Salmonella enterica serovar Typhimurium gene sseK3 is required for intracellular proliferation and virulence

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most significant zoonotic pathogens that poses a threat to humans. Previous studies have identified that Salmonella-secreted effector K3 (SseK3) is a novel translated and secreted protein of S. Typhimurium. The objective of this study was to determine whether deletion of the sseK3 gene can attenuate the virulence of S. Typhimurium. To do this, we constructed an sseK3 deletion mutant using the double-exchange allele of the suicide plasmid pRE112ΔsseK3 and assessed the virulence and intracellular proliferation of the mutant. The sseK3 deletion mutant exhibited adhesion and invasion properties similar to those of wild-type (WT) S. Typhimurium, although the virulence and intracellular proliferation of the mutant were significantly reduced compared to that of the WT strain. Furthermore, the observed increase in the median lethal dose (LD₅₀) reflects a decrease in the pathogenicity of the sseK3 deletion mutant in a murine model. In summary, we concluded that disruption of sseK3 can attenuate the intracellular proliferation and reduce the virulence of S. Typhimurium.

Figure 1

Flowchart of major steps in deletion of sseK3.

Figure 2

A — Identification of the ΔsseK3 in-frame mutation by PCR. M — Marker DL2000; 1 — negative control; 2 — double-crossover ΔsseK3 mutant; 3 — single-crossover ΔsseK3 mutant; and 4 — wild-type (WT). B — Verification of pBR322-sseK3 electroporated into SL1344ΔsseK3. M — Marker DL5000; 1 to 3 — SL1344CΔsseK3; and 4 — negative control.

Figure 3

Biofilm formation assay among wild-type (WT), ΔsseK3 mutant, and sseK3-complemented strains. Data for all groups are presented as mean ± SD. Optical density at 570 nm (OD₅₇₀) values of quantitative microtiter plate among WT, ΔsseK3 mutant, and sseK3-complemented strains with crystal violet staining. Statistical significance was calculated using ANOVA (Bonferroni’s multiple-comparison test) (**P < 0.01). The asterisk (*) indicates that the ΔsseK3 mutant differed significantly from the WT and sseK3-complemented strains.

Figure 4

Biofilm morphology among wild-type (WT), ΔsseK3 mutant, and sseK3-complemented strains.

Figure 5

Analysis of intracellular survival and replication of wild-type (WT), ΔsseK3 mutant, and sseK3-complemented strain in macrophages. The WT, ΔsseK3 mutant, and sseK3-complemented strains were coincubated with cells and the number of bacteria was counted at 0.5 h, 1.5 h, and 4 h. Data for all groups are presented as mean ± SD. Statistical significance was calculated using ANOVA (Bonferroni’s multiple-comparison test). The asterisk (*) indicates a statistically significant difference among the WT, ΔsseK3, and complemented strains at 4 h compared with 1.5 h (*P < 0.05).

Figure 6

Percent survival of mice infected with wild-type (WT) (A) or ΔsseK3 mutant strain (B). The mice were inoculated by intraperitoneal injection and mortality was monitored over 5 wk (n = 12/group). Kaplan-Meier survival curves of mice infected with WT or ΔsseK3 mutant strain. Data were analyzed using the log-rank test. P-values for 1 × 10⁶, 1 × 10⁷, and × 10⁸ were **P < 0.01 when comparing the WT to the ΔsseK3 mutant.