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. 1999 Jun;181(11):3433-7.
doi: 10.1128/JB.181.11.3433-3437.1999.

Growth phase-regulated induction of Salmonella-induced macrophage apoptosis correlates with transient expression of SPI-1 genes

U Lundberg  1 , U Vinatzer, D Berdnik, A von Gabain, M Baccarini
Affiliations

VSports - Growth phase-regulated induction of Salmonella-induced macrophage apoptosis correlates with transient expression of SPI-1 genes

U Lundberg et al. J Bacteriol. 1999 Jun.

Abstract

Invasive Salmonella has been reported to induce apoptosis in a fraction of infected macrophages within 2 to 14 h from the time of infection by a mechanism involving the type III secretion machinery encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we show that bacteria in the transition from logarithmic to stationary phase cause 90% of the macrophages to undergo phagocytosis-independent, caspase-mediated apoptosis within 30 to 60 min of infection. The ability of Salmonella to induce this rapid apoptosis was growth phase regulated and cell type restricted, with epithelial cells being resistant. Apoptosis induction was also abrogated by disruption of the hilA gene (encoding a regulator of SPI-1 genes) and by the expression of a constitutively active PhoPQ. hilA itself and a subset of SPI-1 genes were transiently expressed during aerobic growth in liquid medium. Interestingly, however, hilA was found to be required only for the expression of the prgH gene, while sipB, invA, and invF were expressed in a hilA-independent manner. The expression of SPI-1 genes and the secretion of invasion-associated proteins correlated temporally with the induction of apoptosis and are likely to represent its molecular basis VSports手机版. Thus, growth phase transition regulates the expression and secretion of virulence determinants and represents the most efficient environmental cue for apoptosis induction reported to date. .

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Figures (VSports)

FIG. 1
FIG. 1
Effect of bacterial growth phase on Salmonella-induced macrophage apoptosis. BAC-1.2F5 cells were seeded into 96-well plates (50,000 cells/well) and either left uninfected or infected with S. typhimurium LT2 (34) in different growth phases. Macrophages were infected with an activated culture with a constant MOI of 25. The bacteria were spun onto the macrophages to synchronize infection. (A) Thirty minutes after infection, the number of viable cells was determined by crystal violet staining. The percentage of viable cells (uninfected controls = 100%) and the optical density at 600 nm (OD600) of the bacterial culture used to infect the cells are plotted. The assays were carried out in triplicate. The vertical bars represent the standard deviations of the mean. (B) Time course of macrophage death induced by infection with an activated culture. The percentage of dead cells was determined by trypan blue dye exclusion. (C) Salmonella-induced apoptosis is caspase dependent. Macrophages were infected with Salmonella after preincubation (45 min) with different concentrations of a caspase inhibitor (Z-VAD-CH2F). Thirty minutes after infection, the number of viable cells was determined by crystal violet staining.
FIG. 2
FIG. 2
Salmonella induces apoptosis in macrophages but not in epithelial cells. Macrophages (BAC-1.2F5 and J774 cells and bone marrow-derived macrophages [BMM]) or epithelial cells (HeLa, MDCK, or Caco-2 cells) were seeded into 96-well plates (50,000/well) and infected with activated Salmonella cultures in the appropriate growth phase (MOI of 25). Thirty minutes after infection, the number of viable cells was determined by crystal violet staining. The results are expressed as percentage of viable cells (uninfected controls = 100%). The assays were carried out in quadruplicate. The vertical bars represent the standard deviations of the mean.
FIG. 3
FIG. 3
Effects of different Salmonella mutant strains on macrophage viability. BAC-1.2F5 cells were seeded into 96-well plates (50,000/well) and either left uninfected or infected with different mutant strains of Salmonella in the appropriate growth phase. Infection was performed as described in the legend to Fig. 1A. Thirty minutes after infection, the number of viable cells was determined by crystal violet staining. The results are expressed as percentage of viable cells (uninfected controls = 100%). The assays were carried out in quadruplicate. The vertical bars represent the standard deviations of the mean. Wt, wild type.
FIG. 4
FIG. 4
Analysis of invasion gene expression in wild-type (wt), phoPQ(Con) (phoPQc) and hilA::kan Salmonella strains. Wild-type, phoPQ(Con), and hilA-deficient Salmonella strains were grown at 37°C under continuous agitation. At the time points indicated, RNA was extracted and subjected to RT-PCR analysis with the appropriate primers (see Materials and Methods).
FIG. 5
FIG. 5
Secreted-protein profiles of wild-type and mutant Salmonella strains. Trichloroacetic acid-precipitated proteins from supernatants (3 ml) of activated (∗) or overnight (o/n) cultures were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie blue staining for each of the strains indicated. The molecular masses of invasion-associated proteins are indicated next to the arrows. The hilA mutations were in SL1344 background (15). wt, wild type.
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