Figure 1.
TCA cycle and glyoxylate shunt showing intermediate products and genes encoding enzymes within the pathway.
Deleted genes are shown in bold.
Table 1.
Primers used to construct S. Typhimurium gene deletion mutants.
Figure 2.
Increased intracellular replication of the S. Typhimurium Δmdh, ΔsucAB, ΔsucCD and ΔsdhCDAB strains during infection of resting RAW macrophages.
(A) Intracellular replication assays of S. Typhimurium 4/74, ΔgltA (AT3505) and Δmdh (AT3508) strains during infection of resting RAW macrophages. (B) Intracellular replication assays of S. Typhimurium 4/74, ΔsucAB (AT3448), ΔsucCD (AT3449), and ΔsdhCDAB (AT3475) strains during infection of resting RAW macrophages. The data show the number of viable bacteria (expressed as percentages of the initial inocula) within macrophages at 2 h and 18 h post-infection. Each bar represent the statistical mean from three independent biological replicates and the error bars represent the standard deviation (The significant differences between the parental 4/74 strain and the mutant and complemented strains are shown by asterisks p>0.05, * p<0.05, ** p<0.01, and *** p<0.001.).
Figure 3.
Complementation of the S. Typhimurium ΔsucAB strain in resting RAW macrophages and cytotoxicity assays.
(A) Numbers of viable bacteria (expressed as percentages of the initial inoculum) inside the macrophages at 2 h and 18 h after infection. Each bar indicates the statistical mean for three biological replicates, and the error bars indicate the standard deviations. The significant differences between the parental 4/74 strain and the mutant and complemented strains are shown by asterisks p>0.05, * p<0.05, ** p<0.01, and *** p<0.001. (B) Cytotoxicity assays of S. Typhimurium wild-type, ΔsucCD (AT3449) and complemented ΔsucCD (AT???) strains in RAW macrophages after 18 h infection as a percentage of total LDH release from lysed uninfected macrophages. All cytotoxicity data were obtained from three independent biological replicates.
Figure 4.
Increased intracellular replication of the S. Typhimurium ΔsucCD, ΔsdhCDAB, ΔgltA and Δmdh strains during infection of activated RAW macrophages.
(A) Intracellular replication assays of S. Typhimurium 4/74, ΔsucCD (AT3449), ΔaceA (AT3385) and ΔaceAΔsucCD (AT3496) strains during infection of resting RAW macrophages (B) Intracellular replication assay of S. Typhimurium 4/74, ΔsucAB (AT3448), ΔsucCD, (AT3449), ΔsdhCDAB (AT3475), ΔgltA (AT3505) and Δmdh (AT3508) strains during infection of activated RAW macrophages The data show the number of viable bacteria (expressed as percentages of the initial inocula) within activated macrophages at 2 h and 18 h post-infection. Each bar represent the statistical mean from three independent biological replicates and the error bars represent the standard deviation (The significant differences between the parental 4/74 strain and the mutant strains are shown by asterisks p>0.05, * p<0.05, ** p<0.01, and *** p<0.001).
Figure 5.
Decreased intracellular replication of S. Typhimurium ΔsucCD and ΔgltA strains during infection of HeLa epithelial cells.
Intracellular replication assays of S. Typhimurium 4/74, ΔsucCD (AT3449), ΔgltA (AT3505) and Δmdh (AT3508) strains during infection of HeLa cells. The data show the number of viable bacteria (expressed as percentages of the initial inocula) within macrophages at 2 h and 6 h post-infection. Since S. Typhimurium initiates intracellular replication much earlier in HeLa cells (3–4 h) compared to macrophages (∼8 h post-infection) replication was assessed at 6 h post-infection (Hautefort et al., 2008). Each bar represent the statistical mean from three independent biological replicates and the error bars represent the standard deviation (The significant differences between the parental 4/74 strain and the mutant strains are shown by asterisks p>0.05, * p<0.05, ** p<0.01, and *** p<0.001).
Figure 6.
Succinyl-CoA synthetase and α-ketoglutarate synthetase are required for successful infection of mice.
Colony forming units per gram of tissue recovered from the spleens and livers of BALB/c mice following i.p. infection for 72 h with S. Typhimurium 4/74, ΔsucAB (AT3448) and ΔsucCD (AT3449).