The Ins and Outs of Bacterial Iron Metabolism

Summary

Iron is a critical nutrient for the growth and survival of most bacterial species. Accordingly, much attention has been paid to the mechanisms by which host organisms sequester iron from invading bacteria and how bacteria acquire iron from their environment. However, under oxidative stress conditions such as those encountered within phagocytic cells during the host immune response, iron is released from proteins and can act as a catalyst for Fenton chemistry to produce cytotoxic reactive oxygen species. The transitory efflux of free intracellular iron may be beneficial to bacteria under such conditions. The recent discovery of putative iron efflux transporters in Salmonella enterica serovar Typhimurium is discussed in the context of cellular iron homeostasis.

Introduction

All organisms require transition metals for growth and survival. Approximately one third of all proteins and nearly half of all enzymes that have been structurally characterized contain one or more metal ions (Waldron et al., 2009; Andreini et al., 2008). Iron is the most common redox active metal found in proteins, typically within heme or iron-sulfur prosthetic groups (Beinert et al., 1997; Andreini et al., 2008). Iron is also found in regulatory proteins, which in the enteric bacterium Salmonella enterica includes Fur, Fnr, NorR, SoxR, IscR and NsrR (Ernst et al., 1978; Bagg and Neilands, 1987; Green et al., 1991; Fink et al., 2007; D’Autreaux et al., 2005; Hidalgo and Demple, 1994; Pomposiello and Demple, 2000; Schwartz et al., 2001; Tucker et al., 2008; Karlinsey et al., 2012). While iron-containing proteins are essential for fundamental physiological processes such as respiration, central metabolism and DNA repair, free iron is able to catalyze biomolecular damage to DNA, proteins and lipids via Fenton chemistry. In the Haber-Weiss cycle of reactions, free ferrous iron reacts with hydrogen peroxide (H₂O₂) to produce hydroxyl radicals and ferric iron. Ferric iron can then react with H₂O₂ to produce superoxide and regenerate the original ferrous iron catalyst. To protect against the damage that would result from oxyradical production, iron must be carefully handled within cells to maintain free intracellular iron at low levels.

Iron Withholding and Acquisition in Host-Pathogen Interactions

Most research with respect to iron and infection has focused on mechanisms by which mammalian hosts and pathogens compete for transition metals (Zaharik et al., 2004; Schaible and Kaufmann, 2005). Studies have also indicated the presence of extracellular iron-storage protein homologs in invertebrates such as crabs, worms and insects (Ong et al., 2005; Simonsen et al., 2011; Charlesworth et al., 1997), which have been suggested to play a role in immunity (Simonsen et al., 2011; Beck et al., 2002). The concept of metal withholding in nutritional immunity of higher organisms has been the subject of many recent reviews (Diaz-Ochoa et al., 2014; Cassat and Skaar, 2013; Cerasi et al., 2013; Hood and Skaar, 2012). To acquire necessary iron, bacteria express a variety of uptake systems and produce siderophores to chelate and acquire iron within the host (Figure 1). FeoAB is an uptake system for ferrous iron that consists of a cytosolic protein and inner membrane transporter (Cartron et al., 2006). SitABCD is an ABC family transporter that allows the acquisition of both ferrous iron and manganese (Zhou et al., 1999; Boyer et al., 2002). Iron-chelating siderophores are synthesized by bacteria and secreted using an active efflux mechanism involving inner membrane transporters such as EntS and IroC and the outer membrane protein TolC (Furrer et al., 2002; Crouch et al., 2008; Bleuel et al., 2005). Uptake of iron-bound siderophores also requires specialized proteins such as IroN and the Fep system (Hantke et al., 2003; Crouch et al., 2008). An additional system for iron uptake utilized by some gram negative bacteria but not found in Salmonella is the ferric citrate iron acquisition system Fec (Mahren et al., 2005; Wagegg and Braun, 1981).

Figure 1. Maintenance of Cellular Iron Homeostasis under Deficient and Replete Conditions.

When intracellular iron levels are low (left), cells express siderophores and other iron import systems to acquire iron from the environment. Under iron replete conditions, the iron-sensing transcriptional regulator Fur represses the expression of iron acquisition genes. Available iron is incorporated into iron-sulfur cluster and mononuclear iron proteins, and excess iron is sequestered by ferritin (right). Additional iron can be sequestered by the DNA-binding protein Dps. Under stress conditions in which iron is mobilized from proteins, iron exporters, predicted to be localized in the inner membrane, promote iron efflux to mitigate DNA damage from Fenton chemistry.

Mammalian hosts use the inflammation-induced hormone hepcidin to restrict absorption of dietary iron and degrade ferroportin, which blocks the release of recycled iron stored in macrophages (Ganz, 2013). Host organisms also minimize the availability of extracellular free iron by expressing iron-binding proteins such as transferrin and lactoferrin (Theurl et al., 2005; Weiss and Schett, 2013). Macrophages use the Nramp1 (Slc11A1) metal transporter to restrict iron availability in the phagosomal environment by exporting iron from the phagosome (Atkinson and Barton, 1999; Barton et al., 1999; Blackwell and Searle, 1999; Cellier et al., 2007). The extent of the host-pathogen arms race over iron acquisition is exemplified by the host protein lipocalin-2, which binds the siderophore enterobactin (Flo et al., 2004) but is unable to bind a glucosylated derivative called salmochelin, which then allows Salmonella and certain other enteric bacteria to obtain iron during infection (Neilands, 1995; Fischbach et al., 2005; Crouch et al., 2008; Raffatellu et al., 2009). Lipocalin-2 also promotes iron export from macrophages and modulates macrophage activation (Fritsche et al., 2012; Warszawska et al., 2013).

Studies have shown that iron availability is an important determinant of virulence. Mice with systemic iron deficiency are more resistant to infection (Puschmann and Ganzoni, 1977), whereas acute iron overload in mouse tissues results in enhanced bacterial outgrowth (Sawatzki et al., 1983). Nramp1+ mice are more resistant to infection than Nramp1− organisms (Vidal et al., 1995a; Vidal et al., 1995b). Murine macrophages expressing Nramp1 are better able to restrict the growth of intracellular bacteria (Nairz et al., 2009a; Fritsche et al., 2012). Humans carrying a missense C282Y mutation in the HFE gene develop an iron overload condition known as hemochromatosis due to reduced surface expression and accelerated degradation of the HFE protein (Waheed et al., 1997; Parkkila et al., 2000). The iron overload resulting from this condition confers enhanced susceptibility to extracellular pathogens such as Vibrio vulnificus and Yersinia spp. (Weinberg, 2000; Bullen et al., 1991; Wright et al., 1981; Quenee et al., 2012). Similar iron overload phenotypes have been observed in C282Y and HFE null mice from various genetic backgrounds, providing a model system for the study of this condition and allowing identification of additional genes that might modify the clinical expression of hemochromatosis (Zhou et al., 1998; Levy et al., 1999; Fleming et al., 2001; Levy et al., 2000). Macrophages lacking HFE are paradoxically better able to limit the growth of intracellular pathogens such as Salmonella and Mycobacterium tuberculosis (Olakanmi et al., 2007; Nairz et al., 2009b), and this is most likely attributable to lipocalin-2-mediated redistribution of iron away from the intracellular environment (Nairz et al., 2009b). Thus, HFE controls iron compartmentalization, and an HFE mutation lowers iron availability within macrophages but increases it elsewhere. It has been suggested that typhoid and tuberculosis may have selected for the high prevelance of HFE mutations in some populations (Moalem et al., 2004).

Fenton Chemistry and Iron Toxicity

While it is clear that access to sufficient iron is critical for successful microbial replication within the host, during the course of infection pathogens may encounter stress conditions that raise intracellular free iron levels and promote Fenton chemistry, presenting an entirely different challenge to survival. The Fenton reaction proceeds relatively rapidly at physiological pH and temperature, and small alterations in the concentrations of H₂O₂ or free iron can have a dramatic impact on the amount of radical production and resulting damage (Park et al., 2005). DNA is the most critical target of hydroxyl radical damage, although proteins and lipids can be affected as well.

Intracellular pathogens encounter reactive oxygen species (ROS) and reactive nitrogen species (RNS) inside host macrophages. The macrophage NADPH oxidase NOX2 generates superoxide (O₂^·), which is rapidly converted to H₂O₂. Local H₂O₂ concentrations in phagocytes can reach 100 μM (Park et al., 2005). H₂O₂ is capable of readily diffusing across membranes and into bacterial cells where it can exert a significant physiological impact (Seaver and Imlay, 2001). While bacteria produce multiple catalases and peroxidases to detoxify the H₂O₂ produced as a result of normal metabolism, these defenses can be overwhelmed by the phagocytic oxidative burst with H₂O₂ levels rising to toxic micromolar levels (Gort and Imlay, 1998; Seaver and Imlay, 2001). Both humans and mice lacking a functional NADPH oxidase are significantly more susceptible to infection (Felmy et al., 2013).

H₂O₂ and other reactive oxygen species can harm cells in a variety of ways. One of these is by attacking solvent-exposed iron-sulfur clusters or mononuclear iron centers to result in enzyme inhibition and the release of free iron (Jang and Imlay, 2007; Sobota and Imlay, 2011; Anjem and Imlay, 2012). While iron-sulfur cluster enzymes have been well characterized, the number of mononuclear iron containing enzymes has likely been underestimated due to the challenge of studying these labile proteins under aerobic in vitro conditions (Anjem and Imlay, 2012). Damage to iron-sulfur cluster- and mononuclear iron center-containing proteins leads to a measurable increase in free iron levels within the cell (Keyer and Imlay, 1996). In addition to damaging DNA, proteins and lipids, oxyradicals can mobilize additional free iron.

Immune cells also produce nitric oxide (NO^·) as an antimicrobial defense. While NO· is not as bactericidal as H₂O₂, NO· congeners such as peroxynitrite (ONOO⁻) can release iron from metalloproteins and accelerate damage from H₂O₂ (Woodmansee and Imlay, 2003). Inhibition of respiration by NO· leads to increased NADH levels, which in turn can reduce free flavins, leading to reduction of the free iron pool and enhanced Fenton chemistry (Woodmansee and Imlay, 2003). Organisms lacking both nitric oxide synthase and the phagocyte oxidase are exquisitely sensitive to infection compared to those lacking only one of the two defenses, suggesting they perform complementary functions in vivo (Shiloh et al., 1999).

It is therefore clear that while iron is an essential nutrient for nearly all bacterial species, it can also exert cytotoxic effects under conditions of oxidative and nitrosative stress, such as those encountered within host immune cells. ROS- and RNS-mediated damage to iron-containing proteins leads to transient increases in intracellular free iron, and cells must be able to adapt to changing iron conditions as they move from iron-limited to iron-replete or iron-overload conditions.

Control of iron within cells

Since free intracellular iron is capable of catalyzing damaging chemistry, cells have evolved mechanisms to limit iron toxicity. At the center of this response network is the iron-binding regulator Fur, which acts as an iron sensor to maintain cellular iron at sufficient levels while restricting the presence of free iron. Under iron-replete conditions Fur represses the expression of iron acquisition systems (Hantke, 1981; Ernst et al., 1978) (Figure 1). This repression is relieved during iron starvation when insufficient iron is available to bind to the regulator. Fur also plays a role in regulating iron storage within bacterial cells. Production of the bacterial ferritins FtnA and Bfr is indirectly stimulated by Fur through the actions of the small RNA ryhB (Masse and Gottesman, 2002). The specialized ferritin Dps is capable of both sequestering iron and binding to DNA to shield it from the effects of reactive oxygen species (Almiron et al., 1992; Zhao et al., 2002) (Figure 1). In Salmonella, dps is repressed by Fur under iron-replete conditions, while in other bacteria dps expression has been shown to be RpoS- and OxyR-dependent (Velayudhan et al., 2007; Altuvia et al., 1994). Mutants lacking dps are highly sensitive to H₂O₂ (Velayudhan et al., 2007). Fur plays a critical role in the control of iron acquisition, use and storage, and fur mutants display high rates of mutation and are highly sensitive to oxidative damage from iron overload (Touati et al., 1995). The induction of fur expression by OxyR and SoxRS during oxidative stress enhances the ability of cells to mount a protective response to oxidative stress by modifying iron metabolism (Zheng et al., 1999).

While cells can compensate for elevated free iron levels by reducing iron uptake, upregulating iron storage proteins, and increasing intracellular concentrations of magnesium and manganese to protect against oxidative damage, these responses are likely to be less rapid than the immediate rise in free iron that follows damage to iron-containing proteins. The acute danger presented by the release of intracellular free iron due to ROS and Fenton chemistry has led us to consider alternative defense strategies that bacteria might employ under these conditions. The transient efflux of free iron from the cell could conceivably be of benefit in the presence of ROS and RNS. Removing iron as a Fenton catalyst could prevent further damage until the iron can be safely reacquired and stored, or used to repair damaged iron centers.

Iron Efflux Transporters in Salmonella

We have recently identified two proteins in Salmonella Typhimurium, STM3944 and IceT, whose expression leads to reduced levels of total intracellular iron (Frawley et al., 2013; Velayudhan et al., 2014). Since the storage form of cellular iron is not available to chelators and transporters, it is likely that the reduction in total iron mediated by STM3944 and IceT reflects a reduction in the free iron pool. Both proteins are expressed under stress conditions and appear to exert a protective effect against ROS and RNS.

The STM3944 protein is encoded by the first gene in an operon with a gene encoding the bacterial frataxin homolog CyaY, an iron chaperone that supports iron-sulfur cluster biosynthesis and repair. STM3944 is predicted to encode a four transmembrane domain-containing inner membrane protein in the major facilitator superfamily with homology to the Mn²⁺/Fe²⁺ Nramp1 transporter. Inactivation of stm3944 dramatically enhances the H₂O₂ susceptibility of a cyaY mutant, and this phenotype is catalase-independent (Velayudhan et al., 2014). STM3944 expression leads to reduced free and total iron levels under conditions in which iron homeostasis is disrupted (Velayudhan et al., 2014). These observations suggest that STM3944 plays a role in the efflux of free intracellular iron under stress conditions in which iron storage and [Fe-S] cluster repair are disrupted.

The IceT (Iron citrate efflux Transporter) protein is also a member of the MFS (major facilitator) superfamily with homologs among other γ-proteobacteria as well as certain α- and β-proteobacteria (Frawley et al., 2013). IceT is co-regulated with the MdtABC multidrug efflux transporter and two-component regulator BaeSR (Baranova and Nikaido, 2002; Nishino et al., 2005). Expression of these genes is induced by NO· or by disrupted iron homeostasis (Frawley et al., 2013). IceT expression protects cells from death caused by iron overload and leads to reduced levels of total cellular iron. Expression also leads to secretion of an iron-chelating molecule that has been identified as citrate by biochemical and genetic analyses. Secretion of citrate occurs in excess of iron secretion, thus IceT appears to efflux both iron-bound and free citrate (Frawley et al., 2013).

The efflux of iron and citrate by IceT is an intriguing defense strategy against ROS and RNS, as citrate is both an iron chelator and a key intermediate in the TCA cycle. Elevated levels of citrate appear to be toxic to cells, as bacteria with mutations in aconitase face selective pressure to acquire secondary mutations in citrate synthase and will even secrete citrate by an uncharacterized mechanism (Baumgart et al., 2011; Gruer et al., 1997; Viollier et al., 2001; Varghese et al., 2003). Iron toxicity resulting from deletion of the yeast frataxin YFH1 is exacerbated by overexpression of citrate synthase and can be ameliorated by deletion of the CIT2 citrate synthase (Chen et al., 2002). Elevated citrate levels also exacerbate instability of mitochondrial DNA in an iron-dependent fashion (Farooq et al., 2013). Although the intracellular interactions of iron and citrate have not been precisely characterized, it is clear that elevated levels of citrate and free iron can be detrimental to cells. Secretion of citrate and iron-citrate by IceT removes a Fenton catalyst from the cell, prevents citrate from accumulating intracellularly, and restricts growth. This response confers resistance not only to iron toxicity and H₂O₂ but also to various antibiotics whose actions may be potentiated by Fenton chemistry (Frawley et al., 2013). Unlike STM3944, IceT has no homology to known MFS metal transporters such as Nramp1, nor is it homologous to metal-citrate importers such as those in the CitMHS family despite its function in transport of a citrate-chelated metal ion. IceT and its homologs may therefore represent a novel class of MFS transporters, since the protein appears to be both the first iron excretory system and the first citrate efflux pump to be described in bacteria.

Conclusions

Iron plays a critical role in bacterial physiology as an essential component of metabolic enzymes and regulatory proteins. The ability of iron to transfer electrons at physiological pH makes it both useful and dangerous for cells. As a result, bacteria have evolved a variety of mechanisms to both actively acquire iron from the environment under iron-limiting conditions and to control the availability of intracellular free iron under stress or iron-replete conditions (Figure 1). Much research has focused on mechanisms by which iron and other transition metals are acquired within the host and the global regulation of bacterial iron metabolism by Fur. However, most biologically important metals can be toxic at certain concentrations, and mechanisms of protection against metal toxicity are increasingly appreciated. Efflux transporters for other transition metals, with the exception of magnesium, have been described (Liesegang et al., 1993; Rensing et al., 1997; Stahler et al., 2006; Chao and Fu, 2004; Worlock and Smith, 2002; Long et al., 2012; Waters et al., 2011; Hoch et al., 2012; Guilhen et al., 2013; Delmar et al., 2013; Patel et al., 2014; Padilla-Benavides et al., 2014), and the recent characterization of STM3944 and IceT now adds iron efflux transporters to this list. The efflux of intracellular free iron during stresses that damage iron-containing proteins represents an important and underappreciated aspect of iron homeostasis in bacteria.