Related Experiment Video
Updated: May 14, 2026
Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions
Published on: October 13, 2015
Integrating high-content imaging and chemical genetics to probe host cellular pathways critical for Yersinia pestis
Krishna P Kota1, Brett Eaton, Douglas Lane
1Perkin Elmer, Waltham, MA, USA.
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.
Insights
We developed automated imaging assays to study Yersinia pestis entry into macrophages. Kinase inhibitors were identified that block bacterial invasion and NF-κB activation, offering new therapeutic targets.
Area of Science:
- Microbiology
- Immunology
- Cell Biology
Background:
- The mechanisms governing Yersinia pestis entry and survival within host macrophages are not well understood.
- Identifying host-pathogen interactions is crucial for developing novel antibacterial therapies.
Purpose of the Study:
- To develop automated high-content imaging assays for quantifying Yersinia pestis internalization and host NF-κB activation.
- To identify host-specific molecular targets and potential therapeutic agents against Yersinia pestis infection.
Main Methods:
- Automated high-content imaging assays were developed to measure Y. pestis CO92 internalization by macrophages.
- Host NF-κB activation was quantified using these imaging assays.
- A focused chemical screen was performed to identify kinase inhibitors affecting bacterial entry and NF-κB signaling.
Main Results:
- Specific kinase inhibitors, including a protein Kinase C (PKC) inhibitor, a PI3K inhibitor (wortmannin), and an IκB kinase inhibitor (parthenolide), were identified.
- These inhibitors reduced Y. pestis entry and survival within macrophages and inhibited pathogen-induced NF-κB activation.
- Parthenolide broadly inhibited NF-κB activation, while PI3K and PKC inhibitors showed selectivity for Y. pestis-induced pathways, suggesting phagocytosis as a key NF-κB stimulus.
Conclusions:
- Phagocytosis is a major driver of NF-κB activation during Y. pestis infection.
- Y. pestis entry into macrophages involves protein kinases like PI3K and PKC.
- The developed automated screening platform is applicable to studying other bacteria and identifying novel antibacterial therapeutics.
