Bacterial Iron Homeostasis Research Articles

Transcriptional responses of Neisseria gonorrhoeae to glucose and lactate: implications for resistance to oxidative damage and biofilm formation.

Understanding how bacteria adapt to different environmental conditions is crucial for advancing knowledge regarding pathogenic mechanisms that operate during infection as well as efforts to develop new therapeutic strategies to cure or prevent infections. Here, we investigated the transcriptional response of Neisseria gonorrhoeae, the causative agent of gonorrhea, to L-lactate and glucose, two important carbon sources found in the host environment. Our study revealed extensive transcriptional changes that gonococci make in response to L-lactate, with 37% of the gonococcal transcriptome being regulated, compared to only 9% by glucose. We found that L-lactate induces a transcriptional program that would negatively impact iron transport, potentially limiting the availability of labile iron, which would be important in the face of the multiple hydrogen peroxide attacks encountered by gonococci during its lifecycle. Furthermore, we found that L-lactate-mediated transcriptional response promoted aerobic respiration and dispersal of biofilms, contrasting with an anaerobic condition previously reported to favor biofilm formation. Our findings suggest an intricate interplay between carbon metabolism, iron homeostasis, biofilm formation, and stress response in N. gonorrhoeae, providing insights into its pathogenesis and identifying potential therapeutic targets.IMPORTANCEGonorrhea is a prevalent sexually transmitted infection caused by the human pathogen Neisseria gonorrhoeae, with ca. 82 million cases reported worldwide annually. The rise of antibiotic resistance in N. gonorrhoeae poses a significant public health threat, highlighting the urgent need for alternative treatment strategies. By elucidating how N. gonorrhoeae responds to host-derived carbon sources such as L-lactate and glucose, this study offers insights into the metabolic adaptations crucial for bacterial survival and virulence during infection. Understanding these adaptations provides a foundation for developing novel therapeutic approaches targeting bacterial metabolism, iron homeostasis, and virulence gene expression. Moreover, the findings reported herein regarding biofilm formation and L-lactate transport and metabolism contribute to our understanding of N. gonorrhoeae pathogenesis, offering potential avenues for preventing and treating gonorrhea infections.

A Hemoglobin Bionics-Based System for Combating Antibiotic Resistance in Chronic Diabetic Wounds via Iron Homeostasis Regulation.

Owing to the increased tissue iron accumulation in patients with diabetes, microorganisms may activate high expression of iron-involved metabolic pathways, leading to the exacerbation of bacterial infections and disruption of systemic glucose metabolism. Therefore, an on-demand transdermal dosing approach that utilizes iron homeostasis regulation to combat antimicrobial resistance is a promising strategy to address the challenges associated with low administration bioavailability and high antibiotic resistance in treating infected diabetic wounds. Here, it is aimed to propose an effective therapy based on hemoglobin bionics to induce disturbances in bacterial iron homeostasis. The preferred "iron cargo" is synthesized by protoporphyrin IX chelated with dopamine and gallium (PDGa), and is delivered via a glucose/pH-responsive microneedle bandage (PDGa@GMB). The PDGa@GMB downregulates the expression levels of the iron uptake regulator (Fur) and the peroxide response regulator (perR) in Staphylococcus aureus, leading to iron nutrient starvation and oxidative stress, ultimately suppressing iron-dependent bacterial activities. Consequently, PDGa@GMB demonstrates insusceptibility to genetic resistance while maintaining sustainable antimicrobial effects (>90%) against resistant strains of both S. aureus and E. coli, and accelerates tissue recovery (<20 d). Overall, PDGa@GMB not only counteracts antibiotic resistance but also holds tremendous potential in mediating microbial-host crosstalk, synergistically attenuating pathogen virulence and pathogenicity.

Dps Functions as a Key Player in Bacterial Iron Homeostasis.

Iron plays a vital role in the maintenance of life, being central to various cellular processes, from respiration to gene regulation. It is essential for iron to be stored in a nontoxic and readily available form. DNA binding proteins under starvation (Dps) belong to the ferritin family of iron storage proteins and are adept at storing iron in their hollow protein shells. Existing solely in prokaryotes, these proteins have the additional functions of DNA binding and protection from oxidative stress. Iron storage proteins play a functional role in storage, release, and transfer of iron and therefore are central to the optimal functioning of iron homeostasis. Here we review the multifarious properties of Dps through relevant biochemical and structural studies with a focus on iron storage and ferroxidation. We also examine the role of Dps as a possible candidate as an iron donor to iron-sulfur (Fe-S) clusters, which are ubiquitous to many biological processes.

Mobilization of iron stored in bacterioferritin, a new target for perturbing iron homeostasis and developing antibacterial and antibiofilm molecules

Antibiotic resistance is a global public health threat. The care of chronic infections is complicated by bacterial biofilms. Biofilm embedded cells can be up to 1000-fold more tolerant to antibiotic treatment than planktonic cells. Antibiotic tolerance is a condition which does not involve mutation and enables bacteria to survive in the presence of antibiotics. The antibiotic tolerance of biofilm-cells often renders antibiotics ineffective, even against strains that do not carry resistance-impairing mutations. This review discusses bacterial iron homeostasis and the strategies being developed to target this bacterial vulnerability, with emphasis on a recently proposed approach which aims at targeting the iron storage protein bacterioferritin (Bfr) and its physiological partner, the ferredoxin Bfd. Bfr regulates cytosolic iron concentrations by oxidizing Fe2+ and storing Fe3+ in its internal cavity, and by forming a complex with Bfd to reduce Fe3+ in the internal cavity and release Fe2+ to the cytosol. Blocking the Bfr-Bfd complex in P. aeruginosa cells causes an irreversible accumulation of Fe3+ in BfrB and simultaneous cytosolic iron depletion, which leads to impaired biofilm maintenance and biofilm cell death. Recently discovered small molecule inhibitors of the Bfr-Bfd complex, which bind Bfr at the Bfd binding site, inhibit iron mobilization, and elicit biofilm cell death.

Natural flavonoids disrupt bacterial iron homeostasis to potentiate colistin efficacy.

In the face of the alarming rise in global antimicrobial resistance, only a handful of novel antibiotics have been developed in recent decades, necessitating innovations in therapeutic strategies to fill the void of antibiotic discovery. Here, we established a screening platform mimicking the host milieu to select antibiotic adjuvants and found three catechol-type flavonoids-7,8-dihydroxyflavone, myricetin, and luteolin-prominently potentiating the efficacy of colistin. Further mechanistic analysis demonstrated that these flavonoids are able to disrupt bacterial iron homeostasis through converting ferric iron to ferrous form. The excessive intracellular ferrous iron modulated the membrane charge of bacteria via interfering the two-component system pmrA/pmrB, thereby promoting the colistin binding and subsequent membrane damage. The potentiation of these flavonoids was further confirmed in an in vivo infection model. Collectively, the current study provided three flavonoids as colistin adjuvant to replenish our arsenals for combating bacterial infections and shed the light on the bacterial iron signaling as a promising target for antibacterial therapies.

Stringent Starvation Protein SspA and Iron Starvation Sigma Factor PvdS Coordinately Regulate Iron Uptake and Prodiginine Biosynthesis in Pseudoalteromonas sp. R3.

Organisms need sufficient intracellular iron to maintain biological processes. However, cells can be damaged by excessive iron-induced oxidation stress. Therefore, iron homeostasis must be strictly regulated. In general, bacteria have evolved complex mechanisms to maintain iron homeostasis. In this study, we showed that Pseudoalteromonas sp. R3 has four sets of iron uptake systems. Among these, the siderophore pyoverdine-dependent iron uptake system and the ferrous iron transporter Feo system are more important for iron uptake and prodiginine biosynthesis. Stringent starvation protein SspA positively controls iron uptake and iron-dependent prodiginine biosynthesis by regulating the expression of all iron uptake systems. In turn, the expression of SspA can be induced and repressed by extracellular iron deficiency and excess, respectively. Interestingly, extracytoplasmic function sigma factor PvdS also regulates iron uptake and prodiginine production and responds to extracellular iron levels, exhibiting a similar phenomenon as SspA. Notably, not only do SspA and PvdS function independently, but they can also compensate for each other, and their expression can be affected by the other. All of these findings demonstrate that SspA and PvdS coordinate iron homeostasis and prodiginine biosynthesis in strain R3. More importantly, our results also showed that SspA and PvdS homologs in Pseudomonas aeruginosa PAO1 have similar functions in iron uptake to their counterparts in Pseudoalteromonas, suggesting that coordination between SspA and PvdS on iron homeostasis could be conserved in typical Gram-negative bacteria. Since master regulation of iron homeostasis is extremely important for cell survival, this cross talk between SspA and PvdS may be environmentally significant. IMPORTANCE Both deficiency and excess of intracellular iron can be harmful, and thus, the iron homeostasis needs to be tightly regulated in organisms. At present, the ferric uptake regulator (Fur) is the best-characterized regulator involved in bacterial iron homeostasis, while other regulators of iron homeostasis remain to be further explored. Here, we demonstrated that the stringent starvation protein SspA and the extracytoplasmic function sigma factor PvdS coordinate iron uptake and iron-dependent prodiginine biosynthesis in Pseudoalteromonas sp. R3. These two regulators work independently, but their functions can compensate for the other and their expression can be affected by the other. Moreover, their expression can be activated and repressed by extracellular iron deficiency and excess, respectively. Notably, SspA and PvdS homologs in Pseudomonas aeruginosa PAO1 exhibit similar functions in iron uptake to their counterparts in Pseudoalteromonas, suggesting that this novel fine-tuned mode of iron homeostasis could be conserved in typical Gram-negative bacteria.

Identification and Tetramer Structure of Hemin-Binding Protein SPD_0310 Linked to Iron Homeostasis and Virulence of Streptococcus pneumoniae.

ABSTRACTIron and iron-containing compounds are essential for bacterial virulence and host infection. Hemin is an important supplement compound for bacterial survival in an iron-deficient environment. Despite strong interest in hemin metabolism, the detailed mechanism of hemin transportation in Gram-positive bacteria is yet to be reported. The results of our study revealed that the homologous proteins of SPD_0310 were significantly conservative in Gram-positive bacteria (P < 0.001), and these proteins were identified as belonging to an uncharacterized protein family (UPF0371). The results of thermodynamic and kinetic studies have shown that SPD_0310 has a high hemin-binding affinity. Interestingly, we found that the crystal structure of SPD_0310 presented a homotetramer conformation, which is required for hemin binding. SPD_0310 can interact with many hemin-binding proteins (SPD_0090, SPD_1609, and GAPDH) located on the cell surface, which contributes to hemin transfer to the cytoplasm. It also has a high affinity with other iron transporters in the cytoplasm (SPD_0226 and SPD_0227), which facilitates iron redistribution in cells. More importantly, the knockout of the spd_0310 gene (Δspd_0310) resulted in a decrease in the iron content and protein expression levels of many bacterial adhesion factors. Moreover, the animal model showed that the Δspd_0310 strain has a lower virulence than the wild type. Based on the crystallographic and biochemical studies, we inferred that SPD_0310 is a hemin intermediate transporter which contributes to iron homeostasis and further affects the virulence of Streptococcus pneumoniae in the host. Our study provides not only an important theoretical basis for the in-depth elucidation of the hemin transport mechanism in bacteria but also an important candidate target for the development of novel antimicrobial agents based on metal transport systems.IMPORTANCE Iron is an essential element for bacterial virulence and infection of the host. The detailed hemin metabolism in Gram-positive bacteria has rarely been studied. SPD_0310 belongs to the UPF0371 family of proteins, and results of homology analysis and evolutionary tree analysis suggested that it was widely distributed and highly conserved in Gram-positive bacteria. However, the function of the UPF0371 family remains unknown. We successfully determined the crystal structure of apo-SPD_0310, which is a homotetramer. We found that cytoplasmic protein SPD_0310 with a special tetramer structure has a strong hemin-binding ability and interacts with many iron transporters, which facilitates hemin transfer from the extracellular space to the cytoplasm. The results of detailed functional analyses indicated that SPD_0310 may function as a hemin transporter similar to hemoglobin in animals and contributes to bacterial iron homeostasis and virulence. This study provides a novel target for the development of antimicrobial drugs against pathogenic Gram-positive bacteria.

Host and Bacterial Iron Homeostasis, an Underexplored Area in Tuberculosis Biomarker Research.

Mycobacterium tuberculosis (Mtb) “a human adapted pathogen” has found multiple ways to manipulate the host immune response during infection. The human immune response to Mtb infection is a highly complex cascade of reactions, with macrophages as preferred intracellular location. Interaction with the host through infection gives rise to expression of specific gene products for survival and multiplication within the host. The signals that the pathogens encounter during infection cause them to selectively express genes in response to signals. One strategy to identify Mtb antigens with diagnostic potential is to identify genes that are specifically induced during infection or in specific disease stages. The shortcomings of current immunodiagnostics include the failure to detect progression from latent infection to active tuberculosis disease, and the inability to monitor treatment efficacy. This highlights the need for new tuberculosis biomarkers. These biomarkers should be highly sensitive and specific diagnosing TB infection, specifically distinguishing between latent infection and active disease. The regulation of iron levels by the host plays a crucial role in the susceptibility and outcome of Mtb infection. Of interest are the siderophore biosynthetic genes, encoded by the mbt-1 and mbt-2 loci and the SUF (mobilization of sulphur) operon (sufR-sufB-sufD-sufC-csd-nifU-sufT), which encodes the primary iron-sulphur cluster biogenesis system. These genes are induced during iron limitation and intracellular growth of Mtb, pointing to their importance during infection.

Laser capture microdissection to study Bacillus cereus iron homeostasis gene expression during Galleria mellonella in vivo gut colonization

ABSTRACT Bacillus cereus is a Gram-positive opportunistic pathogen closely related to the entomopathogen, Bacillus thuringiensis, both of which are involved in intestinal infections. Iron is an essential micronutrient for full growth and virulence of pathogens during infection. However, little is known about iron homeostasis during gut infection. Therefore, we aimed to assess the expression of B. cereus genes related to bacterial iron homeostasis, virulence and oxidative stress. The hypothesis is that the expression of such genes would vary between early and later stage colonization in correlation to gut cell damage. To perform the study, a germ-free Galleria mellonella model was set up in order to adapt the use of Laser-capture microdissection (LCM), to select precise areas in the gut lumen from frozen whole larval cryo-sections. Analyses were performed from alive larvae and the expression of targeted genes was assessed byspecific pre-amplification of mRNA followed by quantitative PCR. Firstly, the results reinforce the reliability of LCM, despite a low amount of bacterial RNA recovered. Secondly, bacterial genes involved in iron homeostasis are expressed in the lumen at both 3 and 16 hours post force-feeding. Thirdly, iron gene expression is slightly modulated during gut infection, and lastly, the mRNA of G. mellonella encoding for ferritin and transferrin iron storage and transport are recovered too. Therefore, iron homeostasis should play a role in B. cereus gut colonization. Furthermore, we demonstrate for the first time the value of using LCM for specific in situ gene expression analysis of extracellular bacteria in a whole animal.

Pyoverdine Inhibitors and Gallium Nitrate Synergistically Affect Pseudomonas aeruginosa.

ABSTRACTPseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogen that frequently causes ventilator-associated pneumonia in intensive care units and chronic lung infections in cystic fibrosis patients. The rising prevalence of drug-resistant bacteria demands the exploration of new therapeutic avenues for treating P. aeruginosa infections. Perhaps the most thoroughly explored alternative is to use novel treatments to target pathogen virulence factors, like biofilm or toxin production. Gallium(III) nitrate is one such agent. It has been recognized for its ability to inhibit pathogen growth and biofilm formation in P. aeruginosa by disrupting bacterial iron homeostasis. However, irreversible sequestration by pyoverdine substantially limits its effectiveness. In this report, we show that disrupting pyoverdine production (genetically or chemically) potentiates the efficacy of gallium nitrate. Interestingly, we report that the pyoverdine inhibitor 5-fluorocytosine primarily functions as an antivirulent, even when it indirectly affects bacterial growth in the presence of gallium, and that low selective pressure for resistance occurs. We also demonstrate that the antibiotic tetracycline inhibits pyoverdine at concentrations below those required to prevent bacterial growth, and this activity allows it to synergize with gallium to inhibit bacterial growth and rescue Caenorhabditis elegans during P. aeruginosa pathogenesis.IMPORTANCE P. aeruginosa is one of the most common causative agents for ventilator-associated pneumonia and nosocomial bacteremia and is a leading cause of death in patients with cystic fibrosis. Pandrug-resistant strains of P. aeruginosa are increasingly identified in clinical samples and show resistance to virtually all major classes of antibiotics, including aminoglycosides, cephalosporins, and carbapenems. Gallium(III) nitrate has received considerable attention as an antipseudomonal agent that inhibits P. aeruginosa growth and biofilm formation by disrupting bacterial iron homeostasis. This report demonstrates that biosynthetic inhibitors of pyoverdine, such as 5-fluorocytosine and tetracycline, synergize with gallium nitrate to inhibit P. aeruginosa growth and biofilm formation, rescuing C. elegans hosts during pathogenesis.

Free Rather Than Total Iron Content Is Critically Linked to the Fur Physiology in Shewanella oneidensis.

Ferric uptake regulator (Fur) is a transcriptional regulator playing a central role in iron homeostasis of many bacteria, and Fur inactivation commonly results in pleiotropic phenotypes. In Shewanella oneidensis, a representative of dissimilatory metal-reducing γ-proteobacteria capable of respiring a variety of chemicals as electron acceptors (EAs), Fur loss substantially impairs respiration. However, to date the mechanism underlying the physiological phenomenon remains obscure. This investigation reveals that Fur loss compromises activity of iron proteins requiring biosynthetic processes for their iron cofactors, heme in particular. We then show that S. oneidensis Fur is critical for maintaining heme homeostasis by affecting both its biosynthesis and decomposition of the molecule. Intriguingly, the abundance of iron-containing proteins controlled by H2O2-responding regulator OxyR increases in the fur mutant because the Fur loss activates OxyR. By comparing suppression of membrane-impermeable, membrane-permeable, and intracellular-only iron chelators on heme deficiency and elevated H2O2 resistance, our data suggest that the elevation of the free iron content by the Fur loss is likely to be the predominant factor for the Fur physiology. Overall, these results provide circumstantial evidence that Fur inactivation disturbs bacterial iron homeostasis by altering transcription of its regulon members, through which many physiological processes, such as respiration and oxidative stress response, are transformed.

The battle for iron in enteric infections.

SummaryIron is an essential element for almost all living organisms, but can be extremely toxic in high concentrations. All organisms must therefore employ homeostatic mechanisms to finely regulate iron uptake, usage and storage in the face of dynamic environmental conditions. The critical step in mammalian systemic iron homeostasis is the fine regulation of dietary iron absorption. However, as the gastrointestinal system is also home to >1014 bacteria, all of which engage in their own programmes of iron homeostasis, the gut represents an anatomical location where the inter‐kingdom fight for iron is never‐ending. Here, we explore the molecular mechanisms of, and interactions between, host and bacterial iron homeostasis in the gastrointestinal tract. We first detail how mammalian systemic and cellular iron homeostasis influences gastrointestinal iron availability. We then focus on two important human pathogens, Salmonella and Clostridia; despite their differences, they exemplify how a bacterial pathogen must navigate and exploit this web of iron homeostasis interactions to avoid host nutritional immunity and replicate successfully. We then reciprocally explore how iron availability interacts with the gastrointestinal microbiota, and the consequences of this on mammalian physiology and pathogen iron acquisition. Finally, we address how understanding the battle for iron in the gastrointestinal tract might inform clinical practice and inspire new treatments for important diseases.

Bacillus subtilis Fur Is a Transcriptional Activator for the PerR-Repressed pfeT Gene, Encoding an Iron Efflux Pump.

The physiological relevance of bacterial iron efflux has only recently been appreciated. The Bacillus subtilis P1B4-type ATPase PfeT (peroxide-induced ferrous efflux transporter) was one of the first iron efflux pumps to be characterized, and cells lacking pfeT accumulate high levels of intracellular iron. The pfeT promoter region has binding sites for both PerR, a peroxide-sensing Fur-family metalloregulator, and the ferric uptake repressor Fur. Both Fur and PerR bind DNA with Fe(II) as a cofactor. While reaction of PerR-Fe(II) with peroxide can account for the induction of pfeT under oxidative stress, binding of Fur-Fe(II) would be expected to lead to repression, which is inconsistent with the known role of PfeT as an iron efflux protein. Here, we show that expression of pfeT is repressed by PerR, as anticipated, and induced by Fur in response to Fe(II). Activation by Fur is mediated both by antagonism of the PerR repressor and by direct transcriptional activation, as confirmed using in vitro transcription assays. A similar mechanism of regulation can explain the iron induction of the Listeria monocytogenes PfeT ortholog and virulence factor, FrvA. Mutational studies support a model in which Fur activation involves regions both upstream and downstream of the pfeT promoter, and Fur and PerR have overlapping recognition of a shared regulatory element in this complex promoter region. This work demonstrates that B. subtilis Fur can function as an iron-dependent activator of transcription.IMPORTANCE Iron homeostasis plays a key role at the host-pathogen interface during the process of infection. Bacterial growth restriction resulting from host-imposed iron starvation (nutritional immunity) highlights the importance of iron import during pathogenesis. Conversely, bacterial iron efflux pumps function as virulence factors in several systems. The requirement for iron efflux in pathogens such as Listeria monocytogenes, Streptococcus pyogenes, and Mycobacterium tuberculosis suggests that both import and efflux are needed for cells to successfully navigate rapidly changing levels of iron availability in the host. Here, we provide insight into how iron efflux genes are controlled, an aspect of bacterial iron homeostasis relevant to infectious disease processes.

Transcriptomic Analysis of Streptococcus pyogenes Colonizing the Vaginal Mucosa Identifies hupY, an MtsR-Regulated Adhesin Involved in Heme Utilization.

Streptococcus pyogenes (group A streptococcus [GAS]) is a serious human pathogen with the ability to colonize mucosal surfaces such as the nasopharynx and vaginal tract, often leading to infections such as pharyngitis and vulvovaginitis. We present genome-wide transcriptome sequencing (RNASeq) data showing the transcriptomic changes GAS undergoes during vaginal colonization. These data reveal that the regulon controlled by MtsR, a master metal regulator, is activated during vaginal colonization. This regulon includes two genes highly expressed during vaginal colonization, hupYZ Here we show that HupY binds heme in vitro, affects intracellular concentrations of iron, and is essential for proper growth of GAS using hemoglobin or serum as the sole iron source. HupY is also important for murine vaginal colonization of both GAS and the related vaginal colonizer and pathogen Streptococcus agalactiae (group B streptococcus [GBS]). These data provide essential information on the link between metal regulation and mucosal colonization in both GAS and GBS.IMPORTANCE Colonization of the host requires the ability to adapt to an environment that is often low in essential nutrients such as iron. Here we present data showing that the transcriptome of the important human pathogen Streptococcus pyogenes shows extensive remodeling during in vivo growth, resulting in, among many other differentially expressed genes and pathways, a significant increase in genes involved in acquiring iron from host heme. Data show that HupY, previously characterized as an adhesin in both S. pyogenes and the related pathogen Streptococcus agalactiae, binds heme and affects intracellular iron concentrations. HupY, a protein with no known heme binding domains, represents a novel heme binding protein playing an important role in bacterial iron homeostasis as well as vaginal colonization.

Small Molecule Inhibitors of the BfrB-Bfd Interaction Decrease Pseudomonas aeruginosa Fitness and Potentiate Fluoroquinolone Activity.

The iron storage protein bacterioferritin (BfrB) is central to bacterial iron homeostasis. The mobilization of iron from BfrB, which requires binding by a cognate ferredoxin (Bfd), is essential to the regulation of cytosolic iron levels in P. aeruginosa. This paper describes the structure-guided development of small molecule inhibitors of the BfrB–Bfd protein–protein interaction. The process was initiated by screening a fragment library and followed by obtaining the structure of a fragment hit bound to BfrB. The structural insights were used to develop a series of 4-(benzylamino)- and 4-((3-phenylpropyl)amino)-isoindoline-1,3-dione analogs that selectively bind BfrB at the Bfd binding site. Challenging P. aeruginosa cells with the 4-substituted isoindoline analogs revealed a dose-dependent growth phenotype. Further investigation determined that the analogs elicit a pyoverdin hyperproduction phenotype that is consistent with blockade of the BfrB–Bfd interaction and ensuing irreversible accumulation of iron in BfrB, with concomitant depletion of iron in the cytosol. The irreversible accumulation of iron in BfrB prompted by the 4-substituted isoindoline analogs was confirmed by visualization of BfrB-iron in P. aeruginosa cell lysates separated on native PAGE gels and stained for iron with Ferene S. Challenging P. aeruginosa cultures with a combination of commercial fluoroquinolone and our isoindoline analogs results in significantly lower cell survival relative to treatment with either antibiotic or analog alone. Collectively, these findings furnish proof of concept for the usefulness of small molecule probes designed to dysregulate bacterial iron homeostasis by targeting a protein–protein interaction pivotal for iron storage in the bacterial cell.

Regulation of Iron Uptake by Fine-Tuning the Iron Responsiveness of the Iron Sensor Fur.

Iron is one of most abundant environmental metal ions but is highly limited in organisms. It is an important metal ion as it facilitates various biological processes, including catalysis of metabolic enzymes and DNA biogenesis. In bacteria, the ferric uptake regulator (Fur) protein controls iron uptake by regulating genes coding for iron transporters in response to iron concentration. This iron response is ascribed to Fur's intrinsic affinity for iron because its binding to iron dictates its regulatory function. However, we now report that the pathogen Salmonella achieves a proper response of Fur to changes in environmental iron concentrations via EIIANtr (a nitrogen metabolic phosphotransferase system component). We establish that EIIANtr increases expression of iron transporter-coding genes under low-iron conditions (i.e., nanomolar ranges) in a Fur-dependent manner, which promotes Salmonella growth under such conditions. EIIANtr directly hampers Fur binding to DNA, thereby inducing expression of those genes. This regulation allows Salmonella to express Fur-regulated genes under low-iron conditions. Our findings reveal a potentially widespread control mechanism of bacterial iron uptake systems operating in response to iron availability.IMPORTANCE Iron is a fundamental metal ion for living organisms as it facilitates various biological processes. The ferric uptake regulator (Fur) protein controls iron homeostasis in various bacterial species. It is believed that Fur's iron-dependent regulatory action is sufficient for it to function as an iron sensor. However, we now establish that the bacterial pathogen Salmonella enables Fur to properly reflect changes in surrounding iron availability by fine-tuning its responsiveness to iron. This process requires a protein that hampers Fur DNA binding at low iron concentrations. In this way, Salmonella broadens the range of iron concentrations that Fur responds to. Our findings reveal a potentially widespread control mechanism of bacterial iron homeostasis.

Bfd, a New Class of [2Fe-2S] Protein That Functions in Bacterial Iron Homeostasis, Requires a Structural Anion Binding Site.

Mobilization of iron from bacterioferritin (BfrB) requires specific interactions with a [2Fe-2S] ferredoxin (Bfd). Blocking the BfrB:Bfd interaction results in irreversible iron accumulation in BfrB and iron deficiency in the cytosol [Eshelman, K., et al. (2017) Metallomics 9, 646-659]. The only known Bfd structure, which was obtained in complex with BfrB (Protein Data Bank entry 4E6K ), indicated a new fold and suggested that the stability of Bfd is aided by an anion binding site consisting of R26, R29, and K46. We investigated the Bfd fold using site-directed mutagenesis, X-ray crystallography, and biochemistry in solution. The X-ray structure, which is nearly identical to that of Bfd in the BfrB:Bfd complex, shows that the [2Fe-2S] cluster preorganizes residues at the BfrB:Bfd interface into a structure complementary to the Bfd binding site on BfrB. Studies in solution showed rapid loss of the [2Fe-2S] cluster at a low ionic strength but higher stability with an increasing ionic strength, thus supporting a structural anion binding site. Structures of the R26E and R26E/K46Y mutants are nearly identical to that of Bfd, except for a new network of hydrogen bonds stabilizing the region encompassing the former anion binding site. The stability of the R26E and R26E/K46Y mutants, which is weakly and completely independent of solution ionic strength, respectively, corroborates that Bfd requires an anion binding site. The mutations, which caused only small changes to the strength of the BfrB:Bfd interaction and mobilization of iron from BfrB, indicate that the anion binding site in Bfd serves primarily a structural role.

Bacterial Iron Homeostasis Regulation by sRNAs.

While iron is essential to sustain growth, its excess can be detrimental to the cell by generating highly toxic reactive oxygen species. Regulation of iron homeostasis thus plays a vital role in almost all living organisms. During the last 15 years, the small RNA (sRNA) RyhB has been shown to be a key actor of iron homeostasis regulation in bacteria. Through multiple molecular mechanisms, RyhB represses expendable iron-utilizing proteins, promotes siderophore production, and coordinates Fe-S cluster cofactor biogenesis, thereby establishing a so-called iron-sparing response. In this review, we will summarize knowledge on how sRNAs control iron homeostasis mainly through studies on RyhB in Escherichia coli. The parallel roles and modes of action of other sRNAs in different bacteria will also be described. Finally, we will discuss what questions remain to be answered concerning this important stress response regulation by sRNAs.

Exposure of Escherichia coli to human hepcidin results in differential expression of genes associated with iron homeostasis and oxidative stress.

Hepcidin belongs to the antimicrobial peptide family but has weak activity with regards to bacterial killing. The regulatory function of hepcidin in humans serves to maintain an iron-restricted environment that limits the growth of pathogens; this study explored whether hepcidin affected bacterial iron homeostasis and oxidative stress using the model organism Escherichia coli. Using the Miller assay it was determined that under low iron availability exposure to sub-inhibitory doses of hepcidin (4-12μM) led to 2-fold and 4-fold increases in the expression of ftnA and bfd, respectively (P < 0.05), in both a wild type (WT) and Δfur (ferric uptake regulator) background. Quantitative real-time PCR analysis of oxyR and sodA, treated with 4 or 8 μM of hepcidin showed that expression of these genes was significantly (P < 0.05) increased, whereas expression of lexA was unchanged, indicating that hepcidin likely mediated oxidative stress but did not induce DNA damage.

The iron-stress activated RNA 1 (IsaR1) coordinates osmotic acclimation and iron starvation responses in the cyanobacterium Synechocystis sp. PCC 6803.

In nature, microorganisms are exposed to multiple stress factors in parallel. Here, we investigated the response of the model cyanobacterium Synechocystis sp. PCC 6803 to simultaneous iron limitation and osmotic stresses. Iron is a major limiting factor for bacterial and phytoplankton growth in most environments. Thus, bacterial iron homeostasis is tightly regulated. In Synechocystis, it is mediated mainly by the transcriptional regulator FurA and the iron-stress activated RNA 1 (IsaR1). IsaR1 is an important riboregulator that affects the acclimation of the photosynthetic apparatus to iron starvation in multiple ways. Upon increases in salinity, Synechocystis responds by accumulating the compatible solute glucosylglycerol (GG). We show that IsaR1 overexpression causes a reduction in the de novo GG synthesis rate upon salt shock. We verified the direct interaction between IsaR1 and the 5'UTR of the ggpS mRNA, which in turn drastically reduced the de novo synthesis of the key enzyme for GG synthesis, glucosylglycerol phosphate synthase (GgpS). Thus, IsaR1 specifically interferes with the salt acclimation process in Synechocystis, in addition to its primary regulatory function. Moreover, the salt-stimulated GgpS production became reduced under parallel iron limitation in WT - an effect which is, however, attenuated in an isaR1 deletion strain. Hence, IsaR1 is involved in the integration of the responses to different environmental perturbations and slows the osmotic adaptation process in cells suffering from parallel iron starvation.