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.

Sac1 links phosphoinositide turnover to cryptococcal virulence

“…The samples were then washed to remove unassociated cells, stained with the membrane-impermeant dye Calcofluor White to label externally-associated fungi, fixed, and stained with propidium iodide to label phagocytic cell nuclei. We assessed fungal engulfment as the phagocytic index, or number of internalized fungi (stained with Lucifer Yellow but not Calcofluor White) per host cell (identified by nuclear staining) (24). We define the uptake score as the ratio of a mutant’s phagocytic index to that of wild-type C. neoformans (KN99).…”

“…Assays were performed using a modification of our prior method (24). Host cells were seeded in flat clear bottom black polystyrene TC-treated 96-well plates (Corning) at a density of 10 5 cells/well and incubated for one day at 37°C with 5% CO 2 .…”

“…PBMCs were isolated by Ficoll-Paque centrifugation and differentiated for 6 days. The screen assay was modified from (23). Briefly, opsonized Lucifer Yellow-stained fungi were added to host cells at an MOI of 20 (BMDM) or 30 (HMDM) and the cultures were co-incubated for one hour and washed.…”

“…First, we screened a large collection of 4,692 single gene deletion mutants, representing over eighty percent of the non-essential genes in the cryptococcal genome. Second, we applied a flexible medium-throughput imaging method that avoids pitfalls of earlier studies, such as difficulty distinguishing adherent versus internalized yeast cells (24). Third, we screened using primary cells from both mouse (murine bone marrow-derived macrophages; BMDMs) and human (monocyte derived macrophages; HMDMs), to take advantage of a model system while also examining the system of greatest clinical interest.…”

Contribution of Laccase Expression to Immune Response against Cryptococcus gattii Infection

“…After 2 h of infection, extracellular cryptococci were removed, and macrophage cultures were lysed with sterile distilled water and plated on Sabouraud dextrose agar to analyze CFU. The phagocytic index (PI) was calculated using the following formula: PI ϭ (CFU at 2 hpi/total number of macrophages) ϫ 100 (where hpi is hours postinfection) (42). To determine the intracellular cryptococci (intracellular cryptococcal load [ICL]), the remaining wells were maintained and lysed at 18 and 24 h as Murine model of Cryptococcus infection.…”