Objectives: The control of fungal infections depends on interactions with innate immune cells. Phagocytic cells including macrophages are among the first host cell types to respond to pathogens including Candida albicans, a member of the healthy human microbiome and a major fungal pathogen of immunocompromised individuals. Within a population of interacting macrophages and C. albicans, there are distinct host-pathogen subpopulations reflecting cell specific trajectories and infection outcomes. However, little is known about the molecular mechanisms that control these different fates. We aimed to characterize gene expression patterns that correlate with distinct host and fungal pathogen infection fates.
Methods: We developed an experimental system to isolate the major host-fungal pathogen subpopulations observed during ex vivo infection using fluorescent markers. We separated subpopulations of macrophages infected with live or dead C. albicans from free cells and traced expression variability using RNA-seq and transcriptional analysis of both host and pathogen for each subpopulation and single infected cells across time. 
Results: We show that cell sorting can successfully isolate subpopulations of distinct infection outcomes and examined host and pathogen gene expression in parallel, in sorted subpopulations and in single, infected macrophages. In infected cells, we observed a coordinated, time-dependent shift in gene expression for both host and fungus. The early response in macrophages was established upon exposure to C. albicans prior to engulfment and involved up-regulation of pathways and regulatory genes required for cell migration, pathogen recognition, activation of engulfment, and phagocytosis; this pro-inflammatory response declined during later time points in parallel with expression changes in C. albicans. After phagocytosis, the initial response of C. albicans was to up-regulate genes related to survival in the nutrient-limited and stressful environment within macrophages; at later time points, gene expression shifted to initiate hyphal growth and escape. In addition, we observed that at the single cell level some genes showed bimodal expression levels in both host and fungal pathogen cells that we could not detect in subpopulation samples; we observed that the time shift in expression is asynchronous across the populations and that expression changes in both the host and pathogen in single cells are tightly coupled. 
Conclusion: This work highlights that pairing sorted subpopulation and single-cell RNA-sequencing approaches can trace distinct trajectories during host-fungal pathogen interactions. This system will help us to trace different host-pathogen phenotypes and their distinct expression variability driving drug resistance, host adaptation and immune response.

Revealing parallel host-pathogen transcriptional dynamics using sorted subpopulations and single Candida albicans infected macrophages