UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Dissecting macrophage responses to Salmonella Typhimurium infection Rosenberger, Carrie Melissa

Abstract

Salmonella Typhimurium infection of murine macrophages provides a robust model for studying host-pathogen interactions at a molecular level. Gene array hybridization studies identified changes in the expression of numerous genes not previously recognized to be involved in macrophage response to infection. An overlapping spectrum of genes was expressed in response to virulent S. Typhimurium and purified S. Typhimurium lipopolysaccharide, reinforcing the major role of this bacterial component in stimulating the early response of macrophages to bacterial infection. The infected macrophage gene expression profile was further altered by priming with interferon-γ, indicating that host cell responses depend on the activation state of the cell. These studies identified upregulated expression of MEK1 kinase in macrophages infected by S. Typhimurium, which correlated with increased MEK1 kinase activity during infection. As this kinase plays a key role in regulating macrophage signal transduction and antimicrobial activities, the functional role of MEK1 in Salmonella-infected macrophages was characterized. Inhibiting MEK kinase activity significantly increased intracellular bacterial numbers. In addition, while macrophages exert stress on intracellular Salmonella and impair bacterial cell division to result in long filamentous bacteria, there was a significant decrease in the number of filamentous Salmonella when MEK1 kinase activity was impaired. This filamentous bacterial morphology was also dependent on the production of reactive oxygen intermediates, which function in parallel to MEK signaling. Experiments were performed to characterize the macrophage effector mechanism(s) responsible for impairing the replication of this intracellular pathogen and the consequent bacterial filamentation. Antimicrobial peptides play an important role in the defense against extracellular infections, but the expression of cationic peptides within macrophages as an antibacterial effector mechanism against intracellular pathogens has not been demonstrated. Macrophages indeed express the antimicrobial peptide CRAMP, and this expression was increased following infection and was dependent on the macrophage's production of reactive oxygen intermediates. Studies using CRAMP-deficient mice or synthetic CRAMP peptide determined that CRAMP impairs Salmonella cell division in vivo and in vitro, resulting in long filamentous bacteria. This impaired bacterial cell division was also dependent on intracellular elastase-like serine protease activity, which can proteolytically activate antimicrobial peptides. A peptide-sensitive Salmonella mutant showed enhanced survival within macrophages derived from CRAMP-deficient mice, indicating that Salmonella can sense and respond to cationic peptides in the intracellular environment. Together, these results show that intracellular ROIs and proteases regulate macrophage CRAMP expression and activity to impair the replication of an intracellular bacterial pathogen. In summary, profiling of macrophage gene expression led to characterization of host signal transduction pathways necessary for impairing bacterial replication and the elucidation of novel antibacterial effector mechanisms. These data begin to address the complexity of interactions between macrophage signaling and effector mechanisms triggered by bacterial infection.

Item Media

Item Citations and Data

License

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

Usage Statistics