Iron is an essential element for Escherichia, Salmonella, and Shigella species. The acquisition of sufficient amounts of iron is difficult in many environments, including the intestinal tract, where these bacteria usually reside. Members of these genera have multiple iron transport systems to transport both ferrous and ferric iron. These include transporters for free ferrous iron, ferric iron associated with chelators, and heme. The numbers and types of transport systems in any species reflect the diversity of niches that it can inhabit. Many of the iron transport genes are found on mobile genetic elements or pathogenicity islands, and there is evidence of the spread of the genes among different species and pathotypes. This is notable among the pathogenic members of the genera in which iron transport systems acquired by horizontal gene transfer allow the bacteria to overcome host innate defenses that act to restrict the availability of iron to the pathogen. The need for iron is balanced by the need to avoid iron overload since excess iron is toxic to the cell. Genes for iron transport and metabolism are tightly regulated and respond to environmental cues, including iron availability, oxygen, and temperature. Master regulators, the iron sensor Fur and the Fur-regulated small RNA (sRNA) RyhB, coordinate the expression of iron transport and cellular metabolism genes in response to the availability of iron.
Escherichia, Shigella, and Salmonella are members of the Enterobacteriaceae family and commonly inhabit the vertebrate intestinal tract. Like most other bacteria, they require iron for growth and survival. Because iron can be found in both the ferrous (Fe2+) and ferric (Fe3+) states and can transfer electrons over a wide range of redox potentials (−300 to +700 mV), many cellular enzymes have evolved to use iron as a cofactor. Iron is required for the function of ribonucleotide reductase to produce the precursors for DNA biosynthesis, as a component of cytochromes required for electron transport, and as a cofactor for tricarboxylic acid (TCA) cycle enzymes, among others. The enzymes may use iron in the form of heme, as iron-sulfur clusters, or as an iron atom. In laboratory-grown Escherichia coli, the amount of iron in a cell is in the range of 105 to 106 atoms (1), and low intracellular iron results in growth inhibition or arrest.
Although iron is essential, too much iron is lethal for the cell, especially if it is not chelated or protein bound. Iron acts as a catalyst for Haber-Weiss reactions, generating highly reactive hydroxyl radicals. In the presence of superoxide or hydrogen peroxide, ferric iron is converted to ferrous iron, which reacts with peroxide in the Fenton reaction to produce hydroxyl radicals. Hydroxyl radicals can cause damage or death of the cell by damaging DNA, unsaturated lipids, and proteins (2).
Thus, the uptake of iron by the bacterial cell is tightly regulated (3, 4). There is a balance between the concentration of available iron in the environment and cellular needs, and homeostasis is maintained by regulating iron uptake, use, and storage. When iron is limiting, Enterobacteriaceae turn on genes for iron uptake systems and actively acquire the metal. As the intracellular concentration increases beyond the cell’s immediate needs, these transporters are downregulated, and any excess iron is shunted into iron storage proteins. Similarly, metabolic pathways and processes that have high iron requirements are regulated in response to the availability of iron and other environmental signals that help fine-tune metabolism in response to the levels of iron in the cell. Genes for these metabolic pathways and for iron storage proteins are downregulated in low iron (5). In order to cope with the loss of essential iron-dependent metabolic pathways during iron starvation, cells may upregulate the expression of non-iron-containing isoenzymes that rely on other divalent metals, such as manganese, instead (6).
For bacteria in many environments, the level of free iron is growth limiting, and the acquisition of sufficient iron is problematic. While ferrous iron is relatively soluble, the presence of oxygen results in the formation of insoluble ferric iron. Most oxic environments contain too little free iron to support microbial growth. Furthermore, bacteria colonizing mammalian hosts, as is typical of the Enterobacteriaceae, must compete with their hosts for this essential nutrient (7, 8). As in bacteria, mammalian iron homeostasis is highly regulated, and the majority of intracellular or circulating iron is found in heme, with smaller amounts in iron-sulfur cluster-containing proteins or iron proteins. Nonheme iron being shuttled through the circulation is bound to transferrin. Iron on surfaces, in secretions, and in neutrophils is bound to lactoferrin. Thus, the concentration of free, available iron for microbial growth in mammals is vanishingly low in body fluids, on the order of 10−36 M (7).
Bacteria have evolved a variety of iron acquisition systems to allow them to obtain iron in all the environments in which they reside (Fig. 1). These systems include the transport of ferrous or ferric iron, either free or associated with low-affinity chelators such as citrate or phosphate. Bacteria also synthesize and secrete high-affinity iron chelators (siderophores) that scavenge iron in the extracellular environment. The uptake of the chelated siderophore requires dedicated outer and inner membrane transport proteins specific for the iron-siderophore complex. Bacteria that are host associated or pathogenic often have receptors for host iron complexes such as heme, hemoglobin, or transferrin and can remove the heme or iron from the protein to use within the bacterial cell.
The acquisition of iron in Gram-negative bacteria requires transport across both the outer and inner membranes (Fig. 1). Free ferrous or ferric iron appears to use porins to cross the outer membrane, but larger complexes cannot passively cross the outer membrane or use the general porins. These complexes, such as siderophores or heme, require specific outer membrane receptors. Once the chelate is bound to the receptor, energy must be expended to transport the iron chelate into the periplasm. Since the outer membrane is not energized, the energy for active transport across the outer membrane must originate in the inner membrane. Energy is transferred to receptors in the outer membrane from the energy-transducing complex of TonB, ExbB, and ExbD. Although anchored in the cytoplasmic membrane, TonB can span the periplasmic space to contact the receptors and act as a conduit for energy derived from the cytoplasmic membrane proton motive force (9).
After transiting the outer membrane, the iron chelate is bound to a periplasmic binding protein (PBP) and shuttled to the cytoplasmic membrane for entry into the cell. There are specific cytoplasmic membrane transporters for ferrous iron, ferric iron, and iron bound to siderophores, heme, or citrate. The cytoplasmic membrane permeases consist of a channel-forming protein and an ATPase or other source of energy for active transport (Fig. 1). Within the cell, the iron is released from the carrier, although in the case of heme, the intact molecule may be used by the cell. Because siderophores have a high affinity for ferric, but not ferrous, iron, reduction of the iron in the cytosol aids in its release from the siderophore. Specific enzymes that reduce iron and/or degrade the siderophore are often produced by the bacteria.
REGULATION OF IRON HOMEOSTASIS
The major regulatory factor controlling iron acquisition is an iron binding transcription factor, Fur (ferric uptake regulator) (first identified in E. coli by Hantke [3, 10]). The active form of Fur is a dimer, and each monomer has two metal binding sites: a structural site that binds zinc, which controls dimerization (11), and a regulatory site that binds iron. Recent evidence suggests that iron in the regulatory site is bound as a [2Fe-2S] cluster (12). When intracellular iron is sufficient to satisfy cell needs, the Fur protein will bind to iron, and the active iron-bound dimer then binds to specific operator sequences (Fur boxes) in the DNA (4), usually overlapping the −10 or −35 sequences in the promoter of target genes. In most cases, the binding of Fur at these sites prevents RNA polymerase binding and blocks transcription (Fig. 2); however, Fur has also been found to act less commonly as a positive regulator (13). Fur boxes are often found upstream of genes involved in iron acquisition. The repression of these genes by iron-bound Fur ensures that iron transport is turned off when iron is plentiful in the cell, thereby preventing toxic iron overload. Iron-bound Fur further prevents iron overload by positively regulating the expression of ftnA, encoding the major iron storage protein (13). Fe-Fur alleviates the repressive action of H-NS by competing for binding upstream of ftnA (13). Conversely, when iron is low, Fur cannot bind to its operator sequences to regulate transcription. Thus, the iron transport genes are turned on to initiate iron uptake, while the ftnA iron storage gene is silenced by H-NS (Fig. 2). There are multiple, sometimes overlapping, binding sites for Fur upstream of some Fur-regulated genes, including ftnA (13) and the aerobactin operon (14).
Fur boxes are also found upstream of the fur gene, overlapping the −10 consensus sequence. Fur binds weakly at this site and inhibits fur transcription from pfeoa and pfeob (Fig. 3); this autoregulation may help control the level of Fur in the cell (15). The fur gene is also expressed from upstream promoters, and these are controlled by binding sites for Crp, ArcA, OxyR, and SoxS (15–17). Thus, the level of Fur not only is tightly regulated by the concentration of iron and cellular iron needs but also is influenced by carbon metabolism (Crp), the level of environmental oxygen (ArcA), and oxidative stress (OxyR and SoxS) via the induction of these additional transcripts (Fig. 3).
Studies in E. coli indicated a palindromic 5′-GATAATGATAATCATTATC-3′, 19-bp consensus sequence for the Fur binding site (14, 18), but analysis of a larger number of promoter/operator regions suggests that Fur binding is more complex. The consensus sequence has been proposed to consist of three adjacent repeats of the hexamer 5′-GATAAT-3′ (4, 19). In this model, additional hexamer repeats can extend the binding site, which can be occupied cooperatively by additional Fur dimers (19). The extended binding sites can account for the range of affinities and extent of regulation observed for different Fur targets. Analysis of Fur binding at a bidirectional promoter region suggests that the 19-bp consensus can be structured as overlapping 13-mer sequences, allowing two Fur dimers to interact with opposite faces of the helix (20).
Fur plays a broader role in the cell than solely regulating iron acquisition. A genome-wide analysis of Fur binding and transcriptional regulation in E. coli K-12 showed the direct regulation of at least 81 genes in 42 transcriptional units (21). The regulon includes genes for carbon and energy metabolism and precursors for DNA synthesis. Several targets for Fur regulation are enzymes that have iron cofactors or are involved in cellular processes that require iron. Fur plays an even larger role in pathogenic enteric bacteria. A variety of virulence factors are regulated by Fur, indicating that the low-iron environment of the host serves as an environmental cue to turn on genes required for infection and disease. For example, Shiga toxin, produced by Shigella dysenteriae and some pathogenic E. coli strains, is Fur regulated, and in Salmonella, the type III secretion system encoded on pathogenicity island 1 is regulated by the binding of Fur at the hilD promoter (22, 23).
Another target of Fur repression is the small (90-nucleotide) regulatory RNA RyhB (5, 24) (Fig. 2). RyhB base pairs with the 5′ end of transcripts encoding iron-containing or iron storage proteins and prevents translation, leading to the degradation of the transcript. When iron is abundant, Fur represses the expression of ryhB, allowing transcripts encoding iron-containing and iron storage proteins to be translated. Iron can then be shunted into iron-reliant metabolic pathways (e.g., TCA cycle enzymes and SodB), and any excess iron can be stored (e.g., in bacterioferritin). At low iron concentrations, ryhB is highly expressed, ensuring that many genes encoding iron-dependent metabolism proteins and the iron storage protein Bfr are turned off. This response, termed “iron sparing” (5), reduces the need for iron while the cell is experiencing iron starvation, allowing the cell to reserve its available iron for only the most essential iron-dependent functions. RyhB also affects Fur synthesis. The synthesis of E. coli Fur is coupled to the translation of an upstream open reading frame (ORF) (uof), and the base pairing of RyhB with the Uof mRNA blocks the translation of uof and the downstream fur gene (25) (Fig. 3). Thus, RyhB could moderate the increase in Fur that occurs when iron is limiting and Fur no longer binds upstream.
In most cases, regulation by RyhB is negative. However, in Salmonella, RyhB acts to increase the translation of the iroN mRNA, encoding a component of the Salmonella iron transport system Iro (see the section on salmochelins and Fig. 10C, below). Salmonella encodes two different RyhB small RNAs (sRNAs), RyhB-1 and RyhB-2 (24). The two ryhB genes are found in all sequenced Salmonella species, including the distantly related species Salmonella bongori. The two sRNAs share an identical 33-base region, but the rest of the sequence is not as highly conserved. The two genes have overlapping and separate functions. The expression of both ryhB-1 and ryhB-2 is repressed by Fur, but ryhB-1 is activated by H2O2, while ryhB-2 is activated in stationary phase (26).
E. coli, Salmonella, Shigella, and related bacteria can be found in a variety of environments that differ with respect to the concentrations and forms of iron available to the bacteria. Although members of this family are predominately associated with the vertebrate gastrointestinal tract, they can survive outside the host, and within the host, there are different niches in which these bacteria are found. E. coli is a normal inhabitant of the colon, but pathogenic strains can colonize other parts of the intestine or urinary tract, and some can be found in the blood or cerebrospinal fluid. Pathogenic E. coli strains are often categorized as pathotypes to indicate the location and type of disease that they cause and the presence of specific genes (27). Pathotypes include enterotoxigenic E. coli (ETEC), Shiga-toxin producing E. coli (STEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), adherent-invasive E. coli (AIEC), neonatal meningitis E. coli (NMEC/MNEC), extraintestinal pathogenic E. coli (ExPEC), and uropathogenic E. coli (UPEC) strains, among others. Human-pathogenic strains of Salmonella can spread from the intestine and can disseminate and infect numerous organs. Yersinia, a related member of the Enterobacteriaceae, is also highly invasive and shares many of the iron transport systems found in Salmonella and E. coli. Shigella and enteroinvasive E. coli are adapted for growth in the cytosol of host colonic epithelial cells. All of these areas differ with respect to iron concentrations, the presence of ferrous or ferric iron, levels of oxygen, availability of nutrients, and the sequestration of iron in heme or proteins. The Enterobacteriaceae have evolved to use multiple environmental cues to adapt to iron availability.
Oxygen sensing provides cues about the type and extent of iron availability. In anoxic environments, Fe2+ predominates. Ferrous iron is more soluble and thus is more available to the cell. The intracellular, labile Fe2+ pool was found to be higher for cells growing anaerobically than for aerobically grown cells (28). This leads to an increase in Fur binding to DNA and increased transcriptional repression. Oxygen also regulates specific iron transport systems, adding another layer of regulation to iron acquisition. Enterobacteriaceae in the lumen of the gut grow in the absence of oxygen. However, pathogens that gain access to the surface of intestinal epithelial cells or that invade and replicate within epithelial cells are in a microaerobic environment (17, 29, 30). In Shigella flexneri, oxygen inhibited the expression of the Feo ferrous iron transporter while increasing the transcription of the iuc ferric iron transporter (17). This regulation by oxygen is mediated by two transcription factors, ArcA and the fumarate and nitrate reduction regulatory protein (FNR). These regulatory proteins bind upstream of the start site of transcription and positively or negatively regulate expression in response to oxygen (31). This allows the expression of the feo genes when the bacteria are in an anoxic environment where Fe2+ predominates. In the presence of oxygen, feo is repressed, and the ferric iron transporter iuc is expressed. Oxygen further affects the expression of these genes through an effect on fur expression (Fig. 3); ArcA binds upstream of fur and represses expression under anaerobic conditions (17). Superoxide also regulates fur. It induces a polycistronic mRNA (pfldA) that includes fur (16) (Fig. 3).
Temperature also serves as a signal for the expression of iron acquisition genes. The synthesis of the heme transport protein in Shigella dysenteriae type 1 and pathogenic E. coli is increased at 37°C, a cue indicative of the human environment (32). Additionally, E. coli siderophore synthesis and transport genes (ent, fep, and fes) and ferric citrate transport genes (fec) showed increased expression at 37°C compared to 23°C (33). The ent, fep, and fec genes also respond to carbon sufficiency and are positively controlled by Crp (34). Thus, Fur acts as a master on/off switch to regulate the expression of iron transport genes, and additional transcription factors or posttranscriptional regulation controls individual systems to take advantage of the iron sources most commonly found in that environment.
IRON TRANSPORT SYSTEMS
There are many different iron transporters in the enteric bacteria, and the general rule seems to be the more the better. A summary of the systems found in E. coli, Salmonella, and Shigella is shown in Table 1.
TABLE 1 Iron transport systemsa in E. coli, Shigella, and Salmonella
Yersiniabactin is not produced by human pathogens of S. enterica serovar Enteritidis but is found in some isolates of subspecies III and VI (139).
Ferrous iron transporters. (i) Feo.
The most evolutionarily ancient and widespread of the bacterial iron transporters is Feo (Fig. 4), and this transporter is found in all members of this family. It was first described in E. coli (35, 36) and consists of three proteins, FeoA, -B, and -C. FeoB is a cytoplasmic membrane protein that forms the channel for iron transport. The N-terminal cytoplasmic domain of FeoB has homology with eukaryotic small G proteins, and the GTPase activity is essential for transport (37). FeoA and FeoC are small cytoplasmic proteins whose roles are not fully understood. FeoC is found primarily in the Gammaproteobacteria and is absent from most other bacterial families (38). In Vibrio cholerae, the Feo proteins are present in a large complex, and FeoA was shown to be required for complex formation; FeoC is a component of the complex and is required for iron transport but not for complex formation (39). In the Enterobacteriaceae, FeoC has a cysteine motif suggesting that it might bind iron. It was shown that Klebsiella pneumoniae FeoC contains an iron-sulfur cluster (40), and a suggested function for FeoC includes its acting as an iron-responsive transcription factor. However, these residues are not conserved in all FeoC proteins, and there is no clear evidence of a role for FeoC in the regulation of transcription (41–43). In Salmonella, FeoC plays a role in stabilizing FeoB and protecting it from proteolysis (44).
Free ferrous iron does not appear to require a specific outer membrane receptor. Rather, it crosses the outer membrane through porins (Fig. 4A). The deletion of ompC and ompF prevented growth under conditions where the cells were dependent on Feo for iron transport, and there is strong regulation of feo by the EnvZ-OmpR two-component regulatory system, which also controls porin expression (45).
The feo operon, like other iron transport systems, is regulated by Fur (35), with iron as the corepressor (Fig. 4B). Fe-Fur binds upstream of feoA and represses expression by blocking RNA polymerase binding. In addition to iron, the level of oxygen in the environment influences feo expression. Under anaerobic conditions, where ferrous iron predominates, the feo operon is induced, while the presence of oxygen results in repression. In S. flexneri, the induction of feo when the cells are growing anaerobically is mediated by the oxygen-sensing transcription factors ArcA and FNR (17).
The Sit transporters are members of the periplasmic protein-dependent ABC transporter family and can transport both iron and manganese. The sit genes were first identified in Salmonella (46), and this ABC-type transporter consists of a periplasmic binding protein (SitA), the cytoplasmic membrane complex (SitC and SitD), and an ATPase (SitB) on the cytoplasmic face of the SitCD complex (Fig. 5) (46–49). Salmonella Sit was shown to transport Fe2+ and Mn2+, and under physiological conditions, it appears to be predominantly a manganese transporter (49–51). Similar systems were identified in Shigella (Sit) (30, 52) and Yersinia pestis (Yfe) (53). These three systems share approximately 45 to 75% identity at the amino acid level. The Shigella Sit transporter is also found in pathogenic E. coli strains, including EIEC (54), UPEC (55), and avian-pathogenic E. coli (APEC) (56). This system was determined to be primarily a ferrous iron transporter, although it can also provide manganese to the cell. Both iron and manganese influence the expression of the sit operons; there are binding sites for Fur and MntR upstream of these operons, and these transcription factors act to repress sit in the presence of iron (Fur) or manganese (MntR) (Fig. 5).
Analysis of Shigella and E. coli strains showed that the Shigella sit operon was found in some but not all isolates. Analysis of the location and surrounding DNA sequences indicated that the genes are on islands and have spread horizontally (Fig. 6). In one strain of S. flexneri, for example, the sit operon is flanked by an integrase gene and phage tail fiber genes, and the island is inserted at the selC locus (57). In other isolates, the phage genes are associated with the sit operon, but the upstream genes differ. The sizes and locations of the islands differ widely, even among closely related species (Fig. 6). In UPEC isolates, the sit islands map to icd (Fig. 6) (54), while in other E. coli strains, the genes are plasmid borne (56, 58, 59). The presence of the sit operon is highly correlated with an intracellular lifestyle. Those isolates that can replicate within human epithelial cells, Salmonella, Shigella spp., EIEC, and UPEC, were found to have the sit operon, while sit genes are rarely found in commensal strains or extracellular pathogens. This suggested that the genes might play a role in iron acquisition in the eukaryotic cell cytosol. Evidence for iron acquisition via Sit during intracellular growth was found by analysis of the growth of S. flexneri iron transport mutants. A strain having only the Sit system grew as well as the wild type within the host cell cytoplasm, while those having only Feo or a siderophore showed reduced growth (52). Furthermore, the sit genes have been shown to be expressed during the intracellular growth of S. flexneri (30), UPEC (60), and Salmonella enterica (61).
The Efe ferrous iron transport system was first identified in E. coli strain Nissle (62) and is encoded by a three-gene operon, efeUOB (ycdNOB) (Fig. 7). In E. coli K-12, Efe is not functional because of a frameshift mutation in efeU, but an intact gene is found in other strains, including E. coli O157:H7 (62, 63) and UPEC strain CFT073 (62). Efe is also found in Shigella sonnei (64), Yersinia pestis (65), and some strains of Salmonella enterica (66).
EfeU has homology to Ftr1p, the integral membrane iron-permease component of the Fet3p-Ftr1p iron transporter of Saccharomyces cerevisiae, and it is predicted to be a cytoplasmic membrane protein with 7 transmembrane domains (63, 67) (Fig. 7A). EfeU was able to support the growth of E. coli in the absence of other iron transporters, and in vitro studies using proteoliposomes demonstrated that EfeU is a ferrous iron transporter (62). EfeO and EfeB are periplasmic proteins, and both are required for the function of the Efe transporter (63). However, their precise functions in the Efe system are unknown. EfeO has a large, C-terminal peptidase-M75 domain and a smaller, N-terminal cupredoxin-like domain predicted to have two metal binding sites, with one site predicted to bind Cu2+ (67). This suggests that it might be the equivalent of the Fet3p cupredoxin component of the yeast Fet3p-Ftr1p iron transporter (67). EfeB is found as a homodimer, and the crystal structure shows that each monomer binds heme in a hydrophobic pocket in the C-terminal domain (68). EfeB can remove iron from heme without the degradation of the tetrapyrrole ring (69). The source of energy for the transport of ferrous iron via the Efe system is not known, but a ferroxidation step, such as the one used by Fet3p-Ftr1p, has been proposed as the driving force for transport (67). The expression of efeUOB is regulated by iron and pH. Fur represses expression in the presence of iron (62, 70), and the two-component regulatory system CpxAR controls expression as a function of the pH of the medium. At high pH, the binding of unphosphorylated CpxR represses efe expression (63) (Fig. 7B).
Ferric iron transporters. (i) Ferric dicitrate.
The ability of E. coli to use ferric citrate (ferric dicitrate) as an iron source was identified in E. coli K-12 as a citrate-inducible ferric iron transport system (71). Citrate can act as an iron chelator and provide the metal to some enteric Enterobacteriaceae through the Fec transport system (Fig. 8A). Ferric citrate binds to the outer membrane receptor FecA. The periplasmic binding protein FecB delivers ferric citrate to the ABC transporter that is composed of FecCD (cytoplasmic membrane permease) and FecE (ATPase).
Unlike most enteric iron transporter operons, the fec genes are positively regulated by the iron chelate. Positive regulation is mediated by FecI and FecR. FecI belongs to the extracytoplasmic-function (ECF) sigma factor family, and FecR acts as an anti-sigma factor. Upon the binding of ferric citrate to FecA, FecA transduces the signal to FecR (72), which then releases FecI to bind RNA polymerase (73). This allows the transcription of the fecABCDE operon in response to ferric citrate in the environment (Fig. 8B). FecI and FecR are encoded in an operon immediately upstream of fecABCDE, and both operons are negatively regulated by Fur in the presence of iron (74, 75). The transcriptional regulator SlyA also represses fecIR (76). The fecABCDE promoter region has binding sites for the positive regulators PdhR and Crp (34), coupling iron transport to metabolism, and the negative regulator HypT, which responds to the redox state and sulfoxidation of methionine (77).
The ferric citrate transport system is not widely distributed among the enteric bacteria. Among the E. coli isolates tested, fec genes were more commonly found among UPEC and bacteremic strains than in fecal isolates (78). The presence of the fec genes is variable in the Shigella species. S. sonnei has the Fec system, but it is rarely found in the other Shigella species (57). One isolate of S. flexneri was found to have Fec, and in this strain, the genes map to the Shigella resistance locus (SRL), a pathogenicity island that also carries multiple drug resistance genes (79). Salmonella strains typically lack the fec locus, but analysis of the genome of an S. enterica serovar Tennessee isolate revealed the fec genes in an island that also harbors the lac operon and ORFs that have homology to other metal transporters (80).
Members of the Enterobacteriaceae, like many other bacteria, synthesize and secrete low-molecular-weight iron chelators (siderophores) when stressed for iron. These compounds bind iron with very high affinities and transport it back into the cell. The siderophores produced by Enterobacteriaceae fall into at least three classes: catechol, hydroxamate, and thiazoline containing (Fig. 9).
Enterobactin (enterochelin [Ent]) is a catechol-type siderophore in which three catechols are linked to serines in a cyclic peptide (Fig. 9). This siderophore was initially identified in E. coli (81) and Salmonella (82) and was subsequently found to be widely distributed in this family. Enterobactin, like other peptide-containing siderophores, is synthesized by a nonribosomal peptide synthase from 2,3-dihydroxybenzoic acid (DHB) and l-serine. The biosynthetic enzymes are the products of the entABCDEFGH genes (83–91). YbdZ, a small MbtH-like protein encoded downstream of fes, interacts with EntF and enhances the production of enterobactin (92–94). The mature product is shunted across the cytoplasmic membrane by the enterobactin exporter EntS (P43; YbdA) (95) and finally secreted from the cell by TolC (96).
Enterobactin binds ferric iron with a very high affinity; association constant (Ka) values have been predicted to be as high as 1052 (97). The transport of the iron-siderophore complex into the cells requires a specific outer membrane receptor (FepA), a periplasmic binding protein (FepB), and the cytoplasmic membrane permease (FepCDG) (Fig. 10A). The complex of TonB, ExbB, and ExbD transduces the energy from the cytoplasmic membrane proton motive force to FepA to allow active transport across the outer membrane, and the energy for transport across the cytoplasmic membrane is produced by FepC, the ATPase component of the cytoplasmic membrane permease. In the cytoplasm, Fes, the Fe-enterobactin esterase, cleaves the enterobactin backbone, helping to release the iron from the siderophore. Release is likely aided by the reducing environment of the cytosol; enterobactin has a low affinity for ferrous iron. Linear forms of enterobactin can also be used to transport iron, and these are taken up via the outer membrane receptors Fiu (not shown) and Cir (Fig. 10A) (98, 99).
Because it has such a high affinity for iron, it would be expected that enterobactin would play an important role in the acquisition of iron within the mammalian host by pathogens. However, serum albumin binds enterobactin, significantly reducing its effectiveness (100). Additionally, it was found that lipocalin-2, or NGAL (neutrophil gelatinase-associated lipocalin), tightly binds enterobactin and is bacteriostatic for E. coli under iron-limiting conditions (101–103), giving rise to the alternative name siderocalin for this protein. Siderocalin is secreted by neutrophils and epithelial cells as part of the innate immune inflammatory response (104) and adds to the arsenal of antimicrobial factors produced by the host in response to infection. As might be expected, pathogens have evolved mechanisms to avoid the inhibitory effects of siderocalin. Salmonellae produce salmochelins, enterobactin molecules that are modified by the addition of 1 to 3 glucose moieties (105, 106). This reduces the affinity of enterobactin for siderocalin and promotes the virulence of S. enterica (107–109).
Salmochelin synthesis and transport require the iro genes iroBCDE and iroN (110, 111) in addition to the enterobactin biosynthesis machinery genes (Fig. 10C). IroB is the glucosyltransferase that attaches the glucose moieties to enterobactin (106, 112), and IroC is involved in the secretion of salmochelins from the cell (105, 113). Salmochelins are not recognized by the enterobactin receptor FepA but require the outer membrane receptor IroN (105, 114) for transport into the cell. The uptake of salmochelin across the cytoplasmic membrane has been shown to occur through either the FepBCDG system (113) or IroC (115), which may thus function in both the export and import of salmochelin. IroD and IroE hydrolyze the siderophore (115, 116). IroD, which is found in the cytosol, prefers the ferric salmochelins as the substrate, while the periplasmic IroE cleaves the apo-siderophore to produce linear forms of the salmochelins (116).
The ent genes are organized into 6 clusters with 3 sets of divergent promoters (Fig. 10B). There are multiple binding sites for Fur in these promoter regions, and the operons are negatively regulated by iron. Similarly, there are binding sites for Fur overlapping the −10 sequences of the iroBCDE operon (117) and iroN (118) (Fig. 10C). Additional regulators tie the expression of these genes to the broader metabolism of the cell. There is a binding site for the oxygen-controlled regulator FNR between fepA and fes, and it is predicted to be a positive regulator at this site. Crp binding sites are also present in this region and in the promoter region between fepB and entC, linking expression to carbon metabolism (34). RutB, the master regulator of pyrimidine metabolism in E. coli, binds upstream of fepB with low affinity (119), but a role for RutB in fepB expression has not been established.
Posttranscriptional regulation also regulates the levels of the outer membrane receptors FepA and IroN (Fig. 10B and C). The FepA transcript has binding sites for OmrA and OmrB. These redundant sRNAs are induced by the EnvZ-OmpR two-component regulatory system, and they reduce the synthesis of FepA (120, 121). IroN is regulated by the sRNA RyhB (118). In contrast to most RyhB targets, whose synthesis is decreased by RyhB, IroN requires RyhB for synthesis.
Enterobactin is found in almost all E. coli, Shigella, and Salmonella strains, with the exception of S. flexneri and Shigella boydii (Table 1). Although the genes are present in these Shigella species, deletions, insertions, and mutations have inactivated the genes for the biosynthesis and transport of Ent. The genes for the glucosylation of enterobactin and the transport of the modified siderophore have a much more restricted distribution (Table 1). Salmochelin is found in almost all Salmonella species tested except S. bongori (114). Among Shigella species, the salmochelin locus is found in S. dysenteriae but only rarely in the other species. In E. coli, the salmochelin system has been found in APEC and UPEC strains (122, 123) but is less common among fecal isolates.
Aerobactin, a hydroxamate siderophore that consists of two residues of 6-(N-acetyl-N-hydroxy)lysine linked to citrate (Fig. 9), was first described in Aerobacter aerogenes (81). The siderophore was subsequently identified in many pathogenic E. coli and Shigella isolates (Table 1). The biosynthesis of aerobactin requires the products of the iucABCD genes (124, 125). The secretion of apo-aerobactin out of the cell is not fully understood but may involve the product of the iucA-adjacent shiF gene (126). The iutA gene encodes the outer membrane receptor that is required for aerobactin import (125, 127). Following transport across the outer membrane, iron-loaded aerobactin enters the cell via the generic hydroxamate transport system FhuBCD (128, 129) (Fig. 11).
Although the aerobactin genes are highly conserved, the flanking sequences and their chromosomal locations are not, indicating horizontal gene transfer (Table 1). The aerobactin operon can be plasmid borne (130, 131) or associated with pathogenicity islands (e.g., Shi2 and Shi3) (132–134). The aerobactin genes have been found in most isolates of S. flexneri and S. boydii, which generally lack enterobactin production, but are rarely found in S. sonnei or S. dysenteriae. Among those species that have the genes, their locations can vary. The aerobactin-containing island in S. flexneri is found at the selC locus (133), while in S. boydii, the genes are on a distinct pathogenicity island inserted at pheV (132). Extraintestinal strains of E. coli, including UPEC, APEC, and NMEC, often carry the aerobactin operon, and studies have shown that the presence of aerobactin correlates with virulence in these strains. Among EHEC/STEC strains, the ability to synthesize or utilize aerobactin is absent from O157:H7 type strain EDL933 (135), but the iuc-iut genes are found in strains O157:H7 USDA5905 (GenBank accession number NZ_CP039837) and DEC5E (GenBank accession number NZ_CP038383.1), and they are common among non-O157:H7 EHEC strains as well (136), showing a high degree of variability, even among closely related strains. Interestingly, human isolates of both EHEC strains and K1 invasive strains typically encode either the aerobactin system or a hemolysin (which may allow access to iron from host hemoglobin) but not both (136), suggesting some functional redundancy of these systems with respect to virulence.
The expression of the aerobactin operon is repressed by Fur when iron is abundant, and there is also regulation by oxygen (17, 57). The oxygen-responsive transcription factor ArcA binds to the iuc promoter and prevents transcription when the cells are growing anaerobically (17).
An additional siderophore-mediated iron transport system, yersiniabactin (Ybt) (136) (Fig. 12A), has been identified in some strains of E. coli and Salmonella (138–141). Yersiniabactin was first identified in Yersinia enterocolitica and Y. pseudotuberculosis (142–144) and was later characterized in Y. pestis (137, 145) as part of the high-pathogenicity island (HPI). Yersiniabactin belongs to the thiazoline class of siderophores (Fig. 9), and it is synthesized by a hybrid nonribosomal peptide synthetase/polyketide synthase mechanism (146). Seven gene products are required for yersiniabactin synthesis (high-molecular-weight protein 1 [HMWP1]/Irp1, HMWP2/Irp2, YbtD, YbtE, YbtS, YbtT, and YbtU) (147). It is not known how yersiniabactin is exported from the cell. YbtX is a membrane-embedded protein with some similarity to the enterobactin exporter EntS, but a ybtX mutant is not defective in either yersiniabactin secretion or utilization, and thus, the role of YbtX in yersiniabactin-mediated iron acquisition is unclear (137) (Fig. 12A). The transport of yersiniabactin-bound iron into the cell requires the outer membrane pesticin receptor Psn (FyuA) and the cytoplasmic proteins YbtP and YbtQ (137, 148). The exact mechanism of iron uptake via YbtPQ is not clear. Although classified as an ABC transporter, the YbtPQ permease lacks certain characteristic motifs found in other iron importers, and its structure more closely resembles that of an exporter (149). In addition, a yersiniabactin-specific PBP has not been identified within the ybt locus. It may be encoded elsewhere in the genome, or the YbtPQ permease may have adapted to function without a PBP (137). A role for YbtPQ in the release of the bound metal from the siderophore and the recycling of the empty siderophore to the outside has been proposed (150). Despite these unusual features, YbtPQ is essential for iron import via yersiniabactin, whether by transporting the entire ferri-yersiniabactin complex or just the iron moiety (Fig. 12A). In addition to its role in iron transport, yersiniabactin can bind copper, and this may play a role in copper acquisition, as well as copper detoxification, in UPEC strains (150). Furthermore, yersiniabactin is important for biofilm production by UPEC; the genes are highly upregulated during urine biofilm growth, and an fyuA mutant was defective for biofilm formation (151).
The genes for yersiniabactin synthesis and transport are typically found on the HPI mobile genetic element (152). In Yersinia and some E. coli isolates, including UPEC and EAEC strains, that are positive for yersiniabactin production, the genes are within the HPI, and the island is inserted at an asn tRNA gene (153, 154). In other E. coli strains, including some APEC strains, the ybt genes are located within an island encoding another iron transporter, such as the SitABCD system (Fig. 6).
The three yersiniabactin promoters have binding sites for Fur and the AraC-like transcription factor YbtA (145, 155) (Fig. 12B). Fur represses transcription when iron is present, and YbtA activates transcription when iron is low and yersiniabactin is present in the medium. Increased gene expression dependent upon the presence of the siderophore has not been reported for the other siderophores in enteric bacteria, but there is increased transcription of psn (fyuA) when yersiniabactin is in the medium (145, 156). Regulation at the divergent ybtP-ybtA promoters appears to be more complex than that at the irp2 or fyuA promoter. In the presence of iron, Fur blocks transcription in both directions. When iron is limiting and Fur does not bind, YbtA at low concentrations activates ybtP transcription but inhibits ybtA; at higher concentrations of YbtA, transcription in both directions is repressed (157).
E. coli strains that produce yersiniabactin have been shown to produce a second product of the Ybt biosynthetic pathway (158). This compound, termed escherichelin (Fig. 9), binds Fe3+, but it is unable to supply the iron to E. coli for growth. Rather, this compound may aid E. coli in competing with other organisms in the vicinity. Escherichelin was shown to inhibit the growth of Pseudomonas aeruginosa by inhibiting pyochelin-mediated iron uptake (158).
In addition to producing their own siderophores, the enteric bacteria make receptors for siderophores produced by other organisms. This allows them to steal iron from their neighbors. One receptor commonly found in Enterobacteriaceae is FhuA, which binds the fungal siderophore ferrichrome. This TonB-dependent receptor delivers the siderophore to the periplasm, where it is transported to the cytosol by the FhuBCD PBP-dependent ABC transporter. The FhuBCD permease can transport a variety of hydroxamates, including aerobactin (Fig. 11), rhodotorulic acid, and coprogen, for use by enteric bacteria, but each of these requires a specific outer membrane receptor (128).
Heme, the iron-containing tetrapyrrole found in hemoglobin and other cellular proteins, is the most abundant source of iron in the mammalian host, and it is not surprising that many pathogens have evolved to take advantage of this molecule. Many members of the Enterobacteriaceae produce hemolysins that could aid in the release of hemoglobin from red blood cells, but experimental models are needed to confirm the role of hemolysins in vivo as a mechanism for gaining access to heme in the host. Heme transporters have been characterized in S. dysenteriae and EHEC (135, 159–161) (Fig. 13). The heme transport locus, designated shu in S. dysenteriae, is also present in other pathogenic E. coli strains, including EIEC, EPEC, UPEC, and NMEC (162). The heme transporter is named Chu in E. coli, but the shu and chu genes are almost identical across these organisms, and the Shu terminology is used here to indicate both. These genes are present at the same location in the genome, corresponding to 78 min on the K-12 genome, in all these strains, but there is no evidence for phage genes, insertion elements, or other sequences characteristic of horizontal transmission, and this is not a known hot spot for islands (162). Heme transport systems have been detected in other enteric pathogens that lack the shu locus, but most have not been characterized (162). One additional heme transporter, Hma, has been identified in UPEC strain CFT073 (163).
Shu-mediated heme transport requires the outer membrane receptor ShuA (135, 159, 160), the periplasmic binding protein ShuT (164), and the inner membrane permease proteins ShuU and ShuV (162, 165) (Fig. 13A). ShuS is a cytoplasmic heme binding protein that is required for the efficient use of heme as an iron source and for protection against heme toxicity (166, 167). In E. coli, the ShuS homolog ChuS was shown to be a noncanonical heme oxygenase (168), and the ShuW, ShuX, and ShuY homologs were shown to catalyze the anaerobic degradation of heme (169–171). In addition to serving as an iron source, heme imported through the Shu transport system also provides a source of porphyrin. A hemA mutant, which lacks the ability to synthesize porphyrins, is able to use heme for growth when the Shu transport system is present (160).
There is transcriptional and posttranscriptional regulation of the shu operons. Fur binding sites are present in each of the four promoters (57, 160, 162, 172), blocking transcription when sufficient iron is present. Temperature regulates expression posttranscriptionally. The 5′ untranslated regions (UTRs) of the shuA and shuT transcripts contain FourU RNA thermometers (32, 172). This allows the increased synthesis of the Shu proteins at 37°C, but the FourU thermometer sequences occlude the Shine-Dalgarno sequences at lower temperatures, independent of the concentration of iron.
Roles of iron transporters in E. coli, Shigella, and Salmonella pathogenesis.
Members of the Enterobacteriaceae have multiple iron transport systems (Table 1). Some, such as the heme transporter, provide iron only in a particular environment. Others have overlap or even redundancy in their functions, making it more difficult to determine which systems are essential for growth and pathogenesis.
The least complex among these pathogens with respect to iron transport is S. flexneri 2a. Most S. flexneri 2a strains have only three iron transport systems, the ferrous iron transporters Feo and Sit and the aerobactin (Iuc) siderophore transporter (52, 173). Although genes for enterobactin synthesis and transport are present, insertions, deletions, and mutations have rendered all of the operons nonfunctional (174, 175). In S. flexneri, single, double, and triple mutants were constructed, and their growth was determined in different environments to determine which systems were needed where. The deletion of all three systems (Feo, Sit, and Iuc) prevented the growth of the bacteria in the absence of an exogenously supplied siderophore in the laboratory and within cultured cells, indicating that no other iron transport systems were present (52). Analysis of the growth of the mutants extracellularly or in the intracellular environment following invasion of human epithelial cells showed that the loss of any one of the three systems had only a small impact on growth. However, of the double mutants, only the strain that retained the sit genes was able to grow as well as the wild type in infected epithelial cells (52). Studies in a mouse model also determined that Sit was important for pathogenesis (47). In the laboratory, the strain expressing only aerobactin grew as well as the wild type in the presence of oxygen, and the strain with only Feo grew well anaerobically. Thus, outside the host cell, aerobactin provides iron in aerobic environments, and Feo provides iron when oxygen is limited, such as in the lumen of the gut. The Sit system, which is expressed in the presence of oxygen yet transports ferrous iron, is uniquely adapted to provide iron to bacteria growing within host cells. The cytosol is a reducing environment, but sufficient oxygen to induce the transcription of the sit genes is present (17, 30, 47, 57).
Interestingly, the other species of Shigella, which have the same lifestyle as S. flexneri, have differences in iron transport systems (57). All of them have the Sit system, supporting its role in the acquisition of iron during intracellular growth, but different siderophores are produced (Table 1). S. boydii produces aerobactin but not enterobactin, and S. dysenteriae type 1 produces enterobactin and salmochelin but not aerobactin. All of the Shigella isolates tested produce a siderophore, but it appears that any siderophore, rather than a specific one, is sufficient for Shigella survival.
At the other end of the spectrum are the extraintestinal pathogenic E. coli (ExPEC) strains, which include UPEC and bacteremic strains (Table 1). In addition to the Feo, Sit, and Efe systems for ferrous iron transport, UPEC strain CFT073 encodes at least 14 different TonB-dependent outer membrane receptors (176), and TonB dependent iron transport is required for virulence and infection of the kidney in a mouse model of urinary tract infection (177). The requirement for TonB was at least in part due to the need for siderophore transport, as an iuc ent double mutant also had reduced infectivity compared to the wild type (177). The production of aerobactin, in addition to enterobactin, may provide an advantage at sites of inflammation or in the circulation since aerobactin is not bound by siderocalin. Heme transport is also important for the virulence of UPEC. Strain CFT073, a well-characterized UPEC strain, has two heme receptors, ChuA (177) and Hma (163), both of which are required for virulence. In competition assays with the wild-type strain, the chuA mutant had a greater defect in kidney colonization than the hma mutant, which may be explained by the higher level of expression of chuA than of hma in vivo (163).
Among the Salmonella strains, the enterobactin system is important for the growth of Salmonella enterica serovar Typhi in monocytes (178). Both an enterobactin biosynthesis mutant and a tonB mutant, which is defective for enterobactin transport, have restricted growth in a human monocyte cell line compared to the wild-type parental strain. In contrast, a tonB mutant of S. enterica serovar Typhimurium was unaffected for growth in epithelial or macrophage-like cells, and there was no attenuation of the mutant for mice infected by the intraperitoneal route (179). The tonB mutant was attenuated for infection of mice by the intragastric route, indicating that TonB-dependent receptors, likely siderophore receptors, were required for iron acquisition during infection of the intestinal tract and invasion from this site. Subsequent studies have shown that siderophore synthesis and transport, specifically the salmochelin system, confer a growth advantage to Salmonella in the inflamed intestine (108). Mutation of iroN, the salmochelin receptor, resulted in a competitive disadvantage for S. enterica serovar Typhimurium in the intestine, but the wild type had no advantage over the mutant in lipocalin-2 (siderocalin)-deficient mice (108). Thus, Salmonella infection, which causes inflammation of the intestine, leads to increases in the local levels of siderocalin and the binding of enterobactin produced by bacteria in the gut; S. enterica serovar Typhimurium can glycosylate and linearize enterobactin to form salmochelin, against which siderocalin is ineffective in preventing iron transport.
A role for ferrous iron transport via Feo in enteric bacteria is less clear. An feo mutant of E. coli K-12 was defective in colonizing the mouse intestine (180), and an S. enterica serovar Typhimurium feoB mutant was outcompeted by the wild type in the mouse intestine in a competition assay (179). However, the SalmonellafeoB mutant was as lethal as the wild type following oral or intraperitoneal inoculation of mice (179).
The large number and variety of iron transport systems in these pathogens contribute to their ability to survive in multiple environments in the host: the lumen of the gut, the intracellular environment, the bloodstream, and cerebrospinal fluid. The abilities to use heme iron, acquire intracellular ferrous iron via Sit, modify enterobactin to salmochelin to evade its capture by lipocalin, and produce additional siderophores allow these bacteria to rapidly adapt to the different niches that they encounter in the host. The fact that most of these systems are on pathogenicity islands and have characteristics of horizontally transmitted genes suggests that there is continual evolution of these host-associated species, and the bacteria hold the upper hand in the battle for iron within the host.
Some of the work reported in this review was supported by grants AI016935 and AI091957 to S.M.P. from the National Institute of Allergy and Infectious Diseases.
Figures were prepared in part with BioRender.
We thank Carolyn Fisher for expert editorial assistance.
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