Tracing Conidial Fate and Measuring Host Cell Antifungal Activity Using a Reporter of Microbial Viability in the Lung

Fluorescence can be harnessed to monitor microbial fate and to investigate functional outcomes of individual microbial cell-host cell encounters at portals of entry in native tissue environments. We illustrate this concept by introducing fluorescent Aspergillus reporter (FLARE) conidia that simultaneously report phagocytic uptake and fungal viability during cellular interactions with the murine respiratory innate immune system. Our studies using FLARE conidia reveal stepwise and cell-type-specific requirements for CARD9 and Syk, transducers of C-type lectin receptor and integrin signals, in neutrophil recruitment, conidial uptake, and conidial killing in the lung. By achieving single-event resolution in defined leukocyte populations, the FLARE method enables host cell profiling on the basis of pathogen uptake and killing and may be extended to other pathogens in diverse model host organisms to query molecular, cellular, and pharmacologic mechanisms that shape host-microbe interactions. [Display omitted] ► Construction of fluorescent fungal spores that report on uptake and viability ► Visualization of microbial fate in situ ► Analysis of individual leukocyte-fungal cell interactions in the lung ► Description of fungicidal roles for CARD9 and Syk in neutrophils Coupling fluorescence to microbial viability can be harnessed to trace the fate and cellular associations of fungal conidia within the mammalian lung. Hohl and colleagues develop a fluorescent Aspergillus fumigatus reporter that illustrates this principle and permits leukocyte analysis on the basis of fungal uptake and killing. The approach is used to develop a stepwise model of neutrophil antifungal activity, with cell-type-specific and sequential roles for CARD9 and spleen tyrosine kinase (Syk) in defense against inhaled mold conidia.