Abstract
We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes.
Highlights
Deep and invasive fungal infections caused by Candida species are increasing among immunocompromised individuals
The challenge in developing an in vitro cellular model to perform kinetics studies relied on the fact that the two interacting populations changed over time: the fungal cells grew and divided, leading to an overall increase in the fungal biomass, and the infected phagocytes eventually died, imposing the need to mark each population to monitor their outcome during the interaction
Because of the morphological changes that could occur for the different yeast species during the infection process, variations in the Calcofluor White (CFW) fluorescence were interpreted as variations in the fungal biomass rather than variations in the number of cells
Summary
Deep and invasive fungal infections caused by Candida species are increasing among immunocompromised individuals. Given the evidence that phagocytosis of fungal cells is the first step in the control of infection, developing a cellular model allowing an accurate analysis of the overall interaction involving different Candida species and phagocytes, appears to be of great interest in that way it constitutes an alternative method to in vivo experiments to evaluate virulence of Candida strains. The main objective of this work was to develop a simple and reproducible method for the simultaneous monitoring of the kinetics for phagocyte association to yeasts, phagocyte survival at the individual cell level, and for fungal cells uptake by phagocytes over a 24-hour infection. An accurate evaluation of phagocytosis requires 1) to analyze the phagocyte association to yeast cells and the phagocyte survival simultaneously 2) to distinguish between yeasts that had been internalized by phagocytes from those unphagocytosed, and to measure the uptake of fungal cells by phagocytes while taking into account extracellular yeast multiplication during the infection process. To determine the rate of yeasts internalized in phagocytes, we exploited the ability of the trypan blue, incapable of penetrating into viable phagocytes [5], to quench the fluorescence of the extra-phagocyte CFW-labeled yeasts, in order to detect solely the CFW fluorescence of the internalized yeasts [6]
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