# Microfluidic live‐cell imaging of Aspergillus fumigatus and Candida albicans hyphal growth treated with AmBisome and Caspofungin

## TL;DR

This study uses microfluidics and live-cell imaging to show how two major fungal pathogens respond in real time to antifungal drugs, revealing differences in their survival strategies.

## Contribution

The study introduces a novel microfluidic live-cell imaging method to observe real-time antifungal responses in invasive fungal hyphae.

## Key findings

- AmBisome caused rapid growth arrest and subcellular reorganisation in both Aspergillus fumigatus and Candida albicans.
- Caspofungin induced slower lytic effects, with A. fumigatus showing compensatory growth behaviors unlike C. albicans.
- A. fumigatus exhibited a novel resuscitative growth response after hyphal bursting, while C. albicans transitioned to yeast growth.

## Abstract

Hyphal forms of human pathogenic fungi cause invasive disease in humans, but the hyphal response to antifungals is understudied. 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 (<15 min) and subcellular reorganisation and, in C. albicans, localised expansions of lipid‐like structures from the hyphal perimeter. Responses to Caspofungin exposure were slower, with initial lytic effects occurring after 1.5 or 4 h in A. fumigatus and C. albicans hyphae, respectively. While C. albicans hyphae undergo unsalvageable hyphal lysis in response to Caspofungin, A. fumigatus exhibits several compensatory growth behaviours, including a novel resuscitative growth response, that circumvents 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 be underestimated to arrest/kill the invasive hyphal morphotypes in vitro.

Human pathogenic cause >2 M deaths per year and we have a limited number of antifungals in the clinic to combat these infections. Those drugs are increasingly meeting resistance in killer fungi and our understanding of drug responses are limited. Our main assessment of antifungal resistance comes from end‐point 48 h drug culture of the fungus and is performed by eye for presence of absence of growth in a well. Further, these tests are performed on the pre‐invasive form of the fungus, the yeast or spore (for speed and simplicity), not the invasive filamentous form present during systemic infection of organs in humans.

This work shows how the invasive filamentous form of two major human fungal pathogens respond to two frontline clinical antifungal drugs with real time microscopy. We describe the cell death and/or adaptive growth responses via live‐cell microscopy to understand the morphological and cellular responses over time. We were able to do this by coupling fluorescently engineered pathogens and live‐cell 4D microscopy to microfluidic delivery of culture media and/or antifungal drug. We used the triggerable microfluidics to first establish invasive filamentous growth without drug, and keep the cells in the same focal plane (with shallow roof that kept the filaments growing up out of the focal plane). We then triggered the switch to media with drug(s) to perfuse drug while observing comparative cellular responses live, up to 10 h in two pathogenic fungi.

In both pathogens (Aspergillus fumigatus and Candida albicans) we saw similar immediate responses to one drug (Ambisome), which inhibited filamentous growth almost immediately. In stark contrast, Caspofungin induced different but continued forms of growth to known inhibitory doses against invasive fungal filaments. A. fumigatus (a filamentous mould fungus) appeared hard‐wired to continue filamentous growth by various compensatory regenerative growth forms, including a novel ‘resuscitative’ form we describe which occurs after the drug causes filament bursting. However, C. albicans filaments (a polymorphic yeast) respond by transitioning from filamentous to yeast growth (all filaments eventually burst, leaving only budding yeast). This study details the short‐ and long‐term responses of invasive pathogenic fungal filaments to drugs and highlights that the traditional spore/yeast‐derived inhibitory doses for these fungi may be insufficient for the invasive form of the fungus, where more attention to the filamentous form is needed.