bafilomycin-a1 and Salmonella-Infections--Animal

The two-component regulatory system SsrA-SsrB in Salmonella enterica controls expression of a virulence gene program required for intracellular survival in host cells. SsrA signaling is induced within the acidic host vacuole in which the bacteria reside; however, the mechanism by which SsrA senses this intracellular environment is unknown. Here, we show that the periplasmic sensor domain of SsrA is enriched in histidine residues that increase SsrA signaling below external pH of 6. While no single histidine accounted for the full acid-responsiveness of SsrA, we localized the acid-responsiveness principally to five histidines in the C-terminal end of the periplasmic sensor domain, with input from additional histidines in the N-terminal end of the senor. A sensor mutant lacking critical pH-responsive histidines was defective for acid-promoted activity, yet retained basal activity similar to wild type at neutral pH, indicating that the role of these histidines is to enhance signaling in response to acidification. In support of this, a pH-blind mutant was insensitive to the vacuole acidification blocking activity of bafilomycin, and was attenuated for competitive fitness during infection of mice. Our data demonstrate that SsrA contains a histidine-rich periplasmic sensor that enhances signaling in response to the innate host defense of vacuolar acidification.

Topics: Animals; Bacterial Proteins; Enzyme Inhibitors; Gene Expression Regulation, Bacterial; Genetic Fitness; Histidine; Hydrogen-Ion Concentration; Macrolides; Macrophages; Mice; Mice, Inbred C57BL; Mutagenesis; Periplasm; Protein Kinases; Protein Structure, Tertiary; Salmonella Infections, Animal; Salmonella typhimurium; Signal Transduction; Vacuoles; Virulence

Salmonella species are facultative intracellular pathogens. Following entry into mammalian host cells, they reside in membrane-bound vacuoles, resist killing, and replicate. In this work, we investigated the importance of phagosomal pH in the ability of Salmonella typhimurium to survive and replicate within macrophages. Intraphagosomal pH was measured in situ by recording the fluorescence intensity of a pH-sensitive probe, DM-NERF dextran. The majority of vacuoles containing S. typhimurium (live, heat killed, or formalin fixed) acidified from pH > or = 6.0 to between pH 4.0 and 5.0 within 60 min after formation. In contrast, Mycobacterium avium-containing vacuoles failed to acidify even at later time points. Acidification of S. typhimurium-containing vacuoles was completely blocked by treatment of host cells with bafilomycin A, a specific inhibitor of vacuolar proton-ATPases. Bafilomycin inhibition of vacuolar acidification from the onset of infection significantly decreased the survival of S. typhimurium in macrophages. Furthermore, bafilomycin treatment at 2, 4, 8, or even 12 h postinfection decreased the percentage of recoverable bacteria by up to 20-fold. Loss of bacterial viability was seen with several other reagents which, like bafilomycin, raise the pH of phagosomal compartments but are not directly lethal to the bacteria or host cells. Thus, we conclude that Salmonella-containing phagosomes acidify soon after formation and hypothesize that an acidic environment is necessary for survival and replication of the bacteria within the macrophage.

Topics: Animals; Anti-Bacterial Agents; Cells, Cultured; Dextrans; Enzyme Inhibitors; Fluoresceins; Hydrogen-Ion Concentration; Macrolides; Macrophages; Mice; Phagosomes; Proton-Translocating ATPases; Salmonella Infections, Animal; Salmonella typhimurium; Vacuoles