Macrophages Inhibit Salmonella Typhimurium Replication through MEK/ERK Kinase and Phagocyte NADPH Oxidase Activities

Abstract

Host responses during the later stages of Salmonellamacrophage interactions are critical to controlling infection but have not been well characterized. After 24 h of infection, nearly half of interferon-␥-primed murine RAW 264.7 macrophage-like cells infected by Salmonella enterica serovar Typhimurium contained filamentous bacteria. Bacterial filamentation indicates a defect in completing replication and has been previously observed in bacteria responding to a variety of stresses. To understand whether macrophage gene expression was responsible for this effect on Salmonella Typhimurium replication, we used gene arrays to profile interferon-␥-primed RAW 264.7 cell gene expression following infection. We observed an increase in MEK1 kinase mRNA at 8 h, an increase in MEK protein at 24 h, and measured phosphorylation of MEK's downstream target kinase, ERK1/2, throughout the 24-h infection period. Treatment of cells with MEK kinase inhibitors significantly reduced numbers of filamentous bacteria observed within macrophages after 24 h and increased the number of intracellular colony-forming units. Phagocyte NADPH oxidase inhibitors and antioxidants also significantly reduced bacterial filamentation. Either MEK kinase or phagocyte oxidase inhibitors could be added 4 -8 h after infection and still significantly decrease bacterial filamentation. Oxidase activity appears to mediate bacterial filamentation in parallel to MEK kinase signaling, while inducible nitric-oxide synthase inhibitors had no significant effect on bacterial morphology. In summary, Salmonella Typhimurium infection of interferon-␥-primed macrophages triggers a MEK kinase cascade at later infection times, and both MEK kinase and phagocyte NADPH oxidase activity impair bacterial replication. These two signaling pathways mediate a host bacteriostatic pathway and may play an important role in innate host defense against intracellular pathogens.

Interplay between antibacterial effectors: A macrophage antimicrobial peptide impairs intracellular Salmonella replication

“…1 and data not shown). We described previously that, by using RAW 264.7 murine macro- phage-like cells, macrophage impairment of bacterial cell division is ROI-dependent and reactive nitrogen intermediate-independent (6). This phenotype was observed also in BMDM ( Fig.…”

“…This pathogen counters macrophage antibacterial effectors with acid tolerance and perturbation of endosomal trafficking, to avoid oxidative and nitrosative damage and phagolysosomal degradation (4,5). We have observed that macrophages impair cell division of intracellular S. typhimurium, resulting in the formation of filamentous bacteria with arrested septation (6). This morphology indicates a bacterial stress response and has been observed in bacteria responding to damage from low doses of antibiotics, starvation, and reactive oxygen and nitrogen species (7)(8)(9).…”

“…For immunof luorescence and colonyforming unit (cfu) experiments, BMDM were seeded in 24-well plates (1 ϫ 10 5 cells per well) and infected with S. typhimurium for 20 min, as described (6). Monolayers were washed with PBS, incubated in media containing 100 g͞ml gentamicin (Sigma) for 1.5 h, and maintained with 10 g͞ml gentamicin.…”

“…The Salmonella enterica serovar Typhimurium strains SL1344 and 14028s (Salmonella Genetic Stock Center, Calgary, AB, Canada), 14028s Pho24 PhoP constitutive mutant (F. Heffron, Oregon Health and Sciences University, Portland), 14028s phoP::Tn10 MS7953s PhoP null mutant (S. Miller, University of Washington, Seattle), SL1344 phoP::Tn10 (L. Knodler, Rocky Mountain Laboratories, Hamilton, MT), SL1344 ⌬aroA (B. Stocker, Stanford University, Stanford, CA), and strains expressing pFPV25.1-GFP (S. Meresse, Marseille, France, and S. Falkow, Stanford University) were cultured as described (6). Strains in a SL1344 background were used unless indicated otherwise in the text.…”

Stimulation of Toll-Like Receptor 4 by Lipopolysaccharide During Cellular Invasion by Live Salmonella typhimurium Is a Critical But Not Exclusive Event Leading to Macrophage Responses

“…TTSS effectors are clearly critical in modifying host cell responses, particularly in epithelial cells that lack phagocytic machinery and have a limited capacity for producing inflammatory mediators such as cytokines (2). They have also been described as modulating MAPK function and cellular replication (23). Host macrophage recognition of, and response to, infection therefore involve the complex interaction of bacterial TTSS effectors and host PAMP receptor protein stimulation.…”