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Transfer of T6SSSPI-19 from Salmonella Gallinarum to Salmonella Typhimurium Lacking T6SSSPI-6 Complements its Colonization Defect in Mice

David Pezoa
David Pezoa
, 
Carlos J. Blondel
Carlos J. Blondel
, 
Fernando A. Amaya
Fernando A. Amaya
 y 
Carlos A. Santiviago
Carlos A. Santiviago
   | 14 jun 2023
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Article Category: SHORT COMMUNICATION
Publicado en línea: 14 jun 2023
Páginas: 215 - 219
Recibido: 23 ene 2023
Aceptado: 27 mar 2023
DOI: https://doi.org/10.33073/pjm-2023-017
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© 2023 David Pezoa et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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).
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).

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
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