In a state of flux: new insight into the transport processes that maintain bacterial metal homeostasis.

A new study by Nies et al. (J Bacteriol 206:e00080-24, 2024, https://doi.org/10.1128/jb.00080-24) provides a rich, quantitative data set of zinc accumulation by cells of Cupriavidus metallidurans, including of mutant bacterial strains lacking import or efflux genes, and comparison of zinc accumulation by cells previously starved of metal with those of zinc-replete cells. The data surprisingly demonstrate the concomitant activity of both active metal import and metal efflux systems. They present a flow equilibrium model to describe zinc homeostasis in bacteria.

Role of horizontally transferred copper resistance genes in Staphylococcus aureus and Listeria monocytogenes.

Bacteria have evolved mechanisms which enable them to control intracellular concentrations of metals. In the case of transition metals, such as copper, iron and zinc, bacteria must ensure enough is available as a cofactor for enzymes whilst at the same time preventing the accumulation of excess concentrations, which can be toxic. Interestingly, metal homeostasis and resistance systems have been found to play important roles in virulence. This review will discuss the copper homeostasis and resistance systems in Staphylococcus aureus and Listeria monocytogenes and the implications that acquisition of additional copper resistance genes may have in these pathogens.

The therapeutic and preventive effects of a canine-origin VB12 -producing Lactobacillus on DSS-induced colitis in mice.

Vitamin B12 (VB12 ) plays vital roles as a cofactor in reactions related to biosynthesis and metabolic regulation. Animals with diarrhoea from intestinal inflammation are susceptible to VB12 deficiency due to dysfunctional absorption. No current medications for canine intestinal inflammation can simultaneously act as VB12 supplements. Here we have tested a strain of VB12 -producing Lactobacillus, to investigate its safety in healthy dogs and test for hypothesized therapeutic and preventive effects on murine colitis. Results from enzyme-linked immunosorbent assay, histopathological analysis, and quantitative polymerase chain reaction showed normal physical conditions of healthy dogs given Lactobacillus, and blood biochemical indices showed no significant differences in markers, indicating safety of Lactobacillus to healthy dogs. The microbiota in animals receiving VB12 -producing Lactobacillus probiotic exhibited decreased abundance of Escherichia coli and concomitant increase in Lactobacillus. The probiotic supplement also resulted in downregulation of proinflammatory cytokines in murine colon tissues, reduced myeloperoxidase activity and malondialdehyde level, and significantly increased serum VB12 level and decreased homocysteine in therapeutic and preventive experiments. Moreover, Lactobacillus supplement decreased colonic inflammation and injury, improved gut microbiota, and ameliorated VB12 deficiency as an adjunctive therapy. We conclude this product is potentially beneficial for efficient therapy and prevention of VB12 deficiency form intestinal inflammation in canine clinical practice.

Dissecting the structural and functional roles of a putative metal entry site in encapsulated ferritins.

Encapsulated ferritins belong to the universally distributed ferritin superfamily, whose members function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question because of the differences in organization of the ferroxidase catalytic site and neighboring secondary metal-binding sites. We have previously identified a putative metal-binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron-entry site by means of enzymatic assays, MS, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, although enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.

Targeting metabolism to reverse T-cell exhaustion in chronic viral infections.

CD8 T-cells are an essential component of the adaptive immune response accountable for the clearance of virus-infected cells via cytotoxic effector functions. Maintaining a specific metabolic profile is necessary for these T-cells to sustain their effector functions and clear pathogens. When CD8 T-cells are activated via T-cell receptor recognition of viral antigen, they transition from a naïve to an effector state and eventually to a memory phenotype, and their metabolic profiles shift as the cells differentiate to accomidate different metabolic demands. However, in the context of particular chronic viral infections (CVIs), CD8 T-cells can become metabolically dysfunctional in a state known as T-cell exhaustion. In this state, CD8 T-cells exhibit reduced effector functions and are unable to properly control pathogens. Clearing these chronic infections becomes progressively difficult as increasing numbers of the effector T-cells become exhausted. Hence, reversal of this dysfunctional metabolic phenotype is vital when considering potential treatments of these infections and offers the opportunity for novel strategies for the development of therapies against CVIs. In this review we explore research implicating alteration of the metabolic state as a means to reverse CD8 T-cell exhaustion in CVIs. These findings indicate that strategies targeting dysfunctional CD8 T-cell metabolism could prove to be a promising option for successfully treating CVIs.

Copper tolerance in bacteria requires the activation of multiple accessory pathways.

Copper is a required micronutrient for bacteria and an essential cofactor for redox-active cuproenzymes. Yet, excess copper is extremely toxic, and is exploited as a bacteriocide in medical and biotechnological applications and also by the mammalian immune system. To evade copper toxicity, bacteria not only control intracellular copper homeostasis, but they must also repair the damage caused by excess copper. In this review, we summarize the bacterial cell-wide response to copper toxicity in Enterobacteria. Tapping into the abundant research data on two key organisms, Escherichia coli and Salmonella enterica, we show that copper resistance requires both the direct copper homeostatic response and also the indirect accessory pathways that deal with copper-induced damage. Since patterns of copper response are conserved through the Proteobacteria, we propose a cell-wide view of copper detoxification and copper tolerance that can be used to identify novel targets for copper-based antibacterial therapeutics.

A four-helix bundle stores copper for methane oxidation.

Methane-oxidizing bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase. Certain methanotrophs are also able to switch to using the iron-containing soluble methane monooxygenase to catalyse methane oxidation, with this switchover regulated by copper. Methane monooxygenases are nature's primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and methane monooxygenases have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. Here we discover and characterize a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for particulate methane monooxygenase. Csp1 is a tetramer of four-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realized. Cytosolic homologues of Csp1 are present in diverse bacteria, thus challenging the dogma that such organisms do not use copper in this location.

Conservation of the structural and functional architecture of encapsulated ferritins in bacteria and archaea.

Ferritins are a large family of intracellular proteins that protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. The recently characterized Rhodospirillum rubrum EncFtn displays an unusual structure when compared with classical ferritins, with an open decameric structure that is enzymatically active, but unable to store iron. This EncFtn must be associated with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether this unusual structure is conserved across the family. Here, we characterize EncFtn proteins from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the conserved annular pentamer of dimers topology. Key structural differences are apparent between the homologues, particularly in the centre of the ring and the secondary metal-binding site, which is not conserved across the homologues. Solution and native mass spectrometry analyses highlight that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The ferroxidase activity of EncFtn proteins was confirmed, and we show that while the quaternary structure around the ferroxidase centre is distinct from classical ferritins, the ferroxidase activity is still inhibited by Zn(II). Our results highlight the common structural organization and activity of EncFtn proteins, despite diverse host environments and contexts within encapsulins.

A Superoxide Dismutase Capable of Functioning with Iron or Manganese Promotes the Resistance of Staphylococcus aureus to Calprotectin and Nutritional Immunity.

Staphylococcus aureus is a devastating mammalian pathogen for which the development of new therapeutic approaches is urgently needed due to the prevalence of antibiotic resistance. During infection pathogens must overcome the dual threats of host-imposed manganese starvation, termed nutritional immunity, and the oxidative burst of immune cells. These defenses function synergistically, as host-imposed manganese starvation reduces activity of the manganese-dependent enzyme superoxide dismutase (SOD). S. aureus expresses two SODs, denoted SodA and SodM. While all staphylococci possess SodA, SodM is unique to S. aureus, but the advantage that S. aureus gains by expressing two apparently manganese-dependent SODs is unknown. Surprisingly, loss of both SODs renders S. aureus more sensitive to host-imposed manganese starvation, suggesting a role for these proteins in overcoming nutritional immunity. In this study, we have elucidated the respective contributions of SodA and SodM to resisting oxidative stress and nutritional immunity. These analyses revealed that SodA is important for resisting oxidative stress and for disease development when manganese is abundant, while SodM is important under manganese-deplete conditions. In vitro analysis demonstrated that SodA is strictly manganese-dependent whereas SodM is in fact cambialistic, possessing equal enzymatic activity when loaded with manganese or iron. Cumulatively, these studies provide a mechanistic rationale for the acquisition of a second superoxide dismutase by S. aureus and demonstrate an important contribution of cambialistic SODs to bacterial pathogenesis. Furthermore, they also suggest a new mechanism for resisting manganese starvation, namely populating manganese-utilizing enzymes with iron.

A horizontally gene transferred copper resistance locus confers hyper-resistance to antibacterial copper toxicity and enables survival of community acquired methicillin resistant Staphylococcus aureus USA300 in macrophages.

Excess copper is highly toxic and forms part of the host innate immune system's antibacterial arsenal, accumulating at sites of infection and acting within macrophages to kill engulfed pathogens. We show for the first time that a novel, horizontally gene transferred copper resistance locus (copXL), uniquely associated with the SCCmec elements of the highly virulent, epidemic, community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) USA300, confers copper hyper-resistance. These genes are additional to existing core genome copper resistance mechanisms, and are not found in typical S. aureus lineages, but are increasingly identified in emerging pathogenic isolates. Our data show that CopX, a putative P1B-3 -ATPase efflux transporter, and CopL, a novel lipoprotein, confer copper hyper-resistance compared to typical S. aureus strains. The copXL genes form an operon that is tightly repressed in low copper environments by the copper regulator CsoR. Significantly, CopX and CopL are important for S. aureus USA300 intracellular survival within macrophages. Therefore, the emergence of new S. aureus clones with the copXL locus has significant implications for public health because these genes confer increased resistance to antibacterial copper toxicity, enhancing bacterial fitness by altering S. aureus interaction with innate immunity.

A charge polarization model for the metal-specific activity of superoxide dismutases.

The pathogenicity of Staphylococcus aureus is enhanced by having two superoxide dismutases (SODs): a Mn-specific SOD and another that can use either Mn or Fe. Using 94 GHz electron-nuclear double resonance (ENDOR) and electron double resonance detected (ELDOR)-NMR we show that, despite their different metal-specificities, their structural and electronic similarities extend down to their active-site 1H- and 14N-Mn(ii) hyperfine interactions. However these interactions, and hence the positions of these nuclei, are different in the inactive Mn-reconstituted Escherichia coli Fe-specific SOD. Density functional theory modelling attributes this to a different angular position of the E. coli H171 ligand. This likely disrupts the Mn-H171-E170' triad causing a shift in charge and in metal redox potential, leading to the loss of activity. This is supported by the correlated differences in the Mn(ii) zero-field interactions of the three SOD types and suggests that the triad is important for determining metal specific activity.

Comparison of total ionic strength adjustment buffers III and IV in the measurement of fluoride concentration of teas.

BACKGROUND: Tea is the second most consumed drink in the UK and a primary source of hydration; it is an important source of dietary fluoride (F) for consumers and also abundant in aluminium (Al). Varying ranges of F concentrations in teas have been reported worldwide which may be, in part, due to differences in analytical techniques used to measure this ion. AIM: The effect of using total ionic adjustment buffers (TISAB) III or IV when measuring F concentration of black teas available in the UK was investigated and compared. Based on this evaluation, the effects of three different infusion times, 1 min, 10 min and 1 h, caffeine content and tea form on the F contents of the tea samples were investigated. METHODS: The F concentrations of 47 tea samples were measured directly using a fluoride ion-selective electrode (F-ISE), TISAB III and IV and infusion times of 1 min, 10 min and 1 h. RESULTS: Mean (SD) F concentration of tea samples for all infusion times was statistically significantly higher ( p < 0.001) measured by TISAB IV (4.37 (2.16) mg/l) compared with TISAB III (3.54 (1.65) mg/l). A statistically significant positive correlation ( p < 0.001) was found between Al concentration (mg/l) and differences in F concentration (mg/l) measured using the two TISABs; the difference in F concentration measured by the two TISABs increased with the magnitude of Al concentration. CONCLUSION: Due to higher concentrations of F and Al in teas and their complexing potential, use of TISAB IV facilitates more accurate measurement of F concentration when using an F-ISE and a direct method.

Metalloproteins and metal sensing.

Almost half of all enzymes must associate with a particular metal to function. An ambition is to understand why each metal-protein partnership arose and how it is maintained. Metal availability provides part of the explanation, and has changed over geological time and varies between habitats but is held within vital limits in cells. Such homeostasis needs metal sensors, and there is an ongoing search to discover the metal-sensing mechanisms. For metalloproteins to acquire the right metals, metal sensors must correctly distinguish between the inorganic elements.

Cyanobacterial metallochaperone inhibits deleterious side reactions of copper.

Copper metallochaperones supply copper to cupro-proteins through copper-mediated protein-protein-interactions and it has been hypothesized that metallochaperones thereby inhibit copper from causing damage en route. Evidence is presented in support of this latter role for cyanobacterial metallochaperone, Atx1. In cyanobacteria Atx1 contributes towards the supply of copper to plastocyanin inside thylakoids but it is shown here that in copper-replete medium, copper can reach plastocyanin without Atx1. Unlike metallochaperone-independent copper-supply to superoxide dismutase in eukaryotes, glutathione is not essential for Atx1-independent supply to plastocyanin: Double mutants missing atx1 and gshB (encoding glutathione synthetase) accumulate the same number of atoms of copper per cell in the plastocyanin pool as wild type. Critically, Δatx1ΔgshB are hypersensitive to elevated copper relative to wild type cells and also relative to ΔgshB single mutants with evidence that hypersensitivity arises due to the mislocation of copper to sites for other metals including iron and zinc. The zinc site on the amino-terminal domain (ZiaA(N)) of the P(1)-type zinc-transporting ATPase is especially similar to the copper site of the Atx1 target PacS(N), and ZiaA(N) will bind Cu(I) more tightly than zinc. An NMR model of a substituted-ZiaA(N)-Cu(I)-Atx1 heterodimer has been generated making it possible to visualize a juxtaposition of residues surrounding the ZiaA(N) zinc site, including Asp(18), which normally repulse Atx1. Equivalent repulsion between bacterial copper metallochaperones and the amino-terminal regions of P(1)-type ATPases for metals other than Cu(I) is conserved, again consistent with a role for copper metallochaperones to withhold copper from binding sites for other metals.

Protein-folding location can regulate manganese-binding versus copper- or zinc-binding.

Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).

Tissue differences in BER-related incision activity and non-specific nuclease activity as measured by the comet assay.

DNA repair mechanisms are important for genome stability and to prevent accumulation of DNA damage, which contributes to cellular ageing and cancer development. Study of these physiological processes requires robust and practical assays to quantify DNA repair capacity. The in vitro comet-based assay is a simple, yet reliable, assay for measurement of DNA repair and has been modified recently to quantify DNA incision activity in mouse brain and liver. In this study, we applied this assay to assess DNA incision activity in other mouse tissues, i.e. lung and colon, and found that high, non-specific nuclease activity was a problem when measuring DNA incision activity, especially in the colon. We tested the utility of multiple optimisation steps including addition of aphidicolin, ATP and polyAT and used multiple wash steps, which resulted in modest improvements in performance of the assay. Washing the tissues before protein extraction and decreasing the protein concentration in the assay were the most effective steps in reducing non-specific nuclease activity. Using the comet-based assay with these further modifications, we found that base excision repair incision activity changed with age differently in each tissue. This study shows that non-specific nuclease activity in the comet-based assay for DNA repair is more pronounced in some tissues than others so care should be taken to optimise the protocol when applying the assay to a new tissue. Our data suggest the importance of using control cells (noRo cells incubated with extract) in the assay to assess for non-specific nuclease activity. In conclusion, the comet-based DNA repair assay can be easily adapted to study a range of mammalian tissues.

Mis-regulation of Zn and Mn homeostasis is a key phenotype of Cu stress in Streptococcus pyogenes.

All bacteria possess homeostastic mechanisms that control the availability of micronutrient metals within the cell. Cross-talks between different metal homeostasis pathways within the same bacterial organism have been reported widely. In addition, there have been previous suggestions that some metal uptake transporters can promote adventitious uptake of the wrong metal. This work describes the cross-talk between Cu and the Zn and Mn homeostasis pathways in Group A Streptococcus (GAS). Using a ∆copA mutant strain that lacks the primary Cu efflux pump and thus traps excess Cu in the cytoplasm, we show that growth in the presence of supplemental Cu promotes downregulation of genes that contribute to Zn or Mn uptake. This effect is not associated with changes in cellular Zn or Mn levels. Co-supplementation of the culture medium with Zn or, to a lesser extent, Mn alleviates key Cu stress phenotypes, namely bacterial growth and secretion of the fermentation end-product lactate. However, neither co-supplemental Zn nor Mn influences cellular Cu levels or Cu availability in Cu-stressed cells. In addition, we provide evidence that the Zn or Mn uptake transporters in GAS do not promote Cu uptake. Together, the results from this study strengthen and extend our previous proposal that mis-regulation of Zn and Mn homeostasis is a key phenotype of Cu stress in GAS.

Cellular iron distribution in Bacillus anthracis.

Although successful iron acquisition by pathogens within a host is a prerequisite for the establishment of infection, surprisingly little is known about the intracellular distribution of iron within bacterial pathogens. We have used a combination of anaerobic native liquid chromatography, inductively coupled plasma mass spectrometry, principal-component analysis, and peptide mass fingerprinting to investigate the cytosolic iron distribution in the pathogen Bacillus anthracis. Our studies identified three of the major iron pools as being associated with the electron transfer protein ferredoxin, the miniferritin Dps2, and the superoxide dismutase (SOD) enzymes SodA1 and SodA2. Although both SOD isozymes were predicted to utilize manganese cofactors, quantification of the metal ions associated with SodA1 and SodA2 in cell extracts established that SodA1 is associated with both manganese and iron, whereas SodA2 is bound exclusively to iron in vivo. These data were confirmed by in vitro assays using recombinant protein preparations, showing that SodA2 is active with an iron cofactor, while SodA1 is cambialistic, i.e., active with manganese or iron. Furthermore, we observe that B. anthracis cells exposed to superoxide stress increase their total iron content more than 2-fold over 60 min, while the manganese and zinc contents are unaffected. Notably, the acquired iron is not localized to the three identified cytosolic iron pools.

Old dogs, new tricks: New insights into the iron/manganese superoxide dismutase family.

Superoxide dismutases (SODs) are ancient enzymes of widespread importance present in all domains of life. Many insights have been gained into these important enzymes over the 50 years since their initial description, but recent studies in the context of microbial pathogenesis have resulted in findings that challenge long established dogmas. The repertoire of SODs that bacterial pathogens encode is diverse both in number and in metal dependencies, including copper, copper and zinc, manganese, iron, and cambialistic enzymes. Other bacteria also possess nickel dependent SODs. Compartmentalization of SODs only partially explains their diversity. The need for pathogens to maintain SOD activity across distinct hostile environments encountered during infection, including those limited for essential metals, is also a driver of repertoire diversity. SOD research using pathogenic microbes has also revealed the apparent biochemical ease with which metal specificity can change within the most common family of SODs. Collectively, these studies are revealing the dynamic nature of SOD evolution, both that of individual SOD enzymes that can change their metal specificity to adapt to fluctuating cellular metal availability, and of a cell's repertoire of SOD isozymes that can be differentially expressed to adapt to fluctuating environmental metal availability in a niche.

Interspecies competition in oral biofilms mediated by Streptococcus gordonii extracellular deoxyribonuclease SsnA.

Extracellular DNA (eDNA) is a key component of many microbial biofilms including dental plaque. However, the roles of extracellular deoxyribonuclease (DNase) enzymes within biofilms are poorly understood. Streptococcus gordonii is a pioneer colonizer of dental plaque. Here, we identified and characterised SsnA, a cell wall-associated protein responsible for extracellular DNase activity of S. gordonii. The SsnA-mediated extracellular DNase activity of S. gordonii was suppressed following growth in sugars. SsnA was purified as a recombinant protein and shown to be inactive below pH 6.5. SsnA inhibited biofilm formation by Streptococcus mutans in a pH-dependent manner. Further, SsnA inhibited the growth of oral microcosm biofilms in human saliva. However, inhibition was ameliorated by the addition of sucrose. Together, these data indicate that S. gordonii SsnA plays a key role in interspecies competition within oral biofilms. Acidification of the medium through sugar catabolism could be a strategy for cariogenic species such as S. mutans to prevent SsnA-mediated exclusion from biofilms.