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. 2009 Aug 31;77(11):4750–4760. doi: 10.1128/IAI.00545-09

FIG. 6.

FIG. 6.

Enhanced intracellular replication of Salmonella in TLR-stimulated cells is dependent on functional SPI-2 type 3 secretion. RAW 264.7 cells were treated with PBS, LPS, poly(I:C) (pI:C), PG, or CpG-ODN for 20 h before infection with wild-type S. Typhimurium (control) or a mutant with a defective T3SS, ΔssaR. The intracellular bacterial population was quantified at 2 h postinfection (A), and the relative change in the intracellular bacterial population between 2 to 18 h postinfection was calculated (B). The absolute replication value (ARV) for the control is given above the control bar, and the number of experiments used to produce the data is given above each treatment group. Bars represent the mean, with standard errors normalized to control cells infected with wild-type bacteria. Asterisks denote significant differences from the wild-type control (P < 0.05, one-way analysis of variance). (C) Intracellular transcriptional activity of the sseA virulence operon promoter. RAW 264.7 cells treated with PBS, LPS, or CpG ODN were infected with S. Typhimurium (S. typhim) carrying a chromosomal sseA transcriptional reporter fused to lacZ. Intracellular β-galactosidase activity was measured at 2 h and 10 h postinfection and normalized to the number of intracellular bacteria to facilitate group comparisons. Data are the means with standard errors from triplicate determinations from three separate experiments (P = 0.042 and 0.005 [Mann Whitney test]for LPS and CpG treatments at 2 h compared to the PBS control; P = 0.026 and 0.117 [Mann Whitney test] for LPS and CpG treatments at 10 h compared to the PBS control). (D) The replication advantage of wild-type Salmonella in TLR-primed cells is lost in a genetic mutant with augmented basal activity of SPI-2 transcription. Δhha mutant cells lacking a SPI-2 repressor and with high basal activity of SPI-2 transcription were used to infect TLR-stimulated cells. The replication index was calculated for mutant cells and compared to wild-type cells in which SPI-2 transcription activity is induced inside cells following infection. Data are the means with standard errors from three experiments. The absolute replication value (ARV) for the PBS control is given above the control bars. wt, wild type; cont, control.