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Assay Development for Image-Based Quantification of Intracellular Bacterial Replication and Analysis of the Innate
Alexandra H Miller1, Sharat J Vayttaden1, Souhaila Al-Khodor2
11 Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health , Bethesda, Maryland.
Abstract:
Severe bacterial infection can lead to inflammation, host tissue damage, and ultimately disseminated septic shock. The mammalian innate immune system responds to microbial infection through the detection of invariant pathogen-associated molecular patterns (PAMPs) by a range of pattern recognition receptors (PRRs) expressed by the host cell. A successful immune response involves tightly coordinated signaling from these receptors, leading to a robust transcriptional response producing cytokines and antimicrobial effectors. While the PRR-expressing phagocytes of the host innate immune system function to contain and degrade internalized bacteria through pathways such as selective autophagy, pathogenic bacteria may subvert this process to replicate in the host cell. We describe the development of imaging assays to investigate these host-pathogen interactions through gene perturbation screens, which could lead to the identification of novel effectors of the host response to bacterial infection. We identify markers of coordinated initial signaling in macrophages challenged with ligands to PRRs of the toll-like receptor (TLR) family and compare this response to that induced by intact bacteria of the Burkholderia cenocepacia complex (Bcc), an opportunistic pathogen that causes life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Bcc has been shown to escape the endocytic pathway, activate selective autophagy, and replicate within human macrophages. We demonstrate robust image-based quantification of multiple stages of Bcc infection of macrophages: ubiquitin tagging of cytosolic bacteria, recruitment of selective autophagy effector proteins, and intracellular bacterial replication, and we show perturbation of bacterial replication using drug treatment or siRNA-based gene knockdown. The described panel of imaging assays can be extended to other bacterial infections and pathogenic ligand combinations where high-content siRNA screening could provide significant new insight into regulation of the innate immune response to infection.
Insights
New imaging assays track host-pathogen interactions during bacterial infections. These tools identify ways to disrupt bacterial replication within host cells, offering insights into innate immune responses.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Severe bacterial infections trigger inflammation and septic shock.
- The innate immune system uses pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs).
- Pathogenic bacteria can evade host defenses, like selective autophagy, to replicate intracellularly.
Purpose of the Study:
- To develop and validate imaging assays for studying host-pathogen interactions.
- To identify host factors and bacterial mechanisms involved in intracellular replication.
- To investigate the innate immune response to Burkholderia cenocepacia complex (Bcc) infection.
Main Methods:
- Development of high-content imaging assays for quantifying host-pathogen interactions.
- Gene perturbation screens using siRNA to identify host effectors.
- Analysis of bacterial ubiquitin tagging, selective autophagy recruitment, and intracellular replication.
- Comparison of responses to toll-like receptor (TLR) ligands and Bcc infection in macrophages.
Main Results:
- Established robust image-based quantification of Bcc infection stages in macrophages.
- Identified markers of coordinated signaling upon PRR activation.
- Demonstrated perturbation of Bcc replication via drug treatment and gene knockdown.
- Showcased the utility of imaging assays for studying host-pathogen dynamics.
Conclusions:
- The developed imaging assays provide a powerful platform for dissecting innate immune responses to bacterial infections.
- These assays can identify novel therapeutic targets to combat intracellular bacterial replication.
- The methodology is adaptable for studying diverse host-pathogen interactions and informing treatment strategies.
