The multi-copper-ion oxidase CueO of Salmonella enterica serovar Typhimurium is required for systemic virulence

Abstract

Salmonella enterica serovar Typhimurium possesses a multi-copper-ion oxidase (multicopper oxidase), CueO (also known as CuiD), a periplasmic enzyme known to be required for resistance to copper ions. CueO from S. Typhimurium was expressed as a recombinant protein in Escherichia coli, and the purified protein exhibited a high cuprous oxidase activity. We have characterized an S. Typhimurium cueO mutant and confirmed that it is more sensitive to copper ions. Using a murine model of infection, it was observed that the cueO mutant was significantly attenuated, as indicated by reduced recovery of bacteria from liver and spleen, although there was no significant difference in recovery from Peyer's patches and mesenteric lymph nodes. However, the intracellular survival of the cueO mutant in unprimed or gamma-interferon-primed murine macrophages was not statistically different from that of wild-type Salmonella, suggesting that additional host factors are involved in clearance of the cueO mutant. Unlike a cueO mutant from E. coli, the S. Typhimurium cueO mutant did not show greater sensitivity to hydrogen peroxide and its sensitivity to copper ions was not affected by siderophores. Similarly, the S. Typhimurium cueO mutant was not rescued from copper ion toxicity by addition of the branched-chain amino acids and leucine.

FIG. 1.

CueO is essential for S. Typhimurium copper resistance in vitro. Copper sensitivity and complementation of the S. Typhimurium SL1344 cueO mutant were tested by spotting serial 10-fold dilutions of cultures of the same density onto LB agar plates containing increasing amounts of copper. Pictures were taken after an overnight incubation at 37°C in the presence (A) or absence (B) of oxygen. The data are representative of results of three independent experiments. WT, SL1344; ΔcueO, SL1344 cueO::kan (cueO mutant); ΔcueO-, SL1344 cueO::kan (pWSK29); ΔcueO*, SL1344 cueO::kan (pCueO) (complemented mutant).

FIG. 2.

The S. Typhimurium cueO mutant is partially attenuated in a mouse model of colonization. Colonization of C57BL/6 mice was used to assess the fitness of the SL1344 cueO mutant in vivo. Data points represent CFU isolated from the spleen (A), liver (B), mesenteric lymph nodes (C), and Peyer's patches (D) of 8-week-old female C57BL/6 mice 5 days postinfection. WT (⧫), SL1344; ΔcueO (Δ), SL1344 cueO::kan (cueO mutant); ΔcueO- (□), SL1344 cueO::kan (pWSK29); ΔcueO* (▪), SL1344 cueO::kan (pCueO) (complemented mutant). Significant differences were analyzed by nonparametric Mann-Whitney U test.

FIG. 3.

The survival of the S. Typhimurium cueO mutant in RAW264.7 macrophages is not affected in vitro. Macrophages were untreated or treated with IFN-γ (1 ng/ml) 18 h before infection. RAW264.7 cells were then infected with SL1344 (black bars), the SL1344 cueO mutant (white bars), or the cueO complemented mutant (gray bars) at a multiplicity of infection (MOI) of 10. At 2, 8, and 24 h postinfection, macrophages were lysed and CFU were enumerated. Data are shown as means ± standard deviations of results of experiments performed in triplicate. The bacterial load in macrophages prestimulated with IFN-γ was significantly lower than that in the unstimulated ones at any of the time points (analysis of variance [ANOVA]; P < 0.001), whereas no differences in the survival of the cueO mutant and that of the wild type or the complemented mutant were observed (t tests; P > 0.05).

FIG. 4.

The S. Typhimurium cueO mutant in a siderophore mutant background is still sensitive to copper. Disk diffusion assays were employed to compare the sensitivities of the SL1344 cueO mutant and the SL1344 cueO entC double mutant to CuSO₄. Five microliters of 1 M CuSO₄ was deposited at the center of each MM9 low-iron glycerol agar plate previously seeded with the indicated bacteria. Pictures were taken after an overnight incubation at 37°C. The data presented are representative of results of three independent experiments. WT, SL1344; ΔentC, SL1344 entC::kan; ΔcueO, SL1344 cueO::kan; ΔcueO ΔentC, SL1344 cueO entC::kan; ΔcueO*, SL1344 cueO::kan (pCueO).

FIG. 5.

The copper sensitivity phenotype of S. Typhimurium is not rescued by the addition of isoleucine, leucine, and valine. SL1344 (diamonds; ⧫, ⋄) and SL1344 cueO mutant (triangles; ▴, Δ) were grown in minimal medium at 37°C with 1.5 mM alanine (Ala) (open symbols; ⋄, Δ) or 0.5 mM (each) isoleucine (I), leucine (L), and valine (V) (closed symbols; ⧫, ▴) and the indicated concentrations of CuSO₄. A₅₀₀ was measured at the end of the exponential phase. The data are representative of results of three independent experiments.