To investigate the relevance of zinc in host-pathogen interactions, we have constructedSalmonella entericamutant strains in which theznuAgene, which encodes the periplasmic component of the ZnuABC high-affinity Zn²⁺transporter, was deleted. This mutation does not alter the ability ofSalmonellato grow in rich media but drastically reduces its ability to multiply in media deprived of zinc. In agreement with this phenotype, ZnuA accumulates only in bacteria cultivated in environments poor in zinc. In spite of the nearly millimolar intracellular concentration of zinc, we have found thatznuAis highly expressed in intracellular salmonellae recovered either from cultivated cells or from the spleens of infected mice. We have also observed thatznuAmutants are impaired in their ability to grow in Caco-2 epithelial cells and that bacteria starved for zinc display decreased ability to multiply in phagocytes. A dramatic reduction in the pathogenicity of theznuAmutants was observed inSalmonella-susceptible (BALB/c) orSalmonella-resistant (DBA-2) mice infected intraperitoneally or orally. This study shows that the amount of free metals available for bacterial growth within the infected animal is limited, despite the apparent elevated concentration of free metals within cells and in plasma and suggests thatSalmonellaexploits the ZnuABC zinc transporter to maximize zinc availability in such conditions. These results shed new light on the complex functions of zinc in vertebrate and bacterial physiology and pave the way for a better comprehension of pathogenic mechanisms inSalmonellainfections.

Due to its favourable chemical properties, zinc is used as a structural or catalytic cofactor in a very large number of proteins. Despite the apparent abundance of this metal in all cell types, the intracellular pool of loosely bound zinc ions available for biological exchanges is in the picomolar range and nearly all zinc is tightly bound to proteins. In addition, to limit bacterial growth, some zinc-sequestering proteins are produced by eukaryotic hosts in response to infections. Therefore, to grow and multiply in the infected host, bacterial pathogens must produce high affinity zinc importers, such as the ZnuABC transporter which is present in most Gram-negative bacteria. Studies carried in different bacterial species have established that disruption of ZnuABC is usually associated with a remarkable loss of pathogenicity. The critical involvement of zinc in a plethora of metabolic and virulence pathways and the presence of very low number of zinc importers in most bacterial species mark zinc homeostasis as a very promising target for the development of novel antimicrobial strategies.

The pathways ensuring the efficient uptake of zinc are crucial for the ability of bacteria to multiply in the infected host. To better understand bacterial responses to zinc deficiency, we have investigated the role of the periplasmic protein ZinT in Salmonella enterica serovar Typhimurium. We have found that zinT expression is regulated by Zur and parallels that of ZnuA, the periplasmic component of the zinc transporter ZnuABC. Despite the fact that ZinT contributes to Salmonella growth in media containing little zinc, disruption of zinT does not significantly affect virulence in mice. The role of ZinT became clear using strains expressing a mutated form of ZnuA lacking a characteristic histidine-rich domain. In fact, Salmonella strains producing this modified form of ZnuA exhibited a ZinT-dependent capability to import zinc either in vitro or in infected mice, suggesting that ZinT and the histidine-rich region of ZnuA have redundant function. The hypothesis that ZinT and ZnuA cooperate in the process of zinc recruitment is supported by the observation that they form a stable binary complex in vitro . Although the presence of ZinT is not strictly required to ensure the functionality of the ZnuABC transporter, our data suggest that ZinT facilitates metal acquisition during severe zinc shortage.

Zinc is an essential element for all living cells. Recent studies have shown that the ZnuABC zinc uptake system significantly contributes to the ability of several pathogens to multiply in the infected host and cause disease, suggesting that zinc is scarcely available within different tissues of the host. To better understand the role of zinc in bacterial pathogenicity, we have undertaken a functional characterization of the role of the ZnuABC-mediated zinc uptake pathway in enterohemorrhagic Escherichia coli O157:H7.

Survival of memory B lymphocytes is tightly linked to the integrity of the Bcl-2 protein and is regulated by a nerve growth factor (NGF) autocrine circuit. In factor-starved memory B cells, the addition of exogenous NGF promptly induced p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase (JNK), dephosphorylation. Conversely, withdrawal of endogenous NGF was followed by p38 MAPK activation and translocation onto mitochondria, whereby it combined with and phosphorylated Bcl-2, as assessed by co-immunoprecipitation and kinase assays in vivo and in vitro. Mitochondria isolated from human memory B cells, then exposed to recombinant p38 MAPK, released cytochrome c, as did mitochondria from Bcl-2-negative MDCK cells loaded with recombinant Bcl-2. Apoptosis induced by NGF neutralization could be blocked by the specific p38 MAPK inhibitor SB203580 or by Bcl-2 mutations in Ser-87 or Thr-56. These data demonstrate that the molecular mechanisms underlying the survival factor function of NGF critically rely upon the continuous inactivation of p38 MAPK, a Bcl-2-modifying enzyme.

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.