Investigation of the Role of Genes Encoding Zinc Exporters zntA, zitB, and fieF during Salmonella Typhimurium Infection

Abstract

The transition metal zinc is involved in crucial biological processes in all living organisms and is essential for survival of Salmonella in the host. However, little is known about the role of genes encoding zinc efflux transporters during Salmonella infection. In this study, we constructed deletion mutants for genes encoding zinc exporters (zntA, zitB, and fieF) in the wild-type (WT) strain Salmonella enterica serovar Typhimurium (S. Typhimurium) 4/74. The mutants 4/74ΔzntA and 4/74ΔzntA/zitB exhibited a dramatic growth delay and abrogated growth ability, respectively, in Luria Bertani medium supplemented with 0.25 mM ZnCl₂ or 1.5 mM CuSO₄ compared to the WT strain. In order to investigate the role of genes encoding zinc exporters on survival of S. Typhimurium inside cells, amoeba and macrophage infection models were used. No significant differences in uptake or survival were detected for any of the mutants compared to the WT during infection of amoebae. In natural resistance-associated macrophage protein 1 (Nramp1)-negative J774.1 murine macrophages, significantly higher bacterial counts were observed for the mutant strains 4/74ΔzntA and 4/74ΔzntA/zitB compared to the WT at 4 h post-infection although the fold net replication was similar between all the strains. All four tested mutants (4/74ΔzntA, 4/74ΔzitB, 4/74ΔfieF, and 4/74ΔzntA/zitB) showed enhanced intracellular survival capacity within the modified Nramp1-positive murine RAW264.7 macrophages at 20 h post-infection. The fold net replication was also significantly higher for 4/74ΔzntA, 4/74ΔzitB, and 4/74ΔzntA/zitB mutants compared to the WT. Intriguingly, the ability to survive and cause infection was significantly impaired in all the three mutants tested (4/74ΔzntA, 4/74ΔzitB, and 4/74ΔzntA/zitB) in C3H/HeN mice, particularly the double mutant 4/74ΔzntA/zitB was severely attenuated compared to the WT in all the three organs analyzed. These findings suggest that these genes encoding zinc exporters, especially zntA, contribute to the resistance of S. Typhimurium to zinc and copper stresses during infection.

Keywords: S. Typhimurium; amoebae; infection; macrophages; mice; zinc export.

FIGURE 1

Analysis of the growth of the WT and zinc export mutants in: (A) LB. No statistically significant difference was observed between the WT and mutants. (B) LB supplemented with 0.25 mM ZnCl₂. (C) LB supplemented with 1.5 mM CuSO₄. When LB was supplemented with high concentrations of metals (ZnCl₂ or CuSO₄) the OD values for the mutants 4/74ΔzntA and 4/74ΔzntA/zitB were significantly lower than those observed for the WT at all the time points tested except T0 (P-value < 0.05).

FIGURE 2

Infection of amoebae with WT and zinc export mutants. (A) Uptake by amoebae (percentage of inoculated bacteria that are able to infect). (B) Fold net replication at t = 6 versus t = 1 h. No significant differences were observed between the WT and the mutants neither in percentage of uptake nor in fold net replication at 6 h post-infection compared with 1 h post-infection.

FIGURE 3

Infection of Nramp1-negative macrophages (J774.1) with the WT and zinc export mutants. (A) Uptake by macrophages (percentage of inoculated bacteria that are able to infect). (B) Fold net replication at t = 20 versus t = 1 h. (C) Survival curves. No significant differences were observed neither in the uptake by macrophages nor in fold net replications between the WT and mutants. At 4 h post-infection, the CFU counts for the mutants 4/74ΔzntA and 4/74ΔzntA/zitB were significantly higher than those detected for the WT (P-value < 0.05) although the fold net replications at this time point compared to t = 1 h were not significantly different between WT and mutants (not shown).

FIGURE 4

Infection of Nramp1-positive macrophages (RAW264.7) with WT and zinc export mutants. (A) Uptake by macrophages (percentage of inoculated bacteria that are able to infect). No significant differences were observed in the uptake by macrophages between WT and mutants. (B) Fold net replication at t = 20 versus t = 1 h. Fold net replications of 4/74ΔzntA, 4/74ΔzitB, and 4/74ΔzntA/zitB mutants were significantly higher than that of the WT 4/74 (P-value < 0.05). (C) Survival curves. At t = 20 h, all the mutants showed higher significant CFU counts than the WT (P-value < 0.05).