NOVEL METHODOLOGY TO IDENTIFY HOST AND PATHOGEN DETERMINANTS OF CANDIDA ALBICANS INFECTION
Chrystal Thomas1, Lasya Sreepada2, Biyu Li2, Dawn Thompson2, Christopher Ford2, Aviv Regev2.
1Carnegie Mellon University, Pittsburgh, PA, 2Broad Institute of MIT and Harvard, Cambridge, MA.
Candida albicans comprises a significant portion of the human microbiome, and when invasive, can cause high rates of mortality and morbidity. The first lines of defense in the immune response to invasive C. albicans are macrophages. C. albicans invasion is associated with a phenotypic “switch” from a commensal yeast form to a virulent filamentous form, making it difficult for macrophages to phagocytose them. Little is known about the host-pathogen interaction between C. albicans and the human immune system, and in particular, the transcriptional networks underlying these interactions. This paucity of knowledge, in part, because it is difficult to study this interaction through conventional genetic and immunological approaches. Therefore, we have developed an imaging and RNA-Seq pipeline. Using live-cell microscopy, we discovered significant heterogeneity in the outcome of infection of single macrophages. In some cases, macrophages successfully phagocytized and neutralized the C. albicans, yet in others, C. albicans would filament and burst the macrophages. We explored novel microscopy, fixation, and RNA isolation methods culminating in a pipeline to isolate macrophages infected with C. albicans, separate host-pathogen pairs based on the outcome of infection into distinct subpopulations, and extract RNA from each subpopulation for downstream RNA sequencing. This work will help to better understand the mechanistic and transcriptional basis of infection outcome, ultimately aiding in the identification of targets for therapeutic intervention.