A microscopy imaging assay, developed by Olivier et al., captures the escape of a fungus (C. albicans) from immune cells (macrophages). Left: Fungi (red) contained inside the immune cells. Right: escaped fungal filaments (blue) and the nuclei of dead immune cells (green). Credit: Monash University
Candida albicans is pathogenic yeast that is commonly found in the human body; while it does not usually cause disease, it can lead to life-threatening illness in vulnerable individuals such as ICU, post-surgery and cancer patients. C. albicans is known to evade immune responses by changing form to break out of macrophages. A live-cell imaging assay developed by researchers at Monash University has revealed new information about the pathways involved in the immune escape of C. albicans.
C. albicans transforms from a yeast to a hyphal form to escape from macrophages. The imaging assay is capable of dynamically quantifying both escaped hyphae and permeabilized macrophage membranes in parallel, at high temporal resolution, the authors wrote. The researchers utilized bone marrow-derived macrophages and a C. albicans strain expressing dTomato for the study. Two membrane-impermeable dyes – the fungal cell walls stain calcofluor white (CFW) and the DNA stain DRAQ7 – allowed the team to distinguish between phagocytosed (dTomato+ and CFW-) and escaped (dTomato+ and CFW+) fungal cells, as well as quantify permeabilized macrophages through staining of nuclei with DRAQ7.
The team demonstrated the key role of the toxin candidalysin in hyphal escape, as macrophages infected with a C. albicans strain lacking the candidalysin-encoding gene ECE1 showed lower numbers of escaped hyphae and permeabilized macrophage membranes. The researchers also showed how the pathogen engages the cell death pathway Gasdermin D-mediated pyroptosis to escape, as macrophages lacking the Gasdermin D protein showed lower rates of hyphal escape. Additionally, the study revealed that the formation of macrophage extracellular traps (METs) may provide another escape route for C. albicans, through a cell death pathway known as ETosis, as MET-like structures were associated with escaped hyphae in 87% of cases observed throughout the experiments. This study was published in Cell Reports.
According to corresponding author Ana Traven, targeting C. albicans as it is escaping “presents a promising therapeutic avenue, preventing both the spread of the infection and having the potential to dampen inflammation.”
The newly-developed live-cell imaging platform provides the opportunity to better understand this escape process and determine best strategies for targeting one of the several escape mechanisms revealed.