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.

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.

On the mechanisms of lysis triggered by perturbations of bacterial cell wall biosynthesis.

Inhibition of bacterial cell wall synthesis by antibiotics such as ß-lactams is thought to cause explosive lysis through loss of cell wall integrity. However, recent studies on a wide range of bacteria have suggested that these antibiotics also perturb central carbon metabolism, contributing to death via oxidative damage. Here, we genetically dissect this connection in Bacillus subtilis perturbed for cell wall synthesis, and identify key enzymatic steps in upstream and downstream pathways that stimulate the generation of reactive oxygen species through cellular respiration. Our results also reveal the critical role of iron homeostasis for the oxidative damage-mediated lethal effects. We show that protection of cells from oxygen radicals via a recently discovered siderophore-like compound uncouples changes in cell morphology normally associated with cell death, from lysis as usually judged by a phase pale microscopic appearance. Phase paling appears to be closely associated with lipid peroxidation.

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.

Mirubactin C rescues the lethal effect of cell wall biosynthesis mutations in Bacillus subtilis.

Growth of most rod-shaped bacteria is accompanied by the insertion of new peptidoglycan into the cylindrical cell wall. This insertion, which helps maintain and determine the shape of the cell, is guided by a protein machine called the rod complex or elongasome. Although most of the proteins in this complex are essential under normal growth conditions, cell viability can be rescued, for reasons that are not understood, by the presence of a high (mM) Mg2+ concentration. We screened for natural product compounds that could rescue the growth of mutants affected in rod-complex function. By screening > 2,000 extracts from a diverse collection of actinobacteria, we identified a compound, mirubactin C, related to the known iron siderophore mirubactin A, which rescued growth in the low micromolar range, and this activity was confirmed using synthetic mirubactin C. The compound also displayed toxicity at higher concentrations, and this effect appears related to iron homeostasis. However, several lines of evidence suggest that the mirubactin C rescuing activity is not due simply to iron sequestration. The results support an emerging view that the functions of bacterial siderophores extend well beyond simply iron binding and uptake.

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.

Copper microenvironments in the human body define patterns of copper adaptation in pathogenic bacteria.

Copper is an essential micronutrient for most organisms that is required as a cofactor for crucial copper-dependent enzymes encoded by both prokaryotes and eukaryotes. Evidence accumulated over several decades has shown that copper plays important roles in the function of the mammalian immune system. Copper accumulates at sites of infection, including the gastrointestinal and respiratory tracts and in blood and urine, and its antibacterial toxicity is directly leveraged by phagocytic cells to kill pathogens. Copper-deficient animals are more susceptible to infection, whereas those fed copper-rich diets are more resistant. As a result, copper resistance genes are important virulence factors for bacterial pathogens, enabling them to detoxify the copper insult while maintaining copper supply to their essential cuproenzymes. Here, we describe the accumulated evidence for the varied roles of copper in the mammalian response to infections, demonstrating that this metal has numerous direct and indirect effects on immune function. We further illustrate the multifaceted response of pathogenic bacteria to the elevated copper concentrations that they experience when invading the host, describing both conserved and species-specific adaptations to copper toxicity. Together, these observations demonstrate the roles of copper at the host-pathogen interface and illustrate why bacterial copper detoxification systems can be viable targets for the future development of novel antibiotic drug development programs.

Blocking Polyphosphate Mobilization Inhibits Pho4 Activation and Virulence in the Pathogen Candida albicans.

The ability of pathogenic fungi to obtain essential nutrients from the host is vital for virulence. In Candida albicans, acquisition of the macronutrient phosphate is regulated by the Pho4 transcription factor and is important for both virulence and resistance to host-encountered stresses. All cells store phosphate in the form of polyphosphate (polyP), a ubiquitous polymer comprising tens to hundreds of phosphate residues. Release of phosphate from polyP is one of the first responses evoked in response to phosphate starvation, and here, we sought to explore the importance of polyP mobilization in the pathobiology of C. albicans. We found that two polyphosphatases, Ppn1 and Ppx1, function redundantly to release phosphate from polyP in C. albicans. Strikingly, we reveal that blocking polyP mobilization prevents the activation of the Pho4 transcription factor: following Pi starvation, Pho4 fails to accumulate in the nucleus and induce Pi acquisition genes in ppn1Δ ppx1Δ cells. Consequently, ppn1Δ ppx1Δ cells display impaired resistance to the same range of stresses that require Pho4 for survival. In addition, cells lacking both polyphosphatases are exquisitely sensitive to DNA replication stress, indicating that polyP mobilization is needed to support the phosphate-demanding process of DNA replication. Blocking polyP mobilization also results in significant morphological defects, as ppn1Δ ppx1Δ cells form large pseudohypha-like cells that are resistant to serum-induced hypha formation. Thus, polyP mobilization impacts key processes important for the pathobiology of C. albicans, and consistent with this, we found that blocking this process attenuates the virulence of this important human fungal pathogen. IMPORTANCE Acquisition of the essential macronutrient phosphate is important for the virulence of Candida albicans, a major human fungal pathogen. All cells store phosphate as polyphosphate (polyP), which is rapidly mobilized when phosphate is limiting. Here, we identified the major phosphatases involved in releasing phosphate from polyP in C. albicans. By blocking this process, we found that polyP mobilization impacts many process that contribute to C. albicans pathogenesis. Notably, we found that blocking polyP mobilization inhibits activation of the Pho4 transcription factor, the master regulator of phosphate acquisition. In addition, cell cycle progression, stress resistance, morphogenetic switching, and virulence are all impaired in cells that cannot mobilize polyP. This study therefore provides new insight into the importance of polyP mobilization in promoting the virulence of C. albicans. As phosphate homeostasis strategies differ between fungal pathogen and host, this offers promise for the future development of antifungals.

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.

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.

Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment.

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryo-electron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

Synthetic biology approaches to copper remediation: bioleaching, accumulation and recycling.

One of the current aims of synthetic biology is the development of novel microorganisms that can mine economically important elements from the environment or remediate toxic waste compounds. Copper, in particular, is a high-priority target for bioremediation owing to its extensive use in the food, metal and electronic industries and its resulting common presence as an environmental pollutant. Even though microbe-aided copper biomining is a mature technology, its application to waste treatment and remediation of contaminated sites still requires further research and development. Crucially, any engineered copper-remediating chassis must survive in copper-rich environments and adapt to copper toxicity; they also require bespoke adaptations to specifically extract copper and safely accumulate it as a human-recoverable deposit to enable biorecycling. Here, we review current strategies in copper bioremediation, biomining and biorecycling, as well as strategies that extant bacteria use to enhance copper tolerance, accumulation and mineralization in the native environment. By describing the existing toolbox of copper homeostasis proteins from naturally occurring bacteria, we show how these modular systems can be exploited through synthetic biology to enhance the properties of engineered microbes for biotechnological copper recovery applications.

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.

Hepatoprotective Effects of Selenium-Enriched Probiotics Supplementation on Heat-Stressed Wistar Rat Through Anti-Inflammatory and Antioxidant Effects.

The purpose of this study was to elucidate the effects of selenium-enriched probiotics on the liver of heat-stressed Wistar rats. Ten-week-old male rats were assigned to four groups: control (Con); high temperature (HT); high temperature plus probiotics (HT + P: 1011 CFU/mL Lactobacillus acidophilus and 109 CFU/mL Saccharomyces cerevisiae); or high temperature plus selenium-enriched probiotics (HT + SeP: 0.3 mg/kg Se, 1011 CFU/mL L. acidophilus and 109 CFU/mL S. cerevisiae). The HT, HT + P, and HT + SeP groups were maintained at higher ambient temperature (40-42 °C), while the control group was kept at room temperature (25 °C). After 42 days of thermal exposure, blood and liver tissues were collected and analyzed for morphological and molecular markers of liver physiology. The body weight of rats in the HT group decreased but liver weight and live index were increased. Histological examination showed dilation of liver sinusoids and congestion of interstitial veins in HT group. Moreover, the histomorphology of the liver in HT + P and HT + SeP groups was restored, and the serum AST, ALT, ALP, LDH, and hepatic MDA level decreased significantly, but the serum total protein level and the liver SOD, T-AOC, and GSH-PX activities were increased significantly relative to the HT group. In addition, the mRNA level of Gpx1, SOD1, Nrf2, and Bcl-2 was significantly increased, while the expression level of Bax, IL-6, TNF-α, COX-2, NF-κB, α-SMA, TGFß1, Collagen I, HSP70, and HSP90 was significantly decreased in liver tissues after SeP supplementation. We concluded that SeP can protect Wistar rats from oxidative stress, inflammation, apoptosis, and liver fibrosis induced by heat stress.

Role of Glutathione in Buffering Excess Intracellular Copper in Streptococcus pyogenes.

Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]), this system is encoded by the copYAZ operon. This study demonstrates that although the site of GAS infection represents a Cu-rich environment, inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde-3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of noncognate metal-binding sites by Cu. Surprisingly, the onset of these effects is delayed by ∼4 h even though expression of copZ is upregulated immediately upon exposure to Cu. Further biochemical investigations show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. These results indicate that GSH buffers excess intracellular Cu when the transcriptionally responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance and allows bacteria to maintain metabolism even in the presence of an excess of this metal ion.IMPORTANCE The control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it has been established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and has long been assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this physiological role of glutathione beyond measuring growth of glutathione-deficient mutants in the presence of Cu. Our work with group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.

Fabrication routes via projection stereolithography for 3D-printing of microfluidic geometries for nucleic acid amplification.

Digital Light Processing (DLP) stereolithography (SLA) as a high-resolution 3D printing process offers a low-cost alternative for prototyping of microfluidic geometries, compared to traditional clean-room and workshop-based methods. Here, we investigate DLP-SLA printing performance for the production of micro-chamber chip geometries suitable for Polymerase Chain Reaction (PCR), a key process in molecular diagnostics to amplify nucleic acid sequences. A DLP-SLA fabrication protocol for printed micro-chamber devices with monolithic micro-channels is developed and evaluated. Printed devices were post-processed with ultraviolet (UV) light and solvent baths to reduce PCR inhibiting residuals and further treated with silane coupling agents to passivate the surface, thereby limiting biomolecular adsorption occurences during the reaction. The printed devices were evaluated on a purpose-built infrared (IR) mediated PCR thermocycler. Amplification of 75 base pair long target sequences from genomic DNA templates on fluorosilane and glass modified chips produced amplicons consistent with the control reactions, unlike the non-silanized chips that produced faint or no amplicon. The results indicated good functionality of the IR thermocycler and good PCR compatibility of the printed and silanized SLA polymer. Based on the proposed methods, various microfluidic designs and ideas can be validated in-house at negligible costs without the requirement of tool manufacturing and workshop or clean-room access. Additionally, the versatile chemistry of 3D printing resins enables customised surface properties adding significant value to the printed prototypes. Considering the low setup and unit cost, design flexibility and flexible resin chemistries, DLP-SLA is anticipated to play a key role in future prototyping of microfluidics, particularly in the fields of research biology and molecular diagnostics. From a system point-of-view, the proposed method of thermocycling shows promise for portability and modular integration of funcitonalitites for diagnostic or research applications that utilize nucleic acid amplification technology.

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.

An evolutionary path to altered cofactor specificity in a metalloenzyme.

Almost half of all enzymes utilize a metal cofactor. However, the features that dictate the metal utilized by metalloenzymes are poorly understood, limiting our ability to manipulate these enzymes for industrial and health-associated applications. The ubiquitous iron/manganese superoxide dismutase (SOD) family exemplifies this deficit, as the specific metal used by any family member cannot be predicted. Biochemical, structural and paramagnetic analysis of two evolutionarily related SODs with different metal specificity produced by the pathogenic bacterium Staphylococcus aureus identifies two positions that control metal specificity. These residues make no direct contacts with the metal-coordinating ligands but control the metal's redox properties, demonstrating that subtle architectural changes can dramatically alter metal utilization. Introducing these mutations into S. aureus alters the ability of the bacterium to resist superoxide stress when metal starved by the host, revealing that small changes in metal-dependent activity can drive the evolution of metalloenzymes with new cofactor specificity.

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.

Mass spectrometry reveals the assembly pathway of encapsulated ferritins and highlights a dynamic ferroxidase interface.

Encapsulated ferritins (EncFtn) are a recently characterised member of the ferritin superfamily. EncFtn proteins are sequestered within encapsulin nanocompartments and form a unique biological iron storage system. Here, we use native mass spectrometry and hydrogen-deuterium exchange mass spectrometry to elucidate the metal-mediated assembly pathway of EncFtn.