Integrating high-content imaging and chemical genetics to probe host cellular pathways critical for Yersinia pestis infection

• Published in: PloS one 2013-01, Vol.8 (1), p.e55167-e55167
• Main Authors: Kota, Krishna P, Eaton, Brett, Lane, Douglas, Ulrich, Melanie, Ulrich, Ricky, Peyser, Brian D, Robinson, Camenzind G, Jaissle, James G, Pegoraro, Gianluca, Bavari, Sina, Panchal, Rekha G
• Format: Article
• Language: English
• Subjects: 1-Phosphatidylinositol 3-kinase; Animals; Antibacterial agents; Automation; Bacteria; Bacterial infections; Biochemical genetics; Biology; Cell activation; Cell Line; Cell survival; Chemistry; Drug Evaluation, Preclinical; E coli; Enzyme inhibitors; Escherichia coli; Genetic research; Genetic screening; Genetics; Health aspects; Infections; Infectious diseases; Inhibitors; Internalization; Kinases; Lipopolysaccharides; Machinery and equipment; Macrophages; Macrophages - cytology; Macrophages - drug effects; Macrophages - metabolism; Macrophages - microbiology; Medical research; Medicine; Mice; Microscopy; Molecular Imaging; Molecular machines; NF-kappa B - metabolism; NF-κB protein; Pathogenesis; Pathogens; Phagocytosis; Phagocytosis - drug effects; Phosphatidylinositol 3-Kinases - metabolism; Protein kinase C; Protein Kinase C - metabolism; Protein kinases; Protein Transport - drug effects; R&D; Research & development; Screening; Signal transduction; Signal Transduction - drug effects; Small Molecule Libraries - pharmacology; Survival; TLR2 protein; TLR4 protein; Toll-like receptors; Wortmannin; Yersinia; Yersinia pestis; Yersinia pestis - drug effects; Yersinia pestis - genetics; Yersinia pestis - physiology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0055167

The molecular machinery that regulates the entry and survival of Yersinia pestis in host macrophages is poorly understood. Here, we report the development of automated high-content imaging assays to quantitate the internalization of virulent Y. pestis CO92 by macrophages and the subsequent activation of host NF-κB. Implementation of these assays in a focused chemical screen identified kinase inhibitors that inhibited both of these processes. Rac-2-ethoxy-3 octadecanamido-1-propylphosphocholine (a protein Kinase C inhibitor), wortmannin (a PI3K inhibitor), and parthenolide (an IκB kinase inhibitor), inhibited pathogen-induced NF-κB activation and reduced bacterial entry and survival within macrophages. Parthenolide inhibited NF-κB activation in response to stimulation with Pam3CSK4 (a TLR2 agonist), E. coli LPS (a TLR4 agonist) or Y. pestis infection, while the PI3K and PKC inhibitors were selective only for Y. pestis infection. Together, our results suggest that phagocytosis is the major stimulus for NF-κB activation in response to Y. pestis infection, and that Y. pestis entry into macrophages may involve the participation of protein kinases such as PI3K and PKC. More importantly, the automated image-based screening platform described here can be applied to the study of other bacteria in general and, in combination with chemical genetic screening, can be used to identify host cell functions facilitating the identification of novel antibacterial therapeutics.