Assay development for image-based quantification of intracellular bacterial replication and analysis of the innate immune response to infection

Alexandra H. Miller, Sharat J. Vayttaden, Souhaila M. Al Khodor, Iain D C Fraser

Research output: Contribution to journalArticle

3 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)515-528
Number of pages14
JournalAssay and Drug Development Technologies
Volume13
Issue number9
DOIs
Publication statusPublished - 1 Nov 2015

Fingerprint

Burkholderia cenocepacia
Pattern Recognition Receptors
Innate Immunity
Autophagy
Bacterial Infections
Bacteria
Macrophages
Infection
Small Interfering RNA
Immune System
Ligands
Gene Knockdown Techniques
Host-Pathogen Interactions
Chronic Granulomatous Disease
Bacterial Proteins
Toll-Like Receptors
Phagocytes
Septic Shock
Ubiquitin
Cystic Fibrosis

ASJC Scopus subject areas

  • Medicine(all)
  • Molecular Medicine
  • Drug Discovery

Cite this

Assay development for image-based quantification of intracellular bacterial replication and analysis of the innate immune response to infection. / Miller, Alexandra H.; Vayttaden, Sharat J.; Al Khodor, Souhaila M.; Fraser, Iain D C.

In: Assay and Drug Development Technologies, Vol. 13, No. 9, 01.11.2015, p. 515-528.

Research output: Contribution to journalArticle

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