Contribution of the Twin-Arginine Translocation System to the Intracellular Survival of Salmonella Typhimurium in Dictyostelium discoideum

Abstract

The twin-arginine translocation (Tat) system is a specialized secretion pathway required for bacteria to export fully folded proteins through the cytoplasmic membrane. This system is crucial during Salmonella infection of animal hosts. In this study, we show that Salmonella enterica serovar Typhimurium (S. Typhimurium) requires the Tat system to survive and proliferate intracellularly in the social amoeba Dictyostelium discoideum. To achieve this, we developed a new infection assay to assess intracellular bacterial loads in amoeba by direct enumeration of colony forming units (CFU) at different times of infection. Using this assay we observed that a ΔtatABC mutant was internalized in higher numbers than the wild type, and was defective for intracellular survival in the amoeba at all times post infection evaluated. In addition, we assessed the effect of the ΔtatABC mutant in the social development of D. discoideum. In contrast to the wild-type strain, we observed that the mutant was unable to delay the social development of the amoeba at 2 days of co-incubation. This phenotype correlated with defects in intracellular proliferation presented by the ΔtatABC mutant in D. discoideum after 24 h of infection. All phenotypes described for the mutant were reverted by the presence of a plasmid carrying tatABC genes, indicating that abrogation of Tat system attenuates S. Typhimurium in this model organism. Overall, our results indicate that the Tat system is crucial for S. Typhimurium to survive and proliferate intracellularly in D. discoideum and for virulence in this host. To the best of our knowledge, this is the first report on the relevance of the Tat system in the interaction of any bacterial pathogen with the social amoeba D. discoideum.

Keywords: Dictyostelium; Salmonella; Tat system; infection; intracellular survival; social development; virulence.

FIGURE 1

Adherence and internalization of S. Typhimurium strains in D. discoideum. Individual infection assays were conducted to evaluate the adherence and internalization of different S. Typhimurium strains in D. discoideum AX4. (A) Relative internalization after 1 h of infection at 22°C. (B) Relative adherence after 30 min of co-incubation at 4°C. All values (internalization and adherence) were calculated as CFU_{t =0}/CFU_inoculum and further normalized to the value of the wild-type strain. Graphs show mean values ± SD from at least 7 independent assays. Statistical significance of differences in internalization or adherence between each strain and the wild type was determined using a one-way ANOVA with Dunnett’s test (^∗∗P < 0.01; ^∗∗∗P < 0.001).

FIGURE 2

Qualitative evaluation of cytotoxicity caused by S. Typhimurium strains on D. discoideum. The shape of D. discoideum AX4 cells infected with different strains of S. Typhimurium was monitored at 0, 30, and 60 min of co-incubation at 22°C in Soerensen buffer. Representative images from 3 independent assays carried out at MOIs of ∼100 or ∼1,000 bacteria/amoeba are shown. Scale bar, 20 μm.

FIGURE 3

Intracellular survival of S. Typhimurium strains in D. discoideum. Individual infection assays were conducted to evaluate the intracellular survival of different S. Typhimurium strains in D. discoideum AX4. (A) Relative intracellular survival at different times post infection. All values were calculated as CFU_{t =x}/CFU_{t =0} and further normalized to the value of the wild-type strain at t = 1. (B) Variation in the population of viable amoeba during the infection assay. All values were calculated as cells/mL and further normalized to the value of amoeba infected with the wild-type strain at t = 0. Graphs show mean values ± SD from at least 7 independent assays. Statistical significance of differences in intracellular survival between each strain and the wild type at a given time was determined using a one-way ANOVA with Dunnett’s test (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001). Statistical significance of differences in intracellular survival of a given strain at t = 1 versus t = 24 was determined using a one-way ANOVA with Dunnett’s test (^#P < 0.05; ^###P < 0.001).

FIGURE 4

Social development of D. discoideum co-incubated with S. Typhimurium 14028s derivatives and E. coli B/r. (A) Main phases of the D. discoideum development cycle, including aggregation, elevation and culmination to generate fruiting bodies (scheme adapted from Fey et al., 2007). (B) D. discoideum development after 1 and 2 days of co-incubation with S. Typhimurium strains or E. coli B/r. Representative images from 3 independent assays are shown. Scale bar, 100 μm.