Metal limitation and toxicity at the interface between host and pathogen

Abstract

Metals are required cofactors for numerous fundamental processes that are essential to both pathogen and host. They are coordinated in enzymes responsible for DNA replication and transcription, relief from oxidative stress, and cellular respiration. However, excess transition metals can be toxic due to their ability to cause spontaneous, redox cycling and disrupt normal metabolic processes. Vertebrates have evolved intricate mechanisms to limit the availability of some crucial metals while concurrently flooding sites of infection with antimicrobial concentrations of other metals. To compete for limited metal within the host while simultaneously preventing metal toxicity, pathogens have developed a series of metal regulatory, acquisition, and efflux systems. This review will cover the mechanisms by which pathogenic bacteria recognize and respond to host-induced metal scarcity and toxicity.

Introduction

Maintenance of metal homeostasis is important for both eukaryotes and bacteria. Metals serve as cofactors involved in many processes, including energy generation, electron transfer, DNA replication and transcription. However, many transition metals are toxic at high concentrations; this stems from the ability of some metals to participate as catalysts in redox cycling or the improper metallation of metalloproteins with the incorrect metal. Due to the necessity for invading bacteria to acquire nutrient metals from their environment, vertebrate hosts have developed mechanisms for restricting bioavailability to these metals in a process called nutritional immunity (Weinberg 1975; Oppenheimer 2001; Subramanian Vignesh et al. 2013; Lisher & Giedroc 2013). In response, successful pathogens have evolved the means to sense decreased metal levels and respond with the production of dedicated metal scavenging and import machinery. Conversely, an emerging component of this battle between host and pathogen is host-directed metal intoxication against invading microbes and the ability of pathogens to sufficiently detoxify this assault. Therefore, pathogenic bacteria must be adept at sensing changes in metal levels and reacting to meet the ever-changing environment in the host.

The battle for Fe at the pathogen-host interface

Fe is the most common element by mass on earth and it is required by all forms of life as a cofactor in myriad cellular processes ranging from DNA replication to central metabolism (Andreini et al. 2008). In vertebrates, Fe is predominantly coordinated into a porphyrin ring in the form of heme, and heme is required for transport and storage of oxygen throughout the body. In addition to its critical role in oxygen metabolism, heme is also a cytochrome cofactor in both eukaryotes and bacteria where it participates in energy generating processes. Beyond heme, bacteria require Fe for other processes. Fe-S clusters are needed to populate enzymes where they play roles in electron transfer, protein stabilization, and as sensors of superoxides (Beinert et al. 1997). Furthermore, elemental Fe is necessary to populate many enzymes; for example, the superoxide dismutase SodB in the gastric pathogen Helicobacter pylori requires an Fe cofactor (Ernst et al. 2005). Due to this microbial requirement for Fe, animals have evolved intricate mechanisms to limit this metal in the body as a means to protect against pathogen invasion and colonization.

Host mechanisms for restricting Fe from invading pathogens

Vertebrates have evolved several mechanisms to reduce the availability of ferric Fe (Fe³⁺) as a strategy to restrict microbial growth (Figure 1). At physiological pH, Fe²⁺ is rapidly oxidized to Fe³⁺, which is insoluble and bound by transferrin for transport throughout the body. The exception to this is in the duodenum where the decreased pH caused by gastric acids increases the solubility of Fe³⁺, allowing for its absorption. Lactoferrin, a glycoprotein of the transferrin family, binds Fe³⁺ and is found in milk, tears, and the mucosa of nasal passages. Lactoferrin is also present in the secondary granules of neutrophils, which contributes to lactoferrin being a primary component of the innate immune response to invading pathogens (Sánchez et al. 1992). In addition to transferrin and lactoferrin, neutrophils are also able to secrete hepcidin, the peptide hormone regulator of dietary Fe uptake, in response to Gram-positive and Gram-negative infections, which can occur in a TLR4-dependent manner (Peyssonnaux et al. 2006). This leads to decreased Fe uptake and the subsequent “anemia of inflammation” (Drakesmith & Prentice 2012). Intracellularly, Fe³⁺ is complexed within the storage protein ferritin. This protein serves to regulate the levels of Fe in the host, prevent Fe toxicity, and isolate Fe from intracellular pathogens (Rogers et al. 1990). The host limitation of nutrient Fe necessitates that any microbe capable of colonizing or infecting vertebrates must have mechanisms by which they obtain this important metal.

Figure 1. Fe limitation by the host.

In erythrocytes, Fe is complexed within heme and bound by hemoglobin. Upon erythrocyte lysis, hemoglobin is scavenged by haptoglobin and the complex is recognized by CD163 on macrophages, heme is bound by hemopexin, and free Fe is foraged by transferrin and lactoferrin, which is present in the secondary granules of neutrophils. Neutrophils also release siderocalin which complexes with mammalian and bacterial siderophores. Intracellular Fe stores are maintained in association with ferritin.

Since the most abundant Fe source within vertebrates is heme, many pathogens have evolved mechanisms that exploit heme as a nutrient Fe source. In response, vertebrates have developed a multi-layered approach to sequester heme away from bacteria. Each tetrapyrrole ring of heme supports the coordination of one Fe²⁺ ion. Heme is subsequently complexed within hemoglobin and myoglobin, which are unavailable to circulating bacteria due to the sequestration of these proteins within erythrocytes and muscle tissue, respectively. Each myoglobin binds one heme, whereas each hemoglobin complexes four heme molecules. When hemoglobin is released from erythrocytes, it is quickly bound with high affinity by haptoglobin. CD163 is a scavenger receptor present on macrophages that binds to the haptoglobin-hemoglobin complex (Kristiansen et al. 2001). Similarly, free heme is bound by hemopexin with very high affinity (K_d < 1 pM) (Paoli et al. 1999). Together, haptoglobin and hemopexin prevent pathogen access to free hemoglobin and heme which aids in maintaining very low free Fe levels in the bloodstream.

Bacterial regulation of Fe acquisition

There exist several mechanisms by which bacteria respond to alterations in environmental Fe concentrations (Figure 2). The most common mechanism is through the regulated expression of Fe acquisition systems. The most well studied regulators of bacterial Fe metabolism include the ferric uptake regulator (Fur), the diphtheria toxin repressor (DtxR), and small RNAs. The genomes of most bacterial pathogens generally encode either dtxR or fur. One notable exception to this is Mycobacterium tuberculosis, which expresses homologues of both Fe regulators (Agranoff & Krishna 2004). Both Fur and DtxR function as homodimers and generally act as repressors of transcription in the presence of Fe. Upon Fe release, there is a conformational change in the dimer and the target DNA transcripts are de-repressed (Wakeman & Skaar 2012). The absence of free Fe within vertebrates is one way by which bacterial pathogens sense their exit from the environment and entry into an animal. In this regard, Fur and DtxR serve as sensors that regulate virulence factors in addition to Fe acquisition systems. One example of Fe-regulated virulence factors is the diphtheria toxin produced by Corynebacterium diphtheriae, the causative agent of diphtheria, from which the DtxR class of Fe-responsive regulators gets its name (Boyd et al. 1990; Schmitt & Holmes 1991). The diphtheria toxin is an AB exotoxin, which enters host cells via receptor mediated endocytosis; once in the cytosol, the toxin acts through the inhibition of translation. Vibrio vulnificus is a Gram-negative bacterium that causes diarrhea and vomiting following ingestion of contaminated undercooked seafood. In this organism, the quorum sensing regulator SmcR is regulated by both Fur and quorum sensing small molecules. In conditions of replete Fe, SmcR is repressed by Fur. In the low Fe conditions encountered within the host, Fur no longer binds the promoter of smcR and it is regulated strictly by quorum sensing (Kim et al. 2013a). Similarly, many virulence factors have been shown to be regulated by Fur de-repression during the course of Staphylococcus aureus pathogenesis. Upon entering the host and sensing the Fe-limited environment, S. aureus up-regulates many virulence factors. Among these are the alpha toxin Hla, which leads to erythrocyte hemolysis, and the leukotoxin LukED (Torres et al. 2010). LukED is a pore forming toxin which binds to leukocytes via CCR5 (Alonzo et al. 2013). Together, DtxR and Fur are responsible for sensing the limited Fe environment in the host and altering gene expression to promote pathogen survival and transmission.

Figure 2. Bacterial response to Fe limitation.

In conditions of Fe limitation, DtxR and Fur de-repress Fe-regulated genes. These include Fe and heme uptake systems, siderophore production, export, and acquisition systems, and various virulence factors. The RyhB sRNA is also transcribed upon Fe limitation and generally inhibits translation of target mRNAs, leading to alterations in the central metabolic pathways and decreased ferritin levels inside of the cell.

In addition to the protein regulators of Fe homeostasis, bacteria employ small RNAs to control transcript levels of genes following Fe limitation; these include genes responsible for Fe uptake and virulence, altered central metabolism, and abundance of ferritin within the bacterial cell (Gottesman 2004; Richards & Vanderpool 2011; Oglesby-Sherrouse & Murphy 2013). These sRNAs are conserved throughout the Enterobacteriaceae family. Generally, sRNAs bind to the 5’-untranslated region of target mRNAs resulting in inhibition of ribosome binding and subsequent degradation of the mRNA transcript. In Escherichia coli, the small RNA RyhB is regulated by Fur, and when de-repressed in Fe-poor conditions, leads to decreased levels of the succinate dehydrogenase transcript, (sdhCDAB), genes encoding aconitase and fumarate hydratases (acnA and fumA), ferritin (ftnA and bfr), and superoxide dismutase (sodB) (Massé & Gottesman 2002). Additionally, the E. coli RyhB increases the degradation of msrB transcript but not the msrA transcript (Bos et al. 2013). MsrA and MsrB are both methionine sulfoxide reductases which enzymatically remove oxidative damage from methionine residues. MsrB requires the use of Fe as a cofactor, whereas MsrA does not; thus, in conditions of limiting Fe, RyhB targets msrB for degradation and does not bind msrA (Bos et al. 2013). This strategy enables protection from reactive oxygen species-induced stress without depleting the limited available Fe. The RyhB homolog in Pseudomonas aeruginosa is PrrF; it has also been shown to be regulated by Fur and impact the levels of sodB and sdh (Wilderman et al. 2004). Beyond the transcriptional regulation of the Fe-responsive proteome, sRNAs are emerging as a secondary mechanism by which bacteria adjust to alterations in available Fe levels.

Bacterial Fe acquisition strategies

Pathogens have evolved sophisticated strategies to circumvent the nutritional immunity imposed by the vertebrate host. In order to liberate hemoglobin from erythrocytes, many bacteria secrete hemolysins. These proteins either degrade the erythrocyte membrane or multimerize to form pores leading to osmotic lysis, allowing the pathogen access to free hemoglobin. However, once hemoglobin is released from erythrocytes, bacteria must remove the heme from hemoglobin, transport this molecule into the cytoplasm, and degrade the ring of heme to release free Fe. In Gram-positive pathogens, the most well studied system that performs these functions is the Fe-regulated surface determinant (Isd) system. The prototypical Isd system is found in S. aureus. In this series of proteins, hemoglobin or a hemoglobin-haptoglobin complexes are bound by the cell wall receptors IsdB or IsdH, respectively (Dryla et al. 2003; Torres et al. 2006). Heme is then extracted and passed to the heme receptor, IsdA, where it is shuttled through IsdC across the cell wall before being translocated into the cytoplasm by the ATPase binding cassette (ABC) family transporter IsdEF (Mazmanian et al. 2003; Muryoi et al. 2008).

Gram-negative pathogens can passage heme through a heme-specific outer membrane transporter, which is powered by the proton motive force transferred through a TonB/ExbB/ExbD system (Runyen-Janecky 2013). In uropathogenic E. coli (UPEC), deletion of tonB abolishes the ability of the bacterium to utilize heme, or the siderophores aerobactin and enterobactin as Fe sources (Torres et al. 2001). Furthermore, when the outer membrane heme receptor ChuA is mutated, the ability for UPEC to grow on medium with hemin as the sole Fe source is greatly reduced (Torres et al. 2001). Similarly, Neisseria menigitidis, the causative agent of meningococcal meningitis, preferentially binds human hemoglobin through the outer membrane protein HmbR, extracts heme, and passes the liberated heme across the membrane in a TonB/ExbB/ExbD-dependent manner (Stojiljkovic et al. 1996; Stojiljkovic & Srinivasan 1997). HasA, the secreted heme binding protein from Serratia marcescens, transfers heme to the outer membrane receptor HasR through direct protein-protein interactions in a TonB-independent manner (Izadi-Pruneyre et al. 2006). For the transport of heme across the membrane, S. marcescens employs two TonB proteins, TonBSM and HasB. In addition to transferring free heme into the cell, HasR is capable of passaging heme bound to a hemophore in a process that requires a higher concentration of TonB than the intake of heme alone; the transfer of the hemophore bound heme requires TonBSM and not HasB (Paquelin et al. 2001; Létoffé et al. 2004). The outer membrane transporter of Yersinia enterocolitica, HemR, is capable of also exploiting myoglobin, hemopexin, and haptoglobin-hemoglobin complexes as heme sources (Bracken et al. 1999). Once inside the outer membrane of Gram-negative bacteria, heme is transported into the cytoplasm through ABC type transporters (Runyen-Janecky 2013).

Within the cytoplasm, Fe must be released from the tetrapyrrole ring of heme through the activity of bacterial heme oxygenases. The most well studied family of heme degrading enzymes is the heme oxygenase 1 (HO-1) family whose members are present in both eukaryotes and bacteria. The HO-1 family of heme catabolizing enzymes cleaves the ring and releases Fe²⁺, biliverdin, and carbon monoxide (Wilks 2002; Wilks & Heinzl 2013). Biliverdin and carbon monoxide have anti-oxidative, anti-inflammatory, and signaling properties in the host (Otterbein et al. 2000; Maghzal et al. 2009); at high concentrations, carbon monoxide can also be toxic to the host partially through its ability to bind hemoglobin and myoglobin at a higher affinity than oxygen. Furthermore, carbon monoxide produced by host HO-1 is bactericidal against multiple pathogens and can induce dormancy in the M. tuberculosis life cycle (Nobre et al. 2007; Kumar et al. 2008; Tavares et al. 2012). A second family of heme degrading enzymes that has only been identified in bacteria is the IsdG family. The IsdG family is comprised of structurally similar enzymes that degrade heme to release free Fe. Within this family, the staphylococcal IsdG and IsdI heme oxygenases result in the production of Fe²⁺, staphylobilin, and formaldehyde (Reniere et al. 2010; Haley et al. 2011; Matsui et al. 2013), whereas the M. tuberculosis MhuD heme oxygenase degrades heme to Fe²⁺ and mycobilin (Nambu et al. 2013). Thus far, signaling functions have not been identified for formaldehyde, staphylobilin, biliverdin, or mycobilin in bacteria. Defining the functions of heme oxygenase end-products in bacteria may provide new insights into the evolutionary pressures that gave rise to distinct families of heme degrading enzymes in this group of organisms.

Bacteria also acquire Fe from non-heme sources through the secretion of low molecular weight Fe-binding complexes known as siderophores. Siderophores are secreted from the bacterial cell where they complex Fe before being internalized through dedicated siderophore import systems. Siderophores have a higher affinity for Fe³⁺ than transferrin and lactoferrin and can therefore outcompete these host factors for this critical nutrient. In Vibrio parahaemolyticus, production of the siderophore vibrioferrin is transcriptionally regulated by Fur and post-transcriptionally stabilized through formation of a complex with the RyhB sRNA and Hfq chaperone protein (Tanabe et al. 2013). This is unusual since RyhB typically inhibits the translation of target mRNAs. To combat Fe acquisition by some siderophores, neutrophils release the siderophore binding protein siderocalin (also known as lipocalin-2 and neutrophil gelatinase-associated lipocalin) (Flo et al. 2004). In response, certain bacteria have evolved ‘stealth siderophores’ that are able to evade the host siderocalin. For example, Bacillus anthracis, the causative agent of anthrax, synthesizes petrobactin, which utilizes a less commonly found Fe chelating subunit precluding binding of the siderophore by siderocalin (Abergel et al. 2006). Salmonella enterica serovar Typhimurium secretes salmochelin, a stealth siderophore that is too large to be bound by siderocalin (Crouch et al. 2008; Müller et al. 2009). Whether siderocalin is the only anti-siderophore system produced in response to invading pathogens has yet to be determined; there may exist as-yet-unidentified processes by which the host is able to inhibit or sequester these stealth siderophores, further extending this arms race for nutrient Fe.

Compounds similar to siderophores found in bacteria have been identified in mammals (Correnti & Strong 2012). The Fe-binding properties of these molecules were discovered through their interactions with siderocalin. Catechol is one of these “mammalian siderophores” that interacts with siderocalin with nanomolar affinity when in the presence of Fe, but only low micromolar affinity in the absence of Fe (Bao et al. 2010). A second identified “mammalian siderophore” is 2,5-dihydroxybenzoic acid; this compound is very similar to the Fe binding portion of enterobactin, 2,3-dihydroxybenzoic acid. RNA interference of BDH2, the gene product that catalyzes the final step in the synthesis of this molecule, results in increased levels of cytoplasmic Fe and decreased levels of mitochondrial Fe. This inability to traffic Fe to the mitochondria leads to a marked reduction in heme synthesis (Devireddy et al. 2010). It is currently unclear whether these molecules that interact with siderocalin play a role in infection; additionally, further research is needed to identify other potential host Fe-binding molecules as well as to elucidate their functions in vivo.

In addition to siderophore-mediated Fe acquisition, some bacteria express transferrin and lactoferrin binding proteins that are able to bind and extract Fe from these host proteins. The most well characterized members of these proteins are those found in the Neisseriaceae family (Gray-Owen & Schryvers 1996). In most cases, two transferrin or lactoferrin binding proteins are present—TbpA/B and LbpA/B, respectively. When tbpA, but not tbpB, is inactivated in Neisseria gonorrhoeae, the bacteria are unable to take up Fe from transferrin as the sole Fe source; conversely, tbpB mutants only exhibited decreased binding of transferrin and Fe uptake (Anderson et al. 1994). Indeed, a strain of N. gonorrhoeae inactivated for both tbpA and tbpB was unable to cause urethritis in a human model of infection (Cornelissen et al. 1998). Similarly, LbpA has been shown to be essential for lactoferrin binding and Fe uptake in N. meningitidis (Bonnah & Schryvers 1998). More recently, transferrin and lactoferrin binding proteins have been identified in Gram-positive pathogens. For example, the pneumococcal surface protein A (PspA) is capable of binding human lactoferrin (Hammerschmidt et al. 1999).

Fe homeostasis also affects the composition of the gut microbiota. In both in vitro and in vivo models of Fe depletion, levels of butyrate and propionate are decreased; concurrently, populations of Lactobacilli and Enterobacteriaceae are increased in these conditions (Dostal et al. 2012; Dostal et al. 2013). Additionally, in rats fed low Fe diets, there are decreased numbers of neutrophils infiltrating into the mucosa of the colon (Dostal et al. 2012). Neutrophil levels and production of butyrate and propionate increase upon dietary Fe supplementation. Alterations in inflammation and the dysbiotic composition of commensal microbes could play a role in disease severity and pathogenesis. In a gerbil model of H. pylori infection, animals with decreased dietary Fe developed malignant lesions at a greater rate than animals fed a standard diet (Noto et al. 2013). Furthermore, a probiotic strain of E. coli (Nissle 1917) outcompetes the Fe scavenging abilities of S. Typhimurium through the production of additional siderophores and a heme uptake system. This competition leads to a decrease in Salmonella burden with a concomitant decrease in gut inflammation (Deriu et al. 2013). Taken together, these experiments are demonstrative of the growing evidence exposing a role for dietary Fe and the normal flora in susceptibility to and combatting of intestinal pathogens. Further research is warranted in this expanding field to investigate specific commensal-pathogen interactions, as well as the role of many dietary metals in the course of infections.

Alterations in host Fe homeostasis lead to varied susceptibility to pathogens

Genetic defects in the ability of the host to properly metabolize Fe and synthesize heme can alter the host susceptibility to many infections. Hemochromatosis, a condition of Fe overload in the body, can be the result of both genetic and environmental causes. Hereditary hemochromatosis is an autosomal recessive disorder due to mutations in the HFE gene located on chromosome six in humans. Under normal circumstances, the HFE gene product is membrane localized and regulates the expression of hepcidin and, in this manner, is necessary for normal absorption of Fe. Mutations in HFE can lead to increased absorption of Fe through the intestine; since Fe is generally not secreted via excrement, deposits of the excess Fe are formed in body tissues. Secondary hemochromatosis is Fe overload of non-genetic origin. This can be caused by a chronic need for blood transfusions due to another disorder, such as anemia or thalassemia, or through increased oral dietary or supplemental Fe intake. This increased level of available Fe has been associated with increased susceptibility to a variety of pathogens, including Yersinia enterocolitica (Höpfner et al. 2001), Vibrio vulnificus (Gerhard et al. 2001; Barton & Acton 2009), and Listeria monocytogenes (van Asbeck et al. 1982; Galan et al. 2011). Interestingly, in a murine model of hereditary hemochromatosis, neutrophils from these animals exhibit a decreased ability to respond to lipopolysaccharide induced lung injury, even though levels of circulating neutrophils were equivalent to wildtype mice (Benesova et al. 2012). However, in a different model of hemochromatosis, mice altered in the Hfe gene exhibit improved control of Salmonella enterica serovar Typhi infection within the murine macrophages; this enhanced outcome required functional siderocalin (Nairz et al. 2009). These seemingly differential phenotypes could be attributed to multiple causes, including elevated circulating Fe levels, decreased macrophage Fe content, or as yet unrecognized roles of Hfe in Fe homeostasis and pathogenesis. Additional research is required in order to fully understand the impacts of hemochromatosis on susceptibility and response to infection.

Host sequestration of Zn and Mn to prevent infection

The concept of nutritional immunity has expanded beyond Fe to other metals that are critical to the outcome between host and pathogen, including Zn and Mn (Kehl-Fie & Skaar 2010). It is estimated that six percent of the E. coli proteome has the capacity to bind Zn as a cofactor (Andreini et al. 2008). Both Zn and Mn serve as necessary cofactors for many processes required for growth of bacteria, including enzymes that function in central metabolism. In E. coli, Zn is required for the function of isopentenyl diphosphate isomerase, which catalyzes the conversion between isopentenyl diphosphate and dimethylallyl diphosphate in the mevalonate pathway (Carrigan & Poulter 2003). In Borrelia burgdorferi, the causative agent of Lyme disease, Zn is also needed to populate the enzyme peptide deformylase, which hydrolyzes formyl-L-methioninyl peptides to formate and methionyl peptides (Nguyen et al. 2007). In both Bacillus subtilis and Clostridium perfringens, Mn is necessary for the function of phosphoglycerate mutase, which catalyzes the reaction from 3-phosphoglycerate to 2-phosphoglycerate in glycolysis (Chander et al. 1998). These examples are representative of broad range of bacterial metalloproteins which require Zn and Mn as cofactors.

In addition to the examples listed above, bacteria encode for Zn- and Mn-dependent proteins critical to survival within the host and to medical intervention of infection. Of note, one group of enzymes that require Zn as a cofactor are the metallo-beta-lactamases, including the New Delhi metallo-beta-lactamase 1 (NDM-1) protein (Kim et al. 2011; Kim et al. 2013b). This class of carbapenemases is capable of inactivating beta-lactam antibiotics, one of the treatments of last resort in multi-drug resistant infections. The increasing presence of this antibiotic resistance mechanism is leading to more strains that are extensively- and pan-drug resistant. Additionally, many pathogens require Mn as a cofactor in superoxide dismutases, which protect against oxidative stress imposed by the host (Kehl-Fie et al. 2011). In addition to metallating superoxide dismutases, Mn is also used by some pathogens in place of Fe when experiencing hydrogen peroxide-induced stress. In these instances, the non-reactive Mn is substituted in metalloenzymes for Fe, which is capable of participating in Fenton chemistry (Sobota & Imlay 2011). Thus, Zn and Mn are attractive targets for further nutrient limitation by the host.

Mechanisms of Zn and Mn restriction at sites of infection

There are multiple mechanisms by which vertebrates sequester Zn and Mn from invading pathogens (Figure 3). Within the phagosome, the metal transporter NRAMP1 effluxes both Mn and Fe. Beyond transport of metals away from bacteria, the host also elaborates metal-binding proteins. The most well-studied proteins that are involved in the chelation of Zn and Mn at sites of infection belong to the S100 family of EF-hand, Ca-binding proteins. Several members of this family have been implicated in metal sequestration and induction of an inflammatory response. S100A7 has been shown to function in both of these roles. It is secreted by keratinocytes in the epidermis where it can bind Zn, restricting it from bacteria (Gläser et al. 2005; Mildner et al. 2010). Additionally, it interacts with the receptor for advanced glycation end-products (RAGE), through which it induces a pro-inflammatory response (Shubbar et al. 2012). This inflammatory response contributes to the autoimmune disorder psoriasis, from which S100A7 gets its alternative name, psoriasin. Another S100 family member, S100A12 (also known as calgranulin C), has been shown to bind Cu and Zn in vitro and to be antimicrobial (Moroz et al. 2003; Moroz et al. 2009). However, S100A12 is not encoded in the murine genome which complicates in vivo studies into the function of this protein during the host-pathogen interaction.

Figure 3. Host sequestration of Zn and Mn.

At sites of infection, the host reduces the levels of available Zn and Mn through the secretion of S100 proteins. Neutrophils and epithelial cells release calprotectin, the S100A8/S100A9 heterodimer, allowing for chelation of Zn and Mn. Within the phagosome, macrophages limit the availability of Mn to pathogens through the expression of NRAMP1 proteins, which pump Mn and Fe out of the phagosome and into the cytosol.

Calprotectin, the heterodimer of S100A8 and S100A9 (also known as calgranulin A and B or MRP8 and 14), comprises approximately 50 percent of the neutrophil cytoplasmic protein content (Gebhardt et al. 2006). It has two metal binding sites located at the dimer interface; one of which binds Zn and the other binds Mn or Zn with sub-nanomolar affinity (Kehl-Fie et al. 2011; Brophy et al. 2012). The ability to bind Mn relies on a novel six histidine coordination (Damo et al. 2013). In addition to its prevalence in neutrophils, calprotectin can also be secreted from epithelial cells at mucosal surfaces. Due to the metal sequestration properties, calprotectin is potently antimicrobial against many bacterial and fungal pathogens (Corbin et al. 2008; Urban et al. 2009; Achouiti et al. 2012; Hood et al. 2012; Amich et al. 2013). Similar to S100A7, calprotectin is also pro-inflammatory. Thus far, the S100 proteins are the only host factors known to be recruited to sites of infection that bind non-Fe transition metals and inhibit the ability of invasive pathogens to thrive. Further research is needed to identify other host factors that play a role in this aspect of nutritional immunity.

Bacterial Zn and Mn homeostasis

Similar to the regulation of Fe homeostasis in bacteria, Zn and Mn homeostasis is controlled through transcriptional regulators that bind target DNA in the presence of their respective metal and repress transcription. The Zn uptake regulator (Zur) and a Mn-responsive metalloregulator (MntR) both de-repress their regulons in the absence of Zn or Mn, respectively. Zur is a member of the Fur family of metal-responsive regulators, whereas MntR belongs to the DtxR family. In response to Zn and Mn scarcity, pathogens increase the expression of various cation acquisition systems (Figure 4). ABC family transporters are employed for the acquisition of both Zn and Mn; AdcABC and ZnuABC uptake systems are selective for Zn, whereas MntABC generally transports Mn (Papp-Wallace & Maguire 2006; Bayle et al. 2011; Ilari et al. 2011). AdcABC type transporters are conserved in Gram-positive organisms, whereas ZnuABC systems are found in Gram-negative bacteria; MntABC homologs are found in both Gram-positive and Gram-negative pathogens. In addition to these ABC family transporters, bacteria also express homologs of eukaryotic metal acquisition systems. For example, S. Typhimurium increases transcription of zupT and mntH, encoding a ZIP family Zn transporter and NRAMP family Mn uptake system, respectively (Karlinsey et al. 2010).

Figure 4. Bacterial Zn and Mn acquisition systems.

A) Gram-negative pathogens acquire Zn and Mn through dedicated import systems. Zn is passaged through the outer membrane protein ZnuD, which is powered by a TonB/ExbB/ExbD system. To traverse the inner membrane it is passed through the ABC transporter ZnuABC or the Zip-family transporter ZupT. Mn is moved through the outer membrane protein MnoP in a TonB-independent manner and through the inner membrane ABC transporter MntABC or NRAMP family MntH.

B) Both Zn and Mn are presumed to passively diffuse across the cell wall of Gram-positive organisms. Zn is then transported through the cell membrane by the ABC transporter AdcABC. Similar to Gram-negative bacteria, Mn is passaged through the ABC transporter MntABC or the NRAMP family MntH.

The Zur regulon includes many genes that are not directly involved in Zn uptake. In N. meningitidis, Y. pestis, A. baumannii, and M. tuberculosis, Zur de-repression alters the expression of transcripts encoding for ribosomal proteins (Maciag et al. 2007; Li et al. 2009; Pawlik et al. 2012; Mortensen et al. 2014). Coelibactin, a putative Zn-binding siderophore or ‘zincophore,’ is upregulated in a Zur-responsive manner in the soil bacterium Streptomyces coelicolor (Hesketh et al. 2009; Zhao et al. 2012). Furthermore, in the fungal pathogen Candida albicans, the PRA1 gene has been shown to encode for a secreted protein which is capable of binding Zn and subsequently interacting with the fungal cell surface (Citiulo et al. 2012). With the identification of these potential zincophores in Streptomyces and Candida, it is possible that similar non-Fe siderophores exist in pathogenic bacteria. Indeed, the Y. pestis siderophore yersiniabactin is capable of restoring growth to a Zn-uptake deficient strain (ΔznuBC) in conditions of low Zn in a process that requires the inner membrane protein YbtX (Bobrov et al. 2014).

In M. tuberculosis, Zur regulates genes in the early secretory antigen target 6 (ESAT-6) family; these gene products have been shown to interact with toll-like receptor 2 (TLR2) on macrophages and inhibit their signaling (Maciag et al. 2007). In N. meningitidis, Zur also alters the expression of proteins involved in tRNA modification (Pawlik et al. 2012). Taken together, these studies reveal a role for Zur beyond an increase in Zn transport into the cell to also include various processes required for altered metabolism in Zn-limited environments, and the production of important virulence factors.

Direct roles for Zn and Mn regulation in evading metal sequestration by calprotectin have been identified. In a murine Acinetobacter baumannii pneumonia model of mice co-infected with wildtype and a ΔznuB mutant, the ΔznuB mutant is less able to colonize the lung and disseminate to the liver (Hood et al. 2012). This phenotype is lessened in S100A9-deficient mice. In the inflamed gut, S. Typhimurium is able to outcompete commensal bacteria for limited Zn due to the presence of the high-affinity Zn transporter ZnuABC. In a co-infection model, a ΔznuA mutant colonizes the gut less well than wildtype S. Typhimurium; when repeated in S100A9-deficient mice, both strains were recovered at nearly equal levels (Liu et al. 2012). Additionally, a calprotectin-specific receptor named CpbA has been identified in N. meningitidis. CbpA binds calprotectin in vitro and allows for the use of calprotectin as a Zn source (Stork et al. 2013). Furthermore, CbpA is partially under control of Zur. A role for MntH and MntABC in competing for Mn sequestered by calprotectin has been established in a S. aureus systemic model of infection where a ΔmntHΔmntC strain is less adept at colonizing the liver when compared to wild-type S. aureus (Kehl-Fie et al. 2013). Similar to the Zn studies with A. baumannii, this difference is dependent on the presence of calprotectin. Taken together, these studies demonstrate the necessity for the host to sequester Zn and Mn, and for successful pathogens to express dedicated uptake machinery for these metals.

Disorders in Zn homeostasis and infection

Hyperzincemia and hypercalprotectinemia have been identified at low rates in humans. They are thought to be inherited and have been associated with autoimmune diseases (Saito et al. 2002). As of yet, neither condition has been shown to affect susceptibility to infection; however, in one case, an infant with both hyperzincemia and hypercalprotectinemia exhibited signs of severe anemia and neutropenia (Fessatou et al. 2005). In this disease state, it is possible that the subsequent neutropenia could result in a decreased ability for the body to adequately respond to the initial phases of infection.

Beyond genetic defects in Zn and calprotectin homeostasis, there is growing literature to suggest that dietary Zn and Mn levels may play a role in the ability of the host to respond to infection. Progeny born to turkey hens fed a diet supplemented with Zn-Methionine exhibit increased bursa weight, heightened basophilic response to phytohemagglutinin-P stimulation, and elevated levels of macrophage function following infection with Bordetella avium, the causative agent of bordetellosis in birds (Kidd et al. 2000). Phytohemagglutinin is a plant lectin which, when injected under the skin, recruits basophils and leads to a hypersensitivity response. Concurring with this, progeny from chickens fed diets supplemented with Zn and Mn demonstrate increased thymus weights for all supplemented diets tested, elevated bursa weights for diets supplemented with inorganic sources of Zn and Mn, and intensified basophil hypersensitivity response to phytohemagglutinin-P for diets supplemented with organic Zn and Mn sources (Virden et al. 2004). Finally, calves fed a diet supplemented with organic sources of Zn, Mn, Cu, and Co for fourteen days also have an increased response to phytohemagglutinin-P and exhibit decreased occurrence of respiratory diseases over the 42 day experimental period (George et al. 1997). Taken together, these experiments point towards a role for dietary levels and sources of trace metals in the development and function of the immune system. Additional studies are required to establish whether these metal supplements are only beneficial during development of the immune system or if altered dietary metal levels later in life could lead to differential susceptibility and immune response to various infections.

Metal acquisition systems as potential vaccine targets

In an attempt to create new vaccine strategies with increased efficacy, recent efforts have turned to the generation of neutralizing antibodies against proteins required for survival in the nutrient limited host environment. These therapeutics target cell wall anchored proteins for Gram-positive pathogens and outer membrane proteins for Gram-negative organisms. When the Isd proteins of S. aureus are targeted and the antigens IsdA and IsdB are combined with SdrD and SdrE, mice are protected in a lethal intraperitoneal infection model (Stranger-Jones et al. 2006). More recently, a highly conserved ZnuD outer membrane transporter in N. meningitidis which is up-regulated in the presence of Zn limitation was identified as a potential vaccine target. When immunized with outer membrane vesicles which contain this protein, both mice and guinea pigs produce antibodies which lead to complement-mediated killing of N. meningitidis strains from serogroups A, B, C, and Y (Stork et al. 2010; Hubert et al. 2013). Furthermore, immunization with MntC from S. aureus protects mice challenged with either S. aureus and Staphylococcus epidermidis in a bacteremia model of infection (Anderson et al. 2012). These advances are a proof of principle for the use of proteins expressed following host-mediated metal sequestration as novel candidates for vaccine development.

Cu toxicity as a mechanism to control infections

Although small amounts of Cu are necessary as cofactors for bacterial pathogens, this metal is toxic towards many bacteria and this toxicity is exploited by the host as a potent innate response to infection. The reasons for Cu toxicity are multi-fold. Importantly, due to its multiple oxidation states, Cu⁺ is able to react with hydrogen peroxide to produce reactive oxygen species (Solioz et al. 2010; Dupont et al. 2011). Cu can also outcompete other metals in metalloproteins, thus disrupting their activity (Waldron & Robinson 2009). Within the macrophages, Cu toxicity is induced through the transport of extracellular Cu into the cytoplasm and through ATP7A, a P-type ATPase located on the phagosomal compartment (White et al. 2009; Kim et al. 2012). Successful pathogens have adapted ways to sense and respond to the increased levels of Cu in order to prevent the bactericidal activity of this transition metal.

Cu homeostasis in bacteria

Pathogens recognize Cu influx via two mechanisms. The first is through CueR, a transcriptional regulator of the MerR family. Following binding of Cu, CueR de-represses genes in the Cu response regulon. In M. tuberculosis, this regulator is CsoR (Liu et al. 2007). Additionally, M. tuberculosis encodes RicR, a CsoR paralogue, which aids in resistance of this pathogen to Cu toxicity (Festa et al. 2011). Secondly, bacteria may express two component systems, comprised of a sensor histidine kinase and a response regulator, which are activated in the presence of increased Cu. In Helicobacter pylori, a system such as this is encoded by the genes crdS and crdR. Mutations in this two-component system lead to an inability to activate the Cu-stress response and a subsequent increase in sensitivity to Cu (Waidner et al. 2005).

The response to Cu toxicity involves multiple components. In M. tuberculosis, genes encoding for Cu efflux systems, Cu chaperones, and protection against oxidative stress are all up-regulated (Wolschendorf et al. 2011; Rowland & Niederweis 2012). CueR derepression in S. Typhimurium leads to the expression of CueP, a Cu chaperone, CueO, a multicopper oxidase, and CopA, a P-type ATPase efflux pump (Osman et al. 2010; Achard et al. 2010). Multicopper oxidases convert Cu⁺ to Cu²⁺, the latter of which is unable to participate as a catalyst in Fenton reactions. In addition to the P-type ATPases mentioned above, some bacteria express homologues of the E. coli CusABC, a resistance-nodulation-cell division (RND) family efflux pump (Outten et al. 2001).

Recently, a new function of yersiniabactin, a siderophore found in Yersinia species and strains of uropathogenic E. coli (UPEC), has been identified. Yersiniabactin is capable of forming complexes with Cu(II); this interaction prevents the formation of the toxic Cu(I) (Chaturvedi et al. 2012). Additionally, it was discovered that there were more yersiniabactin-Cu complexes than yersiniabactin-Fe complexes in the urine from patients suffering from cystitis caused by a yersiniabactin-expressing organism. In vitro, yersiniabactin-producing bacteria exhibit increased resistance to elevated Cu levels in the culture medium (Chaturvedi et al. 2012). Together with Cu chaperones and efflux mechanisms, the evolution of yersiniabactin to bind Cu and aid in protection against host-induced Cu toxicity reveals the intricacies that have evolved in the microbial response to host-imposed metal sequestration.

Antimicrobial properties of Ag and its use in preventing infection

Since the Middle Ages, Ag has been used for its antimicrobial properties; in the 1600’s, it was administered systemically as Ag-nitrate to treat cholera (Edwards-Jones 2009). Presently, Ag is employed in wound dressings, especially for burn wounds, and impregnated in implanted devices, like Foley catheters, for the prevention of bacterial infections at wound and implantation sites. Ag toxicity is known as argyria and characterized by blue-grey skin. Due to the fact that Ag⁺ readily binds with phosphate, sulfate, and chloride ions, it must be administered in a way such that there is a low concentration administered constantly. Ag⁺ interacts with the bacterial membrane leading to disruption of membrane permeability and inhibition of the proton motive force (Dibrov et al. 2002; Li et al. 2010). Additionally, it has been shown to inhibit respiration in bacilli (Semeykina & Skulachev 1990). Furthermore, administration of Ag leads to decreases in the pro-inflammatory markers interleukin 12 (IL-12) and tumor necrosis factor alpha (TNF-α) (Bhol & Schechter 2005).

Bacterial mechanisms to resist the effects of Ag are two-fold. First, Gram-negative pathogens missing outer membrane porins are slightly protected from Ag toxicity (Li et al. 1997). Further resistance to Ag occurs through the expression of Ag-specific and promiscuous efflux pumps (Gupta et al. 1999; Nies 2003). There is some concern that Ag resistance determinants may be located on plasmids containing resistance genes to antibiotics and that continual use of Ag will result in the transfer of these plasmids to currently sensitive strains (Percival et al. 2005). However, as of yet, there is little evidence to support this concern.

Concluding Remarks

There have been many advances in the understanding of nutritional immunity at the pathogen-host interface. Specifically, host sequestration of non-Fe transition metals and metal intoxication have broadened the definition beyond Fe restriction. Additionally, continued exploration into the identification and characterization of bacterial sRNAs could lead to a more thorough understanding of the regulation of many cellular processes beyond the response to Fe limitation. There are many other metal and non-metal factors which invading pathogens require that may be subjected to as-yet-undiscovered components of the immune system. Other transition metals necessary for pathogen survival may be sequestered by the host through as-yet-undefined mechanisms. Further analyses into the role of the microbiota in metal homeostasis and infection, as well as more research into the effect of dietary metal intake are needed. Future advances in these areas will lead to a greater understanding of the symbiosis between hosts and their natural flora in the contexts of normal and disease states, and identify roles for altered diets in the development of the immune system and in susceptibility to infection. This increased understanding of the interplay between the microbiota and the host will lead to new ways by which this interaction could be targeted directly to alter disease progression and outcome. Finally, research into ways to augment the natural nutritional immunity of the host may provide insight into new therapeutics or prophylaxis for difficult to treat and drug resistant organisms.