Human Tim8a, Tim8b and Tim13 are auxiliary assembly factors of mature Complex IV.

Human Tim8a and Tim8b are paralogous intermembrane space proteins of the small TIM chaperone family. Yeast small TIMs function in the trafficking of proteins to the outer and inner mitochondrial membranes. This putative import function for hTim8a and hTim8b has been challenged in human models, but their precise molecular function(s) remains undefined. Likewise, the necessity for human cells to encode two Tim8 proteins and whether any potential redundancy exists is unclear. We demonstrate that hTim8a and hTim8b function in the assembly of cytochrome c oxidase (Complex IV). Using affinity enrichment mass spectrometry, we define the interaction network of hTim8a, hTim8b and hTim13, identifying subunits and assembly factors of the Complex IV COX2 module. hTim8-deficient cells have a COX2 and COX3 module defect and exhibit an accumulation of the Complex IV S2 subcomplex. These data suggest that hTim8a and hTim8b function in assembly of Complex IV via interactions with intermediate-assembly subcomplexes. We propose that hTim8-hTim13 complexes are auxiliary assembly factors involved in the formation of the Complex IV S3 subcomplex during assembly of mature Complex IV.

Maternal selenium dietary supplementation alters sociability and reinforcement learning deficits induced by in utero exposure to maternal immune activation in mice.

Maternal immune activation (MIA) during pregnancy increases the risk for the unborn foetus to develop neurodevelopmental conditions such as autism spectrum disorder and schizophrenia later in life. MIA mouse models recapitulate behavioural and biological phenotypes relevant to both conditions, and are valuable models to test novel treatment approaches. Selenium (Se) has potent anti-inflammatory properties suggesting it may be an effective prophylactic treatment against MIA. The aim of this study was to determine if Se supplementation during pregnancy can prevent adverse effects of MIA on offspring brain and behaviour in a mouse model. Selenium was administered via drinking water (1.5 ppm) to pregnant dams from gestational day (GD) 9 to birth, and MIA was induced at GD17 using polyinosinic:polycytidylic acid (poly-I:C, 20 mg/kg via intraperitoneal injection). Foetal placenta and brain cytokine levels were assessed using a Luminex assay and brain elemental nutrients assessed using inductively coupled plasma- mass spectrometry. Adult offspring were behaviourally assessed using a reinforcement learning paradigm, the three-chamber sociability test and the open field test. MIA elevated placental IL-1ß and IL-17, and Se supplementation successfully prevented this elevation. MIA caused an increase in foetal brain calcium, which was prevented by Se supplement. MIA caused in offspring a female-specific reduction in sociability, which was recovered by Se, and a male-specific reduction in social memory, which was not recovered by Se. Exposure to poly-I:C or selenium, but not both, reduced performance in the reinforcement learning task. Computational modelling indicated that this was predominantly due to increased exploratory behaviour, rather than reduced rate of learning the location of the food reward. This study demonstrates that while Se may be beneficial in ameliorating sociability deficits caused by MIA, it may have negative effects in other behavioural domains. Caution in the use of Se supplementation during pregnancy is therefore warranted.

Spatial distribution of trace metals and associated transport proteins during bacterial infection.

Innate immune systems alter the concentrations of trace elements in host niches in response to invading pathogens during infection. This work reports the interplay between d-block metal ions and their associated biomolecules using hyphenated elemental techniques to spatially quantify both elemental distributions and the abundance of specific transport proteins. Here, lung tissues were collected for analyses from naïve and Streptococcus pneumoniae-infected mice fed on a zinc-restricted or zinc-supplemented diet. Spatiotemporal distributions of manganese (55Mn), iron (56Fe), copper (63Cu), and zinc (66Zn) were determined by quantitative laser ablation-inductively coupled plasma-mass spectrometry. The murine transport proteins ZIP8 and ZIP14, which are associated with zinc transport, were also imaged by incorporation of immunohistochemistry techniques into the analytical workflow. Collectively, this work demonstrates the potential of a single instrumental platform suitable for multiplex analyses of tissues and labelled antibodies to investigate complex elemental interactions at the host-pathogen interface. Further, these methods have the potential for broad application to investigations of biological pathways where concomitant measurement of elements and biomolecules is crucial to understand the basis of disease and aid in development of new therapeutic approaches.

Copper(II) Complexes of 2,2'-Bisdipyrrins: Synthesis, Characterization, Cell Uptake, and Radiolabeling with Copper-64.

Complexes prepared with positron-emitting copper-64 are of interest as imaging agents for positron emission tomography (PET). This work investigates the potential of using acyclic tetrapyrrolic 2,2'-bisdipyrrins as ligands to prepare charge-neutral, lipophilic, cell-permeable, redox active complexes with positron-emitting copper-64. The synthesis and characterization of a series of tetrapyrrolic 2,2'-bisdipyrrin copper(II) complexes are reported. Four 2,2'-bisdipyrrin copper(II) complexes were prepared with different functional groups in the meso-position of the ligands. Two of the new copper(II) complexes, one palladium(II) complex, and one nickel(II) complex were characterized by X-ray crystallography, which demonstrated that the copper(II) is in a distorted square planar environment. An investigation of the electrochemical properties of the complexes by cyclic voltammetry revealed that the complexes undergo multiple quasi-reversible processes. A comparison of the cyclic voltammetry of the copper complexes with their palladium(II) analogues suggests that these redox processes are ligand-based and not metal-based. The copper(II) complexes are cell-permeable in A431 mammalian cells and are nontoxic at concentrations of 50 µM. The ligands can be radiolabeled with copper-64 at room temperature.

The Molecular Basis of Acinetobacter baumannii Cadmium Toxicity and Resistance.

Acinetobacter species are ubiquitous Gram-negative bacteria that can be found in water, in soil, and as commensals of the human skin. The successful inhabitation of Acinetobacter species in diverse environments is primarily attributable to the expression of an arsenal of stress resistance determinants, which includes an extensive repertoire of metal ion efflux systems. Metal ion homeostasis in the hospital pathogen Acinetobacter baumannii contributes to pathogenesis; however, insights into its metal ion transporters for environmental persistence are lacking. Here, we studied the impact of cadmium stress on A. baumannii. Our functional genomics and independent mutant analyses revealed a primary role for CzcE, a member of the cation diffusion facilitator (CDF) superfamily, in resisting cadmium stress. We also show that the CzcCBA heavy metal efflux system contributes to cadmium efflux. Collectively, these systems provide A. baumannii with a comprehensive cadmium translocation pathway from the cytoplasm to the periplasm and subsequently the extracellular space. Furthermore, analysis of the A. baumannii metallome under cadmium stress showed zinc depletion, as well as copper enrichment, both of which are likely to influence cellular fitness. Overall, this work provides new knowledge on the role of a broad arsenal of membrane transporters in A. baumannii metal ion homeostasis. IMPORTANCE Cadmium toxicity is a widespread problem, yet the interaction of this heavy metal with biological systems is poorly understood. Some microbes have evolved traits to proactively counteract cadmium toxicity, including Acinetobacter baumannii, which is notorious for persisting in harsh environments. Here, we show that A. baumannii utilizes a dedicated cadmium efflux protein in concert with a system that is primarily attuned to zinc efflux to efficiently overcome cadmium stress. The molecular characterization of A. baumannii under cadmium stress revealed how active cadmium efflux plays a key role in preventing the dysregulation of bacterial metal ion homeostasis, which appeared to be a primary means by which cadmium exerts toxicity upon the bacterium.

Intracellular Accumulation of Staphylopine Can Sensitize Staphylococcus aureus to Host-Imposed Zinc Starvation by Chelation-Independent Toxicity.

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.

Accurate biometal quantification per individual Caenorhabditis elegans.

In the life sciences, small model-organisms are an established research platform. Due to the economy of culturing and maintenance animals such as the roundworm Caenorhabditis elegans, and the fly Drosophila melanogaster, have been instrumental for investigating key genetic pathways, early development, neuronal function, as well as disease pathogenesis and toxicology. Small model organisms have also found utility in the study of inorganic biochemistry, where the role of metal ion cofactors are investigated for numerous fundamental cellular processes. The metabolism and homeostasis of metal ions is also central to many aspects of biology and disease. Accurate quantification of endogenous metal ion content is an important determinant for many biological questions. There is currently no standardised method for quantifying biometal content in individual C. elegans or estimating the variation between individuals within clonal populations. Here, we have determined that ten or more adults are required to quantify physiologically important metals via inductively coupled plasma mass spectrometry (ICP-MS). The accuracy and precision of this method was then compared to synchrotron-based X-ray fluorescence microscopy (XFM) to determine the variation between isogenic, developmentally synchronous C. elegans adults.

Hfe Permease and Haemophilus influenzae Manganese Homeostasis.

Haemophilus influenzae is a commensal of the human upper respiratory tract that can infect diverse host niches due, at least in part, to its ability to withstand both endogenous and host-mediated oxidative stresses. Here, we show that hfeA, a gene previously linked to iron import, is essential for H. influenzae manganese recruitment via the HfeBCD transporter. Structural analyses show that metal binding in HfeA uses a unique mechanism that involves substantial rotation of the C-terminal lobe of the protein. Disruption of hfeA reduced H. influenzae manganese acquisition and was associated with decreased growth under aerobic conditions, impaired manganese-superoxide dismutase activity, reduced survival in macrophages, and changes in biofilm production in the presence of superoxide. Collectively, this work shows that HfeA contributes to H. influenzae manganese acquisition and virulence attributes. High conservation of the hfeABCD permease in Haemophilus species suggests that it may serve similar roles in other pathogenic Pasteurellaceae.

Intracellular zinc protects tumours from T cell-mediated cytotoxicity.

Tumour immune evasion presents a significant challenge to the effectiveness of cancer immunotherapies. Recent advances in high-throughput screening techniques have uncovered that loss of antigen presentation and cytokine signalling pathways are central mechanisms by which tumours evade T cell immunity. To uncover additional vulnerabilities in tumour cells beyond the well-recognized antigen presentation pathway, we conducted a genome-wide CRISPR/Cas9 screen to identify genes that mediate resistance to chimeric-antigen receptor (CAR)-T cells, which function independently of classical antigen presentation. Our study revealed that loss of core-binding factor subunit beta (CBFß) enhances tumour cell resistance to T cell killing, mediated through T cell-derived TNF. Mechanistically, RNA-sequencing and elemental analyses revealed that deletion of CBFß disrupts numerous pathways including those involved in zinc homoeostasis. Moreover, we demonstrated that modulation of cellular zinc, achieved by supplementation or chelation, significantly altered tumour cell susceptibility to TNF by regulating the levels of inhibitor of apoptosis proteins. Consistent with this, treatment of tumour cells with a membrane-permeable zinc chelator had no impact on tumour cell viability alone, but significantly increased tumour cell lysis by CD8+ T cells in a TNF-dependent but perforin-independent manner. These results underscore the crucial role of intracellular zinc in regulating tumour cell susceptibility to T cell-mediated killing, revealing a novel vulnerability in tumour cells that might be exploited for the development of future cancer immunotherapeutics.

Host subversion of bacterial metallophore usage drives copper intoxication.

IMPORTANCE: During infection, bacteria must overcome the dual threats of metal starvation and intoxication. This work reveals that the zinc-withholding response of the host sensitizes S. aureus to copper intoxication. In response to zinc starvation, S. aureus utilizes the metallophore staphylopine. The current work revealed that the host can leverage the promiscuity of staphylopine to intoxicate S. aureus during infection. Significantly, staphylopine-like metallophores are produced by a wide range of pathogens, suggesting that this is a conserved weakness that the host can leverage to toxify invaders with copper. Moreover, it challenges the assumption that the broad-spectrum metal binding of metallophores is inherently beneficial to bacteria.

Host subversion of bacterial metallophore usage drives copper intoxication.

Microorganisms can acquire metal ions in metal-limited environments using small molecules called metallophores. While metals and their importers are essential, metals can also be toxic, and metallophores have limited ability to discriminate metals. The impact of the metallophore-mediated non-cognate metal uptake on bacterial metal homeostasis and pathogenesis remains to be defined. The globally significant pathogen Staphylococcus aureus uses the Cnt system to secrete the metallophore staphylopine in zinc-limited host niches. Here, we show that staphylopine and the Cnt system facilitate bacterial copper uptake, potentiating the need for copper detoxification. During in vivo infection, staphylopine usage increased S. aureus susceptibility to host-mediated copper stress, indicating that the innate immune response can harness the antimicrobial potential of altered elemental abundances in host niches. Collectively, these observations show that while the broad-spectrum metal-chelating properties of metallophores can be advantageous, the host can exploit these properties to drive metal intoxication and mediate antibacterial control. IMPORTANCE: During infection bacteria must overcome the dual threats of metal starvation and intoxication. This work reveals that the zinc-withholding response of the host sensitizes Staphylococcus aureus to copper intoxication. In response to zinc starvation S. aureus utilizes the metallophore staphylopine. The current work revealed that the host can leverage the promiscuity of staphylopine to intoxicate S. aureus during infection. Significantly, staphylopine-like metallophores are produced by a wide range of pathogens, suggesting that this is a conserved weakness that the host can leverage to toxify invaders with copper. Moreover, it challenges the assumption that the broad-spectrum metal binding of metallophores is inherently beneficial to bacteria.

Zinc acquisition and its contribution to Klebsiella pneumoniae virulence.

Klebsiella pneumoniae is a World Health Organization priority pathogen and a significant clinical concern for infections of the respiratory and urinary tracts due to widespread and increasing resistance to antimicrobials. In the absence of a vaccine, there is an urgent need to identify novel targets for therapeutic development. Bacterial pathogens, including K. pneumoniae, require the d-block metal ion zinc as an essential micronutrient, which serves as a cofactor for ~6% of the proteome. During infection, zinc acquisition necessitates the use of high affinity uptake systems to overcome niche-specific zinc limitation and host-mediated nutritional immunity. Here, we report the identification of ZnuCBA and ZniCBA, two ATP-binding cassette permeases that are highly conserved in Klebsiella species and contribute to K. pneumoniae AJ218 zinc homeostasis, and the high-resolution structure of the zinc-recruiting solute-binding protein ZniA. The Znu and Zni permeases appear functionally redundant with abrogation of both systems required to reduce K. pneumoniae zinc accumulation. Disruption of both systems also exerted pleiotropic effects on the homeostasis of other d-block elements. Zinc limitation perturbed K. pneumoniae cell morphology and compromised resistance to stressors, such as salt and oxidative stress. The mutant strain lacking both systems showed significantly impaired virulence in acute lung infection models, highlighting the necessity of zinc acquisition in the virulence and pathogenicity of K. pneumoniae.

The Role of ZntA in Klebsiella pneumoniae Zinc Homeostasis.

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that is a leading cause of healthcare-associated infections, including pneumonia, urinary tract infections, and sepsis. Essential to the colonization and infection by K. pneumoniae is the acquisition of nutrients, such as the transition metal ion zinc. Zinc has crucial structural and catalytic roles in the proteome of all organisms. Nevertheless, in excess, it has the potential to mediate significant toxicity by dysregulating the homeostasis of other transition elements, disrupting enzymatic processes, and perturbing metalloprotein cofactor acquisition. Here, we sought to elucidate the zinc detoxification mechanisms of K. pneumoniae, which remain poorly defined. Using the representative K. pneumoniae AJ218 strain, we showed that the P-type ATPase, ZntA, which is upregulated in response to cellular zinc stress, was the primary zinc efflux pathway. Deletion of zntA rendered K. pneumoniae AJ218 highly susceptible to exogenous zinc stress and manifested as an impaired growth phenotype and increased cellular accumulation of the metal. Loss of zntA also increased sensitivity to cadmium stress, indicating a role for this efflux pathway in cadmium resistance. Disruption of zinc homeostasis in the K. pneumoniae AJ218 ΔzntA strain also impacted manganese and iron homeostasis and was associated with increased production of biofilm. Collectively, this work showed the critical role of ZntA in K. pneumoniae zinc tolerance and provided a foundation for further studies on zinc homeostasis and the future development of novel antimicrobials to target this pathway. IMPORTANCE Klebsiella pneumoniae is a leading cause of healthcare-associated infections, including pneumonia, urinary tract infections, and sepsis. Treatment of K. pneumoniae infections is becoming increasingly challenging due to high levels of antibiotic resistance and the rising prevalence of carbapenem-resistant, extended-spectrum ß-lactamases producing strains. Zinc is essential to the colonization and infection by many bacterial pathogens but toxic in excess. This work described the first dissection of the pathways associated with resisting extracellular zinc stress in K. pneumoniae. This study revealed that the P-type ATPase ZntA was highly upregulated in response to exogenous zinc stress and played a major role in maintaining bacterial metal homeostasis. Knowledge of how this major bacterial pathogen resists zinc stress provided a foundation for antimicrobial development studies to target and abrogate their essential function.

Host-Mediated Copper Stress Is Not Protective against Streptococcus pneumoniae D39 Infection.

Metal ions are required by all organisms for the chemical processes that support life. However, in excess they can also exert toxicity within biological systems. During infection, bacterial pathogens such as Streptococcus pneumoniae are exposed to host-imposed metal intoxication, where the toxic properties of metals, such as copper, are exploited to aid in microbial clearance. However, previous studies investigating the antimicrobial efficacy of copper in vivo have reported variable findings. Here, we use a highly copper-sensitive strain of S. pneumoniae, lacking both copper efflux and intracellular copper buffering by glutathione, to investigate how copper stress is managed and where it is encountered during infection. We show that this strain exhibits highly dysregulated copper homeostasis, leading to the attenuation of growth and hyperaccumulation of copper in vitro. In a murine infection model, whole-tissue copper quantitation and elemental bioimaging of the murine lung revealed that infection with S. pneumoniae resulted in increased copper abundance in specific tissues, with the formation of spatially discrete copper hot spots throughout the lung. While the increased copper was able to reduce the viability of the highly copper-sensitive strain in a pneumonia model, copper levels in professional phagocytes and in a bacteremic model were insufficient to prosecute bacterial clearance. Collectively, this study reveals that host copper is redistributed to sites of infection and can impact bacterial viability in a hypersusceptible strain. However, in wild-type S. pneumoniae, the concerted actions of the copper homeostatic mechanisms are sufficient to facilitate continued viability and virulence of the pathogen. IMPORTANCE Streptococcus pneumoniae (the pneumococcus) is one of the world's foremost bacterial pathogens. Treatment of both localized and systemic pneumococcal infection is becoming complicated by increasing rates of multidrug resistance globally. Copper is a potent antimicrobial agent used by the mammalian immune system in the defense against bacterial pathogens. However, unlike other bacterial species, this copper stress is unable to prosecute pneumococcal clearance. This study determines how the mammalian host inflicts copper stress on S. pneumoniae and the bacterial copper tolerance mechanisms that contribute to maintenance of viability and virulence in vitro and in vivo. This work has provided insight into the chemical biology of the host-pneumococcal interaction and identified a potential avenue for novel antimicrobial development.

Selective ferroptosis vulnerability due to familial Alzheimer's disease presenilin mutations.

Mutations in presenilin 1 and 2 (PS1 and PS2) cause autosomal dominant familial Alzheimer's disease (FAD). Ferroptosis has been implicated as a mechanism of neurodegeneration in AD since neocortical iron burden predicts Alzheimer's disease (AD) progression. We found that loss of the presenilins dramatically sensitizes multiple cell types to ferroptosis, but not apoptosis. FAD causal mutations of presenilins similarly sensitizes cells to ferroptosis. The presenilins promote the expression of GPX4, the selenoprotein checkpoint enzyme that blocks ferroptosis by quenching the membrane propagation of lethal hydroperoxyl radicals. Presenilin γ-secretase activity cleaves Notch-1 to signal LRP8 expression, which then controls GPX4 expression by regulating the supply of selenium into the cell since LRP8 is the uptake receptor for selenoprotein P. Selenium uptake is thus disrupted by presenilin FAD mutations, suppressing GPX4 expression. Therefore, presenilin mutations may promote neurodegeneration by derepressing ferroptosis, which has implications for disease-modifying therapeutics.

The Impact of Chromate on Pseudomonas aeruginosa Molybdenum Homeostasis.

Acquisition of the trace-element molybdenum via the high-affinity ATP-binding cassette permease ModABC is essential for Pseudomonas aeruginosa respiration in anaerobic and microaerophilic environments. This study determined the X-ray crystal structures of the molybdenum-recruiting solute-binding protein ModA from P. aeruginosa PAO1 in the metal-free state and bound to the group 6 metal oxyanions molybdate, tungstate, and chromate. Pseudomonas aeruginosa PAO1 ModA has a non-contiguous dual-hinged bilobal structure with a single metal-binding site positioned between the two domains. Metal binding results in a 22° relative rotation of the two lobes with the oxyanions coordinated by four residues, that contribute six hydrogen bonds, distinct from ModA orthologues that feature an additional oxyanion-binding residue. Analysis of 485 Pseudomonas ModA sequences revealed conservation of the metal-binding residues and ß-sheet structural elements, highlighting their contribution to protein structure and function. Despite the capacity of ModA to bind chromate, deletion of modA did not affect P. aeruginosa PAO1 sensitivity to chromate toxicity nor impact cellular accumulation of chromate. Exposure to sub-inhibitory concentrations of chromate broadly perturbed P. aeruginosa metal homeostasis and, unexpectedly, was associated with an increase in ModA-mediated molybdenum uptake. Elemental analyses of the proteome from anaerobically grown P. aeruginosa revealed that, despite the increase in cellular molybdenum upon chromate exposure, distribution of the metal within the proteome was substantially perturbed. This suggested that molybdoprotein cofactor acquisition may be disrupted, consistent with the potent toxicity of chromate under anaerobic conditions. Collectively, these data reveal a complex relationship between chromate toxicity, molybdenum homeostasis and anaerobic respiration.

Structural and biochemical characterization of Acinetobacter baumannii ZnuA.

Acinetobacter baumannii is a Gram-negative nosocomial pathogen associated with significant disease. Crucial to the survival and pathogenesis of A. baumannii is the ability to acquire essential micronutrients such as Zn(II). Recruitment of Zn(II) by A. baumannii is mediated, at least in part, by the periplasmic solute-binding protein ZnuA and the ATP-binding cassette transporter ZnuBC. Here, we combined genomic, biochemical, and structural approaches to characterize A. baumannii AB5075_UW ZnuA. Bioinformatic analyses using a diverse collection of A. baumannii genomes determined that ZnuA is highly conserved, with the binding site comprised by three strictly conserved histidine residues. The structure of metal-free ZnuA was determined at 2.1 Å resolution, with molecular dynamics analyses revealing loop α2ß2, which harbors the putative Zn(II)-coordinating residue His41, to be highly mobile in the metal-free state. The contribution of the putative binding site histidine residues to Zn(II) interaction was further probed by mutagenesis. Analysis of ZnuA mutant variants was performed by quantitative metal binding assays, differential scanning fluorimetry, and affinity measurements, which showed that all three histidine residues contributed to Zn(II)-recruitment, albeit to different extents. Collectively, these analyses provide insight into the mechanism of Zn(II)-binding by A. baumannii ZnuA and expand our understanding of the functional diversity of Zn(II)-recruiting proteins.

Selenium mediates exercise-induced adult neurogenesis and reverses learning deficits induced by hippocampal injury and aging.

Although the neurogenesis-enhancing effects of exercise have been extensively studied, the molecular mechanisms underlying this response remain unclear. Here, we propose that this is mediated by the exercise-induced systemic release of the antioxidant selenium transport protein, selenoprotein P (SEPP1). Using knockout mouse models, we confirmed that SEPP1 and its receptor low-density lipoprotein receptor-related protein 8 (LRP8) are required for the exercise-induced increase in adult hippocampal neurogenesis. In vivo selenium infusion increased hippocampal neural precursor cell (NPC) proliferation and adult neurogenesis. Mimicking the effect of exercise through dietary selenium supplementation restored neurogenesis and reversed the cognitive decline associated with aging and hippocampal injury, suggesting potential therapeutic relevance. These results provide a molecular mechanism linking exercise-induced changes in the systemic environment to the activation of quiescent hippocampal NPCs and their subsequent recruitment into the neurogenic trajectory.

Dysregulation of Streptococcus pneumoniae zinc homeostasis breaks ampicillin resistance in a pneumonia infection model.

Streptococcus pneumoniae is the primary cause of community-acquired bacterial pneumonia with rates of penicillin and multidrug-resistance exceeding 80% and 40%, respectively. The innate immune response generates a variety of antimicrobial agents to control infection, including zinc stress. Here, we characterize the impact of zinc intoxication on S. pneumoniae, observing disruptions in central carbon metabolism, lipid biogenesis, and peptidoglycan biosynthesis. Characterization of the pivotal peptidoglycan biosynthetic enzyme GlmU indicates a sensitivity to zinc inhibition. Disruption of the sole zinc efflux pathway, czcD, renders S. pneumoniae highly susceptible to ß-lactam antibiotics. To dysregulate zinc homeostasis in the wild-type strain, we investigated the safe-for-human-use ionophore 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2). PBT2 rendered wild-type S. pneumoniae strains sensitive to a range of antibiotics. Using an invasive ampicillin-resistant strain, we demonstrate in a murine pneumonia infection model the efficacy of PBT2 + ampicillin treatment. These findings present a therapeutic modality to break antibiotic resistance in multidrug-resistant S. pneumoniae.

Conformation of the Solute-Binding Protein AdcAII Influences Zinc Uptake in Streptococcus pneumoniae.

Streptococcus pneumoniae scavenges essential zinc ions from the host during colonization and infection. This is achieved by the ATP-binding cassette transporter, AdcCB, and two solute-binding proteins (SBPs), AdcA and AdcAII. It has been established that AdcAII serves a greater role during initial infection, but the molecular details of how the protein selectively acquires Zn(II) remain poorly understood. This can be attributed to the refractory nature of metal-free AdcAII to high-resolution structural determination techniques. Here, we overcome this issue by separately mutating the Zn(II)-coordinating residues and performing a combination of structural and biochemical analyses on the variant proteins. Structural analyses of Zn(II)-bound AdcAII variants revealed that specific regions within the protein underwent conformational changes via direct coupling to each of the metal-binding residues. Quantitative in vitro metal-binding assays combined with affinity determination and phenotypic growth assays revealed that each of the four Zn(II)-coordinating residues contributes to metal binding by AdcAII. Intriguingly, the phenotypic growth impact of the mutant adcAII alleles was, in general, independent of affinity, suggesting that the Zn(II)-bound conformation of the SBP is crucial for efficacious metal uptake. Collectively, these data highlight the intimate coupling of ligand affinity with protein conformational change in ligand-receptor proteins and provide a putative mechanism for AdcAII. These findings provide further mechanistic insight into the structural and functional diversity of SBPs that is broadly applicable to other prokaryotes.