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Zinc is an essential nutrient for the virulence of bacterial pathogens such as Streptococcus pneumoniae. Many Gram-positive bacteria use a two-domain lipoprotein for zinc acquisition, but how this class of metal-recruiting proteins acquire zinc and interact with the uptake machinery has remained poorly defined., Zinc is an essential element in all domains of life. Nonetheless, how prokaryotes achieve selective acquisition of zinc from the extracellular environment remains poorly understood. Here, we elucidate a novel mechanism for zinc-binding in AdcA, a solute-binding protein of Streptococcus pneumoniae. Crystal structure analyses reveal the two-domain organization of the protein and show that only the N-terminal domain (AdcAN) is necessary for zinc import. Zinc binding induces only minor changes in the global protein conformation of AdcA and stabilizes a highly mobile loop within the AdcAN domain. This loop region, which is conserved in zinc-specific solute-binding proteins, facilitates closure of the AdcAN binding site and is crucial for zinc acquisition. Collectively, these findings elucidate the structural and functional basis of selective zinc uptake in prokaryotes.

The host restricts the availability of zinc to prevent infection. To overcome this defense, Staphylococcus aureus and Pseudomonas aeruginosa rely on zincophore-dependent zinc importers. Synthesis of the zincophore staphylopine by S. aureus and its import are both necessary for the bacterium to cause infection. In this study, we sought to elucidate how loss of zincophore efflux impacts bacterial resistance to host-imposed zinc starvation. In culture and during infection, mutants lacking CntE, the staphylopine efflux pump, were more sensitive to zinc starvation imposed by the metal-binding immune effector calprotectin than those lacking the ability to import staphylopine. However, disruption of staphylopine synthesis reversed the enhanced sensitivity phenotype of the ΔcntE mutant to calprotectin, indicating that intracellular toxicity of staphylopine is more detrimental than the impaired ability to acquire zinc. Unexpectedly, intracellular accumulation of staphylopine does not increase the expression of metal importers or alter cellular metal concentrations, suggesting that, contrary to prevailing models, the toxicity associated with staphylopine is not strictly due to intracellular chelation of metals. As P. aeruginosa and other pathogens produce zincophores with similar chemistry, our observations on the crucial importance of zincophore efflux are likely to be broadly relevant. IMPORTANCEStaphylococcus aureus and many other bacterial pathogens rely on metal-binding small molecules to obtain the essential metal zinc during infection. In this study, we reveal that export of these small molecules is critical for overcoming host-imposed metal starvation during infection and prevents toxicity due to accumulation of the metal-binding molecule within the cell. Surprisingly, we found that intracellular toxicity of the molecule is not due to chelation of cellular metals.

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus. WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PASCYT) domain of the histidine kinase WalK. Introducing the WalKH271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PASCYT domain reveal a metal-binding site, in which a zinc ion (Zn2+) is tetrahedrally-coordinated by four amino acids including H271. The WalKH271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn2+-binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon., WalKR is an essential two-component regulator that controls peptidoglycan synthesis in the human pathogen Staphylococcus aureus. Here, the authors provide biochemical, structural, and functional evidence supporting that the binding of a zinc ion inhibits autophosphorylation and thus alters WalKR regulatory activity.

Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S. pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S. pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S. pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S. pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences., Author summary Zinc deficiency affects one-third of the world’s population and is associated with an increased susceptibility to bacterial infection. Despite this, the molecular basis for how zinc deficiency compromises host control of infection remains to be understood. We show that dietary zinc deficiency impacts host tissue zinc abundances and its mobilization during infection by the major respiratory pathogen Streptococcus pneumoniae. Zinc acts as a direct antimicrobial against the pathogen, mobilized by phagocytic cells as a component of the innate immune response. Although immune activation and infiltration of phagocytic cells is unaffected by host zinc status, the lack of antimicrobial zinc compromises bacterial control in zinc deficient hosts. These findings highlight the importance of zinc sufficiency in resisting bacterial infection.

Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.

Sensing platforms that allow rapid and efficient detection of metal ions would have applications in disease diagnosis and study, as well as environmental sensing. Here, we report the first microstructured optical fiber-based biosensor for the reversible and nanoliter-scale measurement of metal ions. Specifically, a photoswitchable spiropyran Zn2+ sensor is incorporated within the microenvironment of a liposome attached to microstructured optical fibers (exposed-core and suspended-core microstructured optical fibers). Both fiber-based platforms retains high selectivity of ion binding associated with a small molecule sensor, while also allowing nanoliter volume sampling and on/off switching. We have demonstrated that multiple measurements can be made on a single sample without the need to change the sensor. The ability of the new sensing platform to sense Zn2+ in pleural lavage and nasopharynx of mice was compared to that of established ion sensing methodologies such as inductively coupled plasma mass spectrometry (ICP-MS) and a commercially available fluorophore (Fluozin-3), where the optical-fiber-based sensor provides a significant advantage in that it allows the use of nanoliter (nL) sampling when compared to ICP-MS (mL) and FluoZin-3 (mu L). This work paves the way to a generic approach for developing surface-based ion sensors using a range of sensor molecules, which can be attached to a surface without the need for its chemical modification and presents an opportunity for the development of new and highly specific ion sensors for real time sensing applications. Refereed/Peer-reviewed

Cadmium is a transition metal ion that is highly toxic in biological systems. Although relatively rare in the Earth’s crust, anthropogenic release of cadmium since industrialization has increased biogeochemical cycling and the abundance of the ion in the biosphere. Despite this, the molecular basis of its toxicity remains unclear. Here we combine metal-accumulation assays, high-resolution structural data and biochemical analyses to show that cadmium toxicity, in Streptococcus pneumoniae, occurs via perturbation of first row transition metal ion homeostasis. We show that cadmium uptake reduces the millimolar cellular accumulation of manganese and zinc, and thereby increases sensitivity to oxidative stress. Despite this, high cellular concentrations of cadmium (~17 mM) are tolerated, with negligible impact on growth or sensitivity to oxidative stress, when manganese and glutathione are abundant. Collectively, this work provides insight into the molecular basis of cadmium toxicity in prokaryotes, and the connection between cadmium accumulation and oxidative stress., The molecular basis for the high toxicity of cadmium is unclear. Here, Begg et al. use the bacterium Streptococcus pneumoniae as a model system, and show that cadmium uptake increases sensitivity to oxidative stress by reducing intracellular concentrations of manganese and zinc through different mechanisms.

Streptococcus pneumoniae requires manganese for colonization of the human host, but the underlying molecular basis for this requirement has not been elucidated. Recently, it was shown that zinc could compromise manganese uptake and that zinc levels increased during infection by S. pneumoniae in all the niches that it colonized. Here we show, by quantitative means, that extracellular zinc acts in a dose dependent manner to competitively inhibit manganese uptake by S. pneumoniae, with an EC50 of 30.2 µM for zinc in cation-defined media. By exploiting the ability to directly manipulate S. pneumoniae accumulation of manganese, we analyzed the connection between manganese and superoxide dismutase (SodA), a primary source of protection for S. pneumoniae against oxidative stress. We show that manganese starvation led to a decrease in sodA transcription indicating that expression of sodA was regulated through an unknown manganese responsive pathway. Intriguingly, examination of recombinant SodA revealed that the enzyme was potentially a cambialistic superoxide dismutase with an iron/manganese cofactor. SodA was also shown to provide the majority of protection against oxidative stress as a S. pneumoniae ΔsodA mutant strain was found to be hypersensitive to oxidative stress, despite having wild-type manganese levels, indicating that the metal ion alone was not sufficiently protective. Collectively, these results provide a quantitative assessment of the competitive effect of zinc upon manganese uptake and provide a molecular basis for how extracellular zinc exerts a ‘toxic’ effect on bacterial pathogens, such as S. pneumoniae.

Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.