An incomplete TCA cycle increases survival of Salmonella Typhimurium during infection of resting and activated murine macrophages

Abstract

Background: In comparison to the comprehensive analyses performed on virulence gene expression, regulation and action, the intracellular metabolism of Salmonella during infection is a relatively under-studied area. We investigated the role of the tricarboxylic acid (TCA) cycle in the intracellular replication of Salmonella Typhimurium in resting and activated macrophages, epithelial cells, and during infection of mice.

Methodology/principal findings: We constructed deletion mutations of 5 TCA cycle genes in S. Typhimurium including gltA, mdh, sdhCDAB, sucAB, and sucCD. We found that the mutants exhibited increased net intracellular replication in resting and activated murine macrophages compared to the wild-type. In contrast, an epithelial cell infection model showed that the S. Typhimurium ΔsucCD and ΔgltA strains had reduced net intracellular replication compared to the wild-type. The glyoxylate shunt was not responsible for the net increased replication of the TCA cycle mutants within resting macrophages. We also confirmed that, in a murine infection model, the S. Typhimurium ΔsucAB and ΔsucCD strains are attenuated for virulence.

Conclusions/significance: Our results suggest that disruption of the TCA cycle increases the ability of S. Typhimurium to survive within resting and activated murine macrophages. In contrast, epithelial cells are non-phagocytic cells and unlike macrophages cannot mount an oxidative and nitrosative defence response against pathogens; our results show that in HeLa cells the S. Typhimurium TCA cycle mutant strains show reduced or no change in intracellular levels compared to the wild-type. The attenuation of the S. Typhimurium ΔsucAB and ΔsucCD mutants in mice, compared to their increased net intracellular replication in resting and activated macrophages suggest that Salmonella may encounter environments within the host where a complete TCA cycle is advantageous.

Figure 1. TCA cycle and glyoxylate shunt showing intermediate products and genes encoding enzymes within the pathway.

Deleted genes are shown in bold.

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).