The ZupT transporter plays an important role in zinc homeostasis and contributes to Salmonella enterica virulence

Abstract

Zinc is an essential metal for cellular homeostasis and function in both eukaryotes and prokaryotes. To acquire this essential nutrient, bacteria employ transporters characterized by different affinity for the metal. Several studies have investigated the role of the high affinity transporter ZnuABC in the bacterial response to zinc shortage, showing that this transporter has a key role in adapting bacteria to zinc starvation. In contrast, the role of the low affinity zinc importer ZupT has been the subject of limited investigations. Here we show that a Salmonella strain lacking ZupT is impaired in its ability to grow in metal devoid environments and that a znuABC zupT strain exhibits a severe growth defect in zinc devoid media, is hypersensitive to oxidative stress and contains reduced levels of intracellular free zinc. Moreover, we show that ZupT also plays a role in the ability of S. Typhimurium to colonize the host tissues. During systemic infections, the single zupT mutant strain was attenuated only in Nramp1(+/+) mice, but competition experiments between znuABC and znuABC zupT mutants revealed that ZupT contributes to metal uptake in vivo independently of the presence of a functional Nramp1 transporter. Altogether, the here reported results show that ZupT plays an important role in Salmonella zinc homeostasis, being involved in metal import both in vitro and in infected animals.

Fig. 1

Transcriptional analysis of S. Typhimurium zupT promoter in bacteria grown overnight in VB-MM or in LB, in presence or absence of 0.01mM ZnSO₄ (VB-MM) or 1mM (LB) ZnSO₄ or 1 mM EDTA or both. Black and grey bars represent the wild type strain (PP150) and znuABC strain (PP152) respectively. Each experiment was repeated at least twice and samples were always assayed in triplicate. P values: * p < 0,05; ** p < 0,01

Fig. 2

Growth of S. Typhimurium on LB-agarose plates. Plates were prepared with standard LB or with LB supplemtend with zinc, iron or manganese.

Fig. 3

Growth curves of S. Typhimurium wild type (■), znuABC (▲), zupT (▼), znuABCzupT (◆). Strains were grown in VB-MM alone (panel A) and VB-MM supplemented with 3 μM zinc (panel B). OD₅₉₅, optical density at 595 nm.

Fig. 4

ZinT::3xFLAG accumulation in the znuA deleted strain (PP137, upper panel) and in znuABC zupT deleted strain (MC113, intermediate panel) grown in VB-MM alone or supplemented with ZnSO₄ as indicated. The lower panel shows the accumulation of CAT-3xFLAG in the control strain SA212

Fig. 5

Zinc import and hydrogen peroxide resistance. S. Typhimurium wild-type, SA186 (znuABC), SA321 (zupT) and SA327 (znuABC zupT) were grown in M9-MM with (gray bars) or without (black bars) the addition of 0.01mM ZnSO₄ and exposed to H₂O₂ for one hour. Samples were assayed in triplicate and the graph shows the average of survival for each strain (percent respect to H₂O₂ unexposed bacteria). P values: ** p < 0,01

Fig. 6

Competition assay in intraperitoneally infected mice. Bacteria were recovered from the spleens of Nramp^+/+ (DBA-2, panel A) and Nramp^−/− (Balb/C, panelB) mice infected with mixed inocula (wild type vs zupT and znuABC vs. znuABCzupT, see legend). P values: * p < 0,05

Fig. 7

Competition assay in orally infected mice. Competitive index in cecal contents (panel A) and in the spleens (panel B) of C57BL6 mice infected with mixed inocula (wild type vs. znuA; wild type vs. zupT; wild type vs. znuA zupT; znuA vs. znuA zupT; see legend). P values: * p < 0,05; ** p < 0,01