An Automated Assay to Measure Phagocytosis of Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis, which kills 200,000 individuals worldwide each year. It is ubiquitous in the environment and is first inhaled into the lungs of the host, where it is taken up by phagocytes. The interaction of these fungal cells with host phagocytes, therefore, is a critical step in the pathogenesis of this disease. One characteristic of this initial step in host-pathogen interactions is the avidity with which fungal cells are taken up by phagocytes, described by the phagocytic index. In this chapter, we detail a high-throughput method of directly assessing the phagocytic index of fungal cells using an imaging-based paradigm. By automating image collection and processing, this method permits rapid assessment of this critical host interaction. © 2019 by John Wiley & Sons, Inc.

Keywords: Cryptococcus neoformans; automation; phagocytic index; phagocytosis.

Figure 1:

Vertical pairs of images represent individual channels from Cytation image data, corresponding to staining with Lucifer Yellow (LY), Calcofluor White (CFW), and propidium iodide (PI); top panel of each pair, unannotated image; bottom panel of each pair, image with detected objects annotated by yellow outlines. The large panel at right shows merged channels without annotation. All fungi are LY-stained, with examples of extracellular fungi (CFW-positive) indicated by open arrowheads and intracellular fungi (CFW-negative) indicated with solid arrowheads. Scale bar, 50 μm.