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Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes
C M Alpuche Aranda1, J A Swanson, W P Loomis
1Infectious Disease Unit, Massachusetts General Hospital, Boston 02114.
Abstract:
Survival of Salmonella typhimurium within macrophage phagosomes requires the coordinate expression of bacterial gene products. This report examines the contribution of phagosomal pH as a signal for expression of genes positively regulated by the S. typhimurium virulence regulators PhoP and PhoQ. Several hours after bacterial phagocytosis by murine bone marrow-derived macrophages, PhoP-activated gene transcription increased 50- to 77-fold. In contrast, no difference in PhoP-activated gene expression was observed after infection of cultured epithelial cells, suggesting that the membrane sensor PhoQ recognized signals unique to macrophage phagosomes. The increase in PhoP-regulated gene expression was abolished when macrophage culture medium contained NH4Cl or chloroquine, weak bases that raise the pH of acidic compartments. Measurements of pH documented that S. typhimurium delayed and attenuated acidification of its intracellular compartment. Phagosomes containing S. typhimurium required 4-5 hr to reach pH < 5.0. In contrast, within 1 hr vacuoles containing heat-killed bacteria were measured at pH < 4.5. The eventual acidification of phagosomes to pH < 5.0 correlated with the period of maximal PhoP-dependent gene expression. These observations implicate phagosome acidification as an intracellular inducer of PhoP-regulated gene expression and suggest that Salmonella survival is dependent on its ability to attenuate phagosome acidification.
Insights
Salmonella typhimurium survival in macrophages depends on controlling phagosome acidity. Delayed acidification activates virulence genes, suggesting this bacterial mechanism is key for intracellular survival.
Area of Science:
- Microbiology
- Cell Biology
- Bacterial Pathogenesis
Background:
- Salmonella typhimurium survival within host macrophages is crucial for infection.
- Virulence gene expression is tightly regulated by bacterial sensors like PhoP and PhoQ.
- The intracellular environment of macrophages, particularly phagosomal pH, is a potential signaling cue.
Purpose of the Study:
- To investigate the role of phagosomal pH in regulating Salmonella typhimurium virulence gene expression.
- To determine if pH changes within macrophage phagosomes signal for PhoP- and PhoQ-mediated gene activation.
- To understand how Salmonella manipulates its phagosomal environment for survival.
Main Methods:
- Phagocytosis of Salmonella typhimurium by murine bone marrow-derived macrophages.
- Measurement of PhoP-activated gene transcription levels.
- Manipulation of macrophage phagosome pH using weak bases (NH4Cl, chloroquine).
- Direct measurement of phagosomal pH over time.
Main Results:
- PhoP-activated gene transcription increased significantly (50-77 fold) in macrophages but not epithelial cells.
- Treatment with weak bases abolished the increase in PhoP-regulated gene expression.
- Salmonella typhimurium delayed and attenuated the acidification of its phagosome, reaching pH < 5.0 after 4-5 hours.
- In contrast, phagosomes with heat-killed bacteria acidified rapidly (within 1 hour to pH < 4.5).
- Maximal PhoP-dependent gene expression correlated with the eventual acidification of the phagosome.
Conclusions:
- Phagosome acidification acts as an intracellular inducer for PhoP-regulated virulence gene expression in Salmonella.
- Salmonella typhimurium's ability to delay and attenuate phagosome acidification is essential for its survival within macrophages.
- The PhoQ sensor likely detects macrophage-specific signals related to phagosomal pH.