| Summary: | 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. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate
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