Cell painting assays for profiling responses in Mycobacterium tuberculosis-infected macrophages after metabolic perturbation

Abstract Tuberculosis (TB) is the leading infectious disease killer in the world, and a lack of accessible, easy treatments contributes to its growing burden. Repurposing approved treatments for other conditions, including metabolic inhibitors under consideration for use in cancer therapy, as host-directed therapies may improve TB treatments. Detailed preliminary studies to identify mechanisms that underly drug function need to be performed before these drugs can be used in patients but there is a lack of tools to investigate metabolic perturbations in target cells. This problem is particularly acute for treatments that have uncharacterized effects on host cells or host-pathogen interactions, or for adherent cells where cell morphology and migration patterns may be factors that are modulated by treatments. Cell painting assays are microscopy-based analyses that use quantitative imaging data to generate insight into how drugs and infections perturb host cells. Here, we sought to optimize a cell painting assay to investigate changes in macrophage morphology and antimicrobial activity after metabolic perturbation and Mycobacterium tuberculosis(Mtb) infection. Our objective was to apply the quantification and analysis pipelines to macrophages that were infected with fluorescent Mtb reporter strains to identify cellular responses after inhibition of fatty acid oxidation, oxidative phosphorylation, or glycolysis. Moreover, we compared inhibitors that target different points in the glycolysis pathway to define how these points differentially affect macrophage antimicrobial activity. Our work provides a framework for using cell painting assays as tools for dissecting host-pathogen interactions in metabolically perturbed cells. Supported by grants from NIH grants AI167710 and #LINK#AI164970#ENDLINK#