Fig. 1.
In vivo competition between wild-type Salmonella Typhimurium and a ΔT6SSSPI-6 mutant complemented in trans with R995, R995 + SPI6 or R995 + SPI-19.
A) Genetic organization of Salmonella Typhimurium T6SSSPI-6 and Salmonella Gallinarum T6SSSPI-19 gene clusters. Genes encoding core components of each T6SS are shown in yellow, and the effector and immunity protein-encoding genes are shown in red and blue, respectively. B) For competition assays, groups of six- to eight-week-old female BALB/c mice were orally infected with 106 CFU of a 1 : 1 mixture of WT/R995 with ΔT6SSSPI-6/R995 (blue), ΔT6SSSPI-6/R995+SPI-6 (red) or ΔT6SSSPI-6/R995+SPI-19 (green). After four days of infection, mice were euthanized, and the cecum, liver and spleen were aseptically removed and homogenized in sterile PBS. The bacterial load recovered from each organ was determined by plating serial tenfold dilutions on LB agar plates with appropriate antibiotics. Data show the mutant to wild-type CFU ratio normalized to the inoculum and expressed as log10. Bars represent mean values ± standard error. Statistical significance was determined using a one-way ANOVA test followed by Tukey’s multiple comparisons tests (*p < 0.05; ****p < 0.0001; ns – not significant).
Table I
Bacterial strains and plasmids used in this study.
| Strain or plasmid | Features | Source or reference |
|---|---|---|
| Salmonella Typhimurium | ||
| 14028s | Wild-type, virulent strain | Laboratory collection |
| ΔT6SSSPI-6 | 14028s Δ(STM14_0312-STM14_0351)::Cam | Pezoa et al. 2013 |
| ΔphoN | 14028s Δ(STM14_5193)::Kan | Pezoa et al. 2013 |
| ΔphoN/R995 | 14028s Δ(STM14_5193)::Kan harboring plasmid R995 | Pezoa et al. 2013 |
| ΔT6SSSPI-6/R995 | 14028s Δ(STM14_0312-STM14_0351)::Cam harboring plasmid R995 | Pezoa et al. 2013 |
| ΔT6SSSpI-6/R995+SPI-6 | 14028s Δ(STM14_0312-STM14_0351)::Cam harboring plasmid R995+SPI-6 | Pezoa et al. 2013 |
| ΔT6SSSpI-6/R995+SPI-19 | 14028s Δ(STM14_0312-STM14_0351)::Cam harboring plasmid R995+SPI-19 | Pezoa et al. 2013 |
| Plasmids | ||
| R995 | Self-transmissible, broad-host range IncP plasmid | Wilson et al. 2004 |
| R995+SPI-6 | T6SSSPI-6 gene cluster (STM14_0312-STM14_0351) from Salmonella Typhimurium 14028s cloned in plasmid R995 | Pezoa et al. 2013 |
| R995+SPI-19 | T6SSSPI-19 gene cluster (SG1021-SG1056) from Salmonella Gallinarum 287/91 cloned in plasmid R995 | Blondel et al. 2010 |