Abstract
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.
Highlights
The bacterial invasion of mammalian cells is often visualized directly by microscopy or quantitated by flow cytometry using fluorescently labeled bacteria [1]
We developed high-throughput automated imaging assays to quantitate bacterial phagocytosis and activation of the host Nuclear Factor-kB (NF-kB) signaling pathway during the infection of macrophages by virulent Yersinia pestis CO92, a Gram-negative facultative pathogen that is the causative agent of plague [16]
RAW264.7 macrophages were infected with the virulent Y. pestis CO92 at multiplicity of infection (MOI) of 30:1
Summary
The bacterial invasion of mammalian cells is often visualized directly by microscopy or quantitated by flow cytometry using fluorescently labeled bacteria [1]. Modulation of host responses to bacterial infection are often studied using global approaches such as microarrays or semi-quantitative low-throughput biochemical methods such as Western blots. Traditional genetic-based methods have been applied to identify bacterial and host cell-specific factors that are required for intracellular pathogenesis. While all these approaches provide significant insight into the biology of disease pathogenesis, they are not amenable for the highthroughput screening of small molecules or siRNAs to identify antibacterial compounds or cellular targets that are involved in host-pathogen interactions. Image-based phenotypic screens have recently been adapted for functional genomic studies and to discover novel antimicrobials for intracellular bacterial pathogens such as Chlamydia, Salmonella and Mycobacteria [12,13,14,15]
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