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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 4/74Δ zntA  and 4/74Δ zntA/zitB  exhibited a dramatic growth delay and abrogated growth ability, respectively, in Luria Bertani medium supplemented with 0.25 mM ZnCl 2  or 1.5 mM CuSO 4  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 4/74Δ zntA  and 4/74Δ zntA/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 (4/74Δ zntA , 4/74Δ zitB , 4/74Δ fieF , and 4/74Δ zntA/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 4/74Δ zntA , 4/74Δ zitB , and 4/74Δ zntA/zitB  mutants compared to the WT. Intriguingly, the ability to survive and cause infection was significantly impaired in all the three mutants tested (4/74Δ zntA , 4/74Δ zitB , and 4/74Δ zntA/zitB ) in C3H/HeN mice, particularly the double mutant 4/74Δ zntA/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.

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