Vomocytosis is a process that occurs when internalized
fungal pathogens
escape from phagocytes without compromising the viability of the pathogen
and the host cell. Manual quantification of time-lapse microscopy
videos is currently used as the standard to study pathogen behavior
and vomocytosis incidence. However, human-driven quantification of
vomocytosis (and the closely related phenomenon, exocytosis) is incredibly
burdensome, especially when a large volume of cells and interactions
needs to be analyzed. In this study, we designed a MATLAB algorithm
that measures the extent of colocalization between the phagocyte and
fungal cell (
Cryptococcus neoformans
; CN) and rapidly
reports the occurrence of vomocytosis in a high throughput manner.
Our code processes multichannel, time-lapse microscopy videos of cocultured
CN and immune cells that have each been fluorescently stained with
unique dyes and provides quantitative readouts of the spatiotemporally
dynamic process that is vomocytosis. This study also explored metrics,
such as the rate of change of pathogen colocalization with the host
cell, that could potentially be used to predict vomocytosis occurrence
based on the quantitative data collected. Ultimately, the algorithm
quantifies vomocytosis events and reduces the time for video analysis
from over 1 h to just 10 min, a reduction in labor of 83%, while simultaneously
minimizing human error. This tool significantly minimizes the vomocytosis
analysis pipeline, accelerates our ability to elucidate unstudied
aspects of this phenomenon, and expedites our ability to characterize
CN strains for the study of their epidemiology and virulence.