Supplementary data are available at Bioinformatics online.

In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, a morphological profiling assay multiplexing six fluorescent dyes imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multi-well plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Then, automated image analysis software identifies individual cells and measures ~1,500 morphological features (various measures of size, shape, texture, intensity, etc.) to produce a rich profile suitable for detecting subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes two weeks; feature extraction and data analysis take an additional 1-2 weeks.

Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by modulating host pathways. Given the existing experience with some of our identified compounds for other therapeutic indications, further clinically-directed study of these compounds is merited.

No abstract