviours, including a novel resuscitative growth form, that circumvent lytic events to maintain apical and sub-apical hyphal growth. This study reveals how the differing biologies of the two pathogens affected outcomes and contributes to the highly disparate rates of antifungal efficacy amongst commonly used drugs, where spore/yeast-derived inhibitory doses may underestimate the dose required to arrest/kill the invasive hyphal morphotypes of fungal pathogens in vitro.