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Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection
Published on: January 8, 2020
Non-apoptotic toxicity of Pseudomonas aeruginosa toward murine cells
Sanhita Roy1, Tracey Bonfield, Alan M Tartakoff
1Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio, USA.
Abstract:
Although P. aeruginosa is especially dangerous in cystic fibrosis (CF), there is no consensus as to how it kills representative cell types that are of key importance in the lung. This study concerns the acute toxicity of the sequenced strain, PAO1, toward a murine macrophage cell line (RAW 264.7). Toxicity requires brief contact with the target cell, but is then delayed for more than 12 h. None of the classical toxic effectors of this organism is required and cell death occurs without phagocytosis or acute perturbation of the actin cytoskeleton. Apoptosis is not required for toxicity toward either RAW 264.7 cells or for alveolar macrophages. Transcriptional profiling shows that encounter between PAO1 and RAW 264.7 cells elicits an early inflammatory response, followed by growth arrest. As an independent strategy to understand the mechanism of toxicity, we selected variant RAW 264.7 cells that resist PAO1. Upon exposure to P. aeruginosa, they are hyper-responsive with regard to classical inflammatory cytokine production and show transient downregulation of transcripts that are required for cell growth. They do not show obvious morphologic changes. Although they do not increase interferon transcripts, when exposed to PAO1 they dramatically upregulate a subset of the responses that are characteristic of exposure to g-interferon, including several guanylate-binding proteins. The present observations provide a novel foundation for learning how to equip cells with resistance to a complex challenge.
Insights
Pseudomonas aeruginosa PAO1 toxicity in macrophages is delayed and does not require classical effectors. Resistant cells show enhanced inflammatory responses and altered growth-related gene expression, offering insights into cellular defense mechanisms.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Pseudomonas aeruginosa (P. aeruginosa) poses a significant threat in cystic fibrosis (CF).
- The precise mechanisms by which P. aeruginosa induces cell death in critical lung cell types remain unclear.
- Understanding P. aeruginosa's cytotoxic effects on macrophages is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the acute toxicity of P. aeruginosa strain PAO1 towards murine macrophage cell line RAW 264.7.
- To elucidate the mechanisms underlying P. aeruginosa-induced cell death, independent of classical toxic effectors.
- To identify cellular responses and genetic alterations associated with resistance to P. aeruginosa.
Main Methods:
- Exposure of RAW 264.7 murine macrophages to P. aeruginosa PAO1.
- Analysis of cell death kinetics and morphology.
- Transcriptional profiling to assess gene expression changes.
- Selection and characterization of P. aeruginosa-resistant RAW 264.7 cell variants.
Main Results:
- P. aeruginosa PAO1-induced toxicity is delayed (>12h) and does not require classical toxic effectors, phagocytosis, or actin cytoskeleton perturbation.
- Cell death occurs independently of apoptosis in both RAW 264.7 cells and alveolar macrophages.
- Transcriptional profiling revealed an early inflammatory response followed by growth arrest in susceptible cells.
- Resistant macrophage variants exhibited hyper-responsiveness to inflammatory cytokines and transient downregulation of growth-related transcripts, with upregulation of interferon-gamma-inducible genes.
Conclusions:
- P. aeruginosa employs a novel mechanism of delayed toxicity against macrophages.
- Cellular resistance involves enhanced inflammatory signaling and specific interferon-gamma-like responses.
- These findings provide a foundation for developing strategies to enhance cellular resistance against P. aeruginosa infection.
