Abstract
Background Cryptococcus neoformans causes serious disease in immunocompromised individuals, leading to over 600,000 deaths per year worldwide. Part of this impact is due to the organism's ability to thwart what should be the mammalian hosts' first line of defense against cryptococcal infection: internalization by macrophages. Even when C. neoformans is engulfed by host phagocytes, it can survive and replicate within them rather than being destroyed; this ability is central in cryptococcal virulence. It is therefore critical to elucidate the interactions of this facultative intracellular pathogen with phagocytic cells of its mammalian host.Methodology/Principal FindingsTo accurately assess initial interactions between human phagocytic cells and fungi, we have developed a method using high-throughput microscopy to efficiently distinguish adherent and engulfed cryptococci and quantitate each population. This method offers significant advantages over currently available means of assaying host-fungal cell interactions, and remains statistically robust when implemented in an automated fashion appropriate for screening. It was used to demonstrate the sensitivity of human phagocytes to subtle changes in the cryptococcal capsule, a major virulence factor of this pathogen.Conclusions/SignificanceOur high-throughput method for characterizing interactions between C. neoformans and mammalian phagocytic cells offers a powerful tool for elucidating the relationship between these cell types during pathogenesis. This approach will be useful for screens of this organism and has potentially broad applications for investigating host-pathogen interactions.
Highlights
Cryptococcus neoformans is an opportunistic fungal pathogen of mammals, which causes life-threatening illness in severely immunocompromised hosts
We began with a mouse macrophage-like cell line, J774.16, which has been extensively evaluated for interactions with C. neoformans
Current ways to assess fungal-host cell interactions, such as protocols that rely on lowthroughput imaging techniques and limited reagents [37,61], are not ideal for screens
Summary
Cryptococcus neoformans is an opportunistic fungal pathogen of mammals, which causes life-threatening illness in severely immunocompromised hosts. Cryptococcosis affects close to one million people annually and kills over 600,000 of them, mainly in sub-Saharan Africa [2] This virulence is mediated by multiple factors, but prominent among them is the ability to form an anti-phagocytic polysaccharide capsule [3]. Fungi can persist and replicate in the alveolar spaces, or they may encounter host macrophages and become internalized [4,5,6] These infected macrophages may remain in the lungs or leave the pulmonary system, allowing fungal dissemination. Cryptococcus neoformans causes serious disease in immunocompromised individuals, leading to over 600,000 deaths per year worldwide Part of this impact is due to the organism’s ability to thwart what should be the mammalian hosts’ first line of defense against cryptococcal infection: internalization by macrophages. It is critical to elucidate the interactions of this facultative intracellular pathogen with phagocytic cells of its mammalian host
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