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Iron acquisition and regulation systems in Streptococcus species Ruiguang Ge*a and Xuesong Sun*b Gram-positive Streptococcus species are responsible for millions of cases of meningitis, bacterial pneumonia, endocarditis, erysipelas and necrotizing fasciitis. Iron is essential for the growth and survival of Streptococcus in the host environment. Streptococcus species have developed various mechanisms to uptake iron from an environment with limited available iron. Streptococcus can directly extract iron
Received 13th January 2014, Accepted 13th March 2014
from host iron-containing proteins such as ferritin, transferrin, lactoferrin and hemoproteins, or indirectly
DOI: 10.1039/c4mt00011k
by relying on the employment of specialized secreted hemophores (heme chelators) and small siderophore molecules (high affinity ferric chelators). This review presents the most recent discoveries in
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the iron acquisition system of Streptococcus species – the transporters as well as the regulators.
Introduction Almost all organisms need iron for a wide range of cellular pathways, including electron transport, oxygen activation, peroxide reduction, amino acid and nucleoside synthesis, and photosynthesis. An environmental concentration of 10 8 M iron is commonly required for the growth of most bacteria.1,2 However in aqueous neutral and aerobic environments, Fe3+ forms stable ferric oxide hydrate complexes (Fe2O3nH2O), leading to a free a
Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China. E-mail: [email protected]; Fax: +86-20-39332973; Tel: +86-20-39332973 b Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institutes of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China. E-mail: [email protected]; Fax: +86-20-85226165; Tel: +86-20-85226165
Ruiguang Ge
Ruiguang Ge received his PhD in 2006 with Profs. Hongzhe Sun and Jian-Dong Huang from The University of Hong Kong. After postdoctoral training at Washington University in St. Louis with Prof. Dan Goldberg, he joined the Faculty of Sun Yat-Sen University where he is currently an associate professor of Biochemistry and Molecular Biology. His research focuses on metalloproteins and metallodrugs.
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Fe3+ concentration of around 10 10 M.3 The availability of iron to bacterial pathogens in the host is further restricted due to the iron-binding proteins.4 In serum the free iron concentration is calculated to be around 10 24 M due to sequestration by transferrins and lactoferrins.5 Therefore bacterial pathogens have to develop various ways to overcome the limited availability of iron in the host environment. One way is to replace the iron in cellular pathways with abundantly available metal ions, such as copper, as in the case of Borrelia burgdorferi.6 However, this strategy is probably only feasible for pathogenic bacteria which do not require many of the biosynthetic enzymes that use iron as an essential cofactor. Instead, most of the bacterial pathogens have developed direct or indirect high-affinity iron uptake systems. Direct mechanisms comprise the uptake of iron either from host iron-containing proteins (ferritin, transferrin or lactoferrin) or from heme-containing proteins (hemoglobin, haptoglobin or hemopexin). Indirect strategies use secreted hemophores to
Xuesong Sun is currently a full professor in College of Life Science and Technology of Jinan University. She received her PhD in 2006 with Profs. Qing-Yu He and Hongzhe Sun from The University of Hong Kong. After postdoctoral training at The University of Hong Kong, she joined Jinan University as an associate professor in 2008. Her research interests include metalloproteins and metalloproteomics. Xuesong Sun
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Table 1
Metallomics
Iron uptake systems of the Streptococcus species
Organism
Transporter
Substrate
Ref.
S. pyogenes
Fhu/FtsABCD/SiuADBG MtsABC SiaABC/HtsABC Shr Shp
Heme/siderophore Fe2+ Heme Heme Heme
59 and 60 16, 18 and 19 29–31 38 31, 36, 40, 44 and 45
S. pneumoniae
PiuBCDA/Pit1BCDA/FhuDBGC PiaABCD/Pit2ABCD PitBCDA
Siderophore/heme Siderophore Siderophore
65–67 and 69 67 65
S. equi
EqbHIJ Shr/Shp/HtsA
Siderophore Heme
70 47
S. iniae
MtsABC FtsABCD
Heme Heme
25 50
S. agalactiae
MtsABC FhuCDBG PefAB PefRCD
Fe2+/Mn2+ Siderophore Heme Heme
17 62 53 53
acquire heme or small siderophore molecules (normally o1 kDa) as high affinity ferric chelators.7 The uptake of iron from either direct or indirect sources requires specific receptors, such as ATP-binding protein cassette (ABC) transporters on the cell surface to facilitate the transmembrane delivery.8 Both Gram-positive and Gram-negative bacteria have evolved similar strategies but different tactics in the iron acquisition, possibly due to the different cellular architecture.9 For example, Gram-negative bacteria use siderophore-binding outer membrane receptors, such as E. coli FepA to transport ferric-siderophore into the periplasmic space as driven by the TonB–ExbB–ExbD energy-transducing system.10 Afterwards, the ferric-siderophores are imported into the cytosol via the inner membrane by ABC transporters. In contrast, the Gram-positive bacteria can directly uptake ferric-siderophores from the environments through the ABC transporters. Streptococcus is a genus of Gram-positive pathological bacteria. For example, S. pyogenes is responsible for streptococcal toxic shock, rheumatic fever, rheumatic heart disease, pharyngitis and bacteremia.11 S. pneumoniae is the most common cause of bacterial pneumonia and a frequent cause of septicemia and meningitis.12 S. agalactiae is the leading cause of invasive infection among newborns possibly via aspiration of the vaginal content or the amniotic fluid during delivery.13 S. iniae causes serious infections such as meningoencephalitis and generalized septicemia with considerable incidence of disease and mortality in cultured fish worldwide.14 The iron trafficking mechanisms in these Gram-positive Streptococcus species have been a hot topic for some time.15 The present review focuses on the most recent reports and discoveries in the field of streptococcal iron acquisition and regulation systems.
conditions, and is taken up nonspecifically by the ubiquitous divalent metal ABC transporter. However, in most microbial habitats, Fe2+ is oxidized to Fe3+ enzymatically or through reactions with molecular oxygen. As listed in Table 1, MtsABC (divalent metal transporter of Streptococcus) is the only one proposed to be a ferrous iron transporter in two Streptococcus species.16–19 MtsA is a lipoprotein tethered to the outside of cell membrane with an N-terminal lipid anchor and is functionally similar to the periplasmic ferric-ion binding protein (Fbp) in Gram-negative bacteria (Fig. 1).20,21 With the ATP-binding protein in MtsABC providing energy, MtsA interacts with the integral membrane component for the transmembrane iron uptake. MtsA was supposed to primarily bind to Fe2+ under physiologically relevant conditions.16,22 Considering that the
Ferrous iron transporter
Fig. 1 A schematic overview of the major iron, heme and siderophore acquisition systems in S. pyogenes. Ferric iron is reduced to the ferrous form and is transported by the MtsABC transporter; heme is delivered into cells via the Shr/Shp/SiaABC system; and Fch iron is transported through the FtsABCD transporter. Intracellular iron is temporarily stored in Dpr (ferrous or ferric iron) or AhpC (heme).
The cellular uptake of iron is restricted to its physiologically relevant species, Fe2+ (ferrous iron) and Fe3+ (ferric iron). Fe2+ is soluble in aqueous solution under anaerobic or reducing
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S. pyogenes genome encodes a ferredoxin23 which may reduce environmental Fe3+ to Fe2+, it is possible that MtsA may work as a specific receptor targeting Fe2+ for iron acquisition. This was further supported by a study with the S. agalactiae MtsA homologue that MtsA was coordinately regulated by both manganese and ferrous ions.17 The order of MtsA affinity for metal ions is as follows: Fe2+ ((2.23 0.16) 105 M 1) 4 Fe3+ ((8.36 1.05) 104 M 1) 4 Cu2+ ((5.97 0.22) 104 M 1) 4 Mn2+ ((1.84 0.11) 104 M 1) 4 Zn2+ ((1.30 0.03) 104 M 1).16 Bicarbonate was required as a synergistic anion for the stable iron binding in MtsA,16 similar to the iron binding characteristics of human transferrin in which bicarbonate is needed for Fe3+ coordination.24 This is different from the active site of FbpA where the synergistic anion is monodentate and labile, e.g. phosphate at neutral pH and carbonate at higher pH values.21 The ferric binding site is composed of four amino acid side-chains: aspartate, histidine and two tyrosines in human transferrin, and glutamate, histidine and two tyrosines in FbpA.21 However in the MtsA crystal structure we determined, Fe was bound to the side chains of His68, His140, Glu206 and Asp281, without any hint of the presence of bicarbonate in the metal binding.22 This suggests that the anion may be displaced during the final stages of metal binding during the crystallization. Although the MtsAs in S. pyogenes and S. agalactiae were supposed to be responsible for ferrous transport, the S. iniae homologue was found to be involved in heme utilization.25 This suggests that MtsAs may have differential roles in the iron uptake, which is strain dependent.
Heme transporter Heme is the most abundant source of iron in the body. One method of heme uptake is via ABC transporters.26 The heme is first bound by a heme-binding protein (Hbp), and then is transferred to the permease for passage through the cell membrane. During the process, ATPase provides the energy through the hydrolysis of ATP. In some hemoproteins, iron is hexa-coordinated with four ligands from protoporphyrin IX and two axial ligands from the side chains of His, Lys, Tyr, Met or Cys, resulting in the low spin iron states.27,28 In S. pyogenes, a 10-gene operon named sia (Streptococcus iron acquisition) or hts (heme transport S. pyogenes) locus mediates the iron uptake from hemoproteins.29–31 The suppression of sia resulted in reduced iron uptake and decreased hemoglobin binding. The sia locus encodes a hemoprotein receptor Shr, a heme binding protein Shp and an ABC transporter SiaABC. Shr and Shp are two NEAT (near-iron transporter) type receptors and are anchored to the cell membrane, contributing to bacterial adherence and virulence.32 Each NEAT domain has around 120 amino acids and adopts an eight-b-strand sandwich structure.33 Shr has a unique functional N-terminal domain (NTD) that interacts with methemoglobin and two distinctive heme-binding NEAT domains separated by a series of LRRs (leucine-rich repeats). Besides heme-binding, NEAT2 also binds extracellular matrix components34,35 and exhibits novel heme iron-reduction ability.34 Shr and Shp do not have the axial ligand (mostly tyrosine) to
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acquire and transfer heme, commonly found in the ligandbinding pocket of other NEAT domain-containing proteins. The NEAT domain in Shp uses bis-methionyl (Met66/Met153) for heme ligation,36 while the binding ligands in NEAT1 and NEAT2 in Shr remain unknown. Shr extracts heme from host hemoproteins and delivers it to a NEAT module in the adjacent Shp on the surface.37,38 The transfer is through a biphasic process and only partial with around 63% of Shr heme transferred to Shp.37 Further study with stopped-flow suggested that holoNEAT1 quickly delivered heme to apoShp, while holoNEAT2 is transferred only slowly and negligibly to apoShp.39 The heme in the NEATs of Shr can be transported in between from NEAT1 to NEAT2 by a fast and reversible process,39 suggesting that NEAT1 is responsible for quick heme delivery to Shp and NEAT2 serves as a temporary storage for heme on the bacterial surface (Fig. 2). Heme is then further transferred to SiaABC proteins for the subsequent transport across the membrane. Shp cannot directly extract heme from hemoglobin as the heme transfer from hemoglobin to apo-Shp is similar to the dissociation rate constants of heme from hemoglobin.38 The crystal structure of the heme-binding domain of Shp exhibited an immunoglobulin-like b-sandwich fold (Fig. 3). This domain is located in the N-terminus.36 The N-terminus is functionally active by itself, although inclusion of the C-terminal domain speeds up the process to around 10-folds. Besides the biaxial ligands of Met66 and Met153 in Shp, hydrogen bonds between N2 of heme O and Ne of Arg155 are formed to stabilize the structure. In the SiaABC transporter, SiaA is a heme-binding lipoprotein, and is associated with SiaB and SiaC, the membrane permease and ATPase, respectively. ESR studies revealed that SiaA has a bisligated heme center,40 with the biaxial ligands to be methionine (Met79) and histidine (His229).41,42 The heme iron in SiaA is identified to be six-coordinate and low-spin through characterization by Raman, magnetic circular dichroism and
Fig. 2 Scheme for the heme acquisition by the Shr/Shp/SiaABC system in S. pyogenes. Shr obtains heme upon binding of hemoglobin to the NTD domain. The heme in the NEAT1 of Shr is rapidly transferred to Shp in the presence of high amounts of environmental heme. The NEAT2 is a temporary storage place for heme. When environmental heme availability is limited, heme from NEAT2 is transferred to NEAT1 for the subsequent transfer to Shp. The heme in Shp is further transported to SiaA protein of the SiaABC transporter for the subsequent transmembrane transport. The zoomed-in area shows the intermediate state of heme transfer from Shp to SiaA, with the axial heme ligands highlighted.
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Once internalized, heme may bind to AhpC in a 1 : 1 ratio with a Kd of 0.5 mM.54 AhpC has roles in the intracellular fate of heme due to the following findings: (1) AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo; (2) the bacterium with AhpC inactivated displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase; and (3) heme is protected from chemical degradation in vitro upon AhpC binding.54
Siderophore transporter Fig. 3 Bis-methionyl coordination in Shp and heme stacking in the crystal interface (PDB ID: 2Q7A). Heme packing is shown between the bound and the exogenous hemes. Hydrogen bonds formed between Arg155 and a propionate on the bound heme molecules.
nuclear magnetic resonance.41 The biaxial ligands Met79 and His229 of SiaA have differential roles with His229 as the key residue for heme binding,42 further supported by competitive binding experiments with exogenous imidazole, cyanide and CO.43 Shp can rapidly transfer heme to SiaA in a concerted two-step process: holoShp forms a complex with apoSiaA prior to heme transfer.40,44,45 Further study implies that the axial residues of Met79 and His229 of SiaA displace the Met66 and Met153 residues of Shp, respectively, during the heme transfer reaction.46 The axial ligand replacement during the heme transfer between holoShp and apoSiaA displays a first-order kinetic phase, indicating that the displacement of the first Met ligand is rate limiting and the two new coordination bonds in SiaA form roughly at the same time.40,45 The heme transportation system in Streptococcus species other than S. pyogenes is still far from clear. The Shr/Shp/ HtsABC(SiaABC) system was found to be important for heme acquisition in S. equi,47,48 albeit detailed mechanisms have not been established. Two membrane proteins from S. pneumonia were identified to be potential heme acquisition proteins through a proteomic work on the proteins purified from heme-affinity chromatography and competitive binding experiments with heme and iron.49 MtsABC25 and FtsABCD50 transporters were supposed to be involved in heme utilization in S. iniae. The internalized heme was important to the activation of the respiratory chain in S. agalactiae in the presence of exogenous menaquinone or quinone.51,52 S. agalactiae cells with an activated respiratory chain displayed improved survival in an aerobic environment and greater virulence in a murine septicemia model.51 Although the heme acquisition system is unknown for S. agalactiae, two efflux operons (pefAB and pefRCD)53 and one intracellular heme binding protein (alkyl hydroperoxide reductase AhpC)54 were identified to be important for heme and protoporphyrin IX homeostasis. The bacterium with both pefAB and pefRCD inactivated displayed accrued sensitivity to heme and protoporphyrin IX.53 The porphyrins accumulate intracellularly.
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Siderophores are small organic molecules with variable Fe3+ affinities (ranging over 30 orders of magnitude).55–58 Siderophores are classified into catecholates (e.g. enterobactin), hydroxamates (e.g. ferrichrome (Fch)) and hydroxycarboxylate (e.g. staphyloferrin A) based on their structures. The iron utilization as mediated by siderophores consists of a series of steps: siderophore synthesis, apo-siderophore secretion and the subsequent transmembrane transportation through holo-siderophore specific transporters or through ferrous transporters such as MtsABC. S. pyogenes genome encodes three ABC transporters, HtsABC/SiaABC, MtsABC, and FtsABCD (ferrichrome transporter system)/SiuADBG (Streptococcal iron uptake).59,60 In a study conducted by Montanez and coworkers, SiuADBG was named to emphasize its role in iron uptake and was proposed to be involved in heme utilization by S. pyogenes.60 In a later report by Lei and coworkers, the ABC transporter was renamed as FtsABCD,59 as this transporter was found to be important for Fe3+–Fch uptake. During Fch utilization, Fch is captured by the lipoprotein FtsB in 1 : 1 stoichiometry and then transferred to the permease FtsCD for subsequent transmembrane delivery. FtsA is an ATPase which provides energy for the Fch transfer. Fch binding in FtsB occurs in two steps with the C-terminal Trp204 and the N-terminal Tyr137 to be two essential residues.61 One hydroxamate-type siderophore transporter FhuCDBG was identified in S. agalactiae, containing one putative ATPase (FhuC, at the inner side of the cytoplasmic membrane), a cell surface binding lipoprotein (FhuD) and two transmembrane proteins (FhuB and FhuG, at the outer and inner cytoplasmic membranes respectively).62 FhuD binds hydroxamate-type siderophores with high affinities (e.g. for Fe3+–desferroxamine interaction, Kd = 50 nM).62 The role of FhuD in the acquisition of siderophore iron was also established through Fe accumulation assays. In S. pneumoniae, three ABC transporters are involved in siderophore acquisition, i.e., piuBCDA/pit1BCDA/fhuDBGC,63,64 piaABCD/pit2ABCD56,57 and pitBCDA.65 In S. pneumoniae, all the four fhu genes have the same transcription direction with the homologues in S. agalactiae but are arranged as fhuDBGC.66 This siderophore system was first described as pit2 (pneumococcal iron transporter) and pia (pneumococcal iron acquisition).65,67 FhuD of S. pneumoniae is more closely related to the fhuD homologue of E. coli than to other Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus.66 Therefore the fhu operon of S. pneumoniae is probably acquired by gene transfer as evidenced by the presence of a 27 kb region with characteristics of a
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Gram-negative bacterial pathogenicity island,67 a hint that the acquisition played a significant role in the evolution of this pathological bacterium. The membrane-anchored substratebinding protein PiaA is composed of an N-terminal and a C-terminal domain bridged by an a-helix.68 Fch is bound at the inter-domain cleft and stabilized by a number of amino acids: (1) two hydrogen bonds between Arg231 and carbonyl oxygen atoms of two hydroxamic acid moieties from Fch; (2) one hydrogen bond between the hydroxyl group of Tyr225 and the carbonyl oxygen atom of the third hydroxamic acid moiety; (3) three hydrogen bonds between the backbone of Fch and the side chains of Trp158 and Asn83; (4) hydrophobic interactions between the methylene carbon atoms of the hydroxyornithine moieties with the hydrophobic barrel formed by Met213, Trp223, Tyr225 and Phe255 from the C-terminal and Trp63, Tyr84 and Trp158 from the N-terminal domain.68 Besides siderophore uptake, piu and pia were also identified as responsible for hemoglobin utilization of S. pneumoniae,69 indicating that they may have variable substrates. In S. equi, a siderophore named equibactin is produced by a non-ribosomal peptide synthetase system within the locus eqbA-N.70 The production of equibactin was abolished in the absence of eqbE, eqbG and eqbM and was reduced in the absence of eqbN. Therefore the equibactin locus containing all of the genes unique to S. equi is required for the biosynthesis. EqbK and EqbL share 29% amino acid sequence identity with YbtP and YbtiQ of Y. pestis which are involved in siderophoremediated iron import and are structurally similar to the ABC transporters associated with iron acquisition.71 The ABC transporters encoded by the genes of eqbH, eqbI and eqbJ are required for the associated iron import.
Iron regulation Bacteria need to achieve intracellular iron homeostasis to fulfill their metabolic functions and to prevent the toxic effects attributed to the oxygen radicals produced by excessive iron (Fenton reaction: H2O2 + Fe2+ - OH + OH + Fe3+).72 Metaldependent transcription regulators, Fur or DtxR, are commonly found in bacteria to maintain iron homeostasis.73 Although the sequence homology between Fur and DtxR is modest, crystallographic data indicated substantial structural similarities,74,75 all sharing an N-terminal DNA binding domain and a dimerization domain with two metal binding sites per subunit. In S. pyogenes, PerR is a Fur type peroxide stress response regulator (Fig. 4).76–78 Oxidative conditions stimulate the oxidation of selected histidine residues of PerR and the subsequent release of the regulator from the DNA.77 One transcriptome study of the wild type and perR mutant indicates that PerRdependent genes were almost exclusively down-regulated and are involved in purine and deoxyribonucleotide biosynthesis, iron and heme homeostasis and amino acid/peptide transport.79 The regulated iron and heme homeostasis genes include the mts ferrous iron transporter, fhu/fts heme/Fch transport system, shr/shp/sia heme acquisition system,79 and the only
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Fig. 4 A schematic overview of the iron regulation system in S. pyogenes. PerR and MtsR coordinate together to control the iron uptake system in S. pyogenes.
ferritin-like iron storage protein Dpr/MrgA.80,81 Dpr expression was elevated upon treatment with millimolar concentrations of iron. Further study indicated that PerR binds the promoter region of dpr and increased iron concentrations and decreased PerR binding to the promoter region of dpr,80 thus resulting in an elevated expression of Dpr. DNase I footprinting revealed that S. pyogenes PerR binds to a single site upstream of the promoter for the gene encoding AhpC,81 the S. agalactiae homologue involved in intracellular heme homeostasis.54 However the regulation of AhpC was revealed to be independent of PerR.81 A structural study of the S. pyogenes PerR revealed that PerR exists as a homodimer with two metal-binding sites per subunit: one structural zinc site and one regulatory metal site.82 The regulatory site has an N-terminal HXH metal-binding motif83,84 fully conserved among PerRs from Streptococcus species and unique from other Fur family proteins. The integrity of this site is critical to bacterial pathology.82 A second S. pyogenes metal-dependent transcription regulator protein, MtsR, belongs to the DtxR family of metallo-repressors, and is located upstream and divergently transcribed from the mts gene cluster.85 PerR and MtsR coordinate the expression of genes involved in metal uptake and homeostasis as well as genes essential for the production of infection and pathology. MtsR directly represses the sia and mts transporters30,86 and the heme binding protein ahpC87 under conditions of high metal levels. MtsR has a more robust control of mtsA expression than of siaA, possibly attributed to its higher binding affinity for the mts promoter region.30,86 An mtsR mutant derivative of serotype M49 strain NZ131 has an elevated level of intracellular iron and is more sensitive to oxidative stress and less virulent in zebrafish infection models.86 A transcriptome analysis of the wild type and the mtsR inactivated mutant S. pyogenes revealed 44 up-regulated genes and 20 down-regulated genes,88 indicating that MtsR has an extensive regulatory role in S. pyogenes. The activated genes contribute to nucleotide metabolism, transport,
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cell envelope and regulation. The repressed genes are involved in nucleotide metabolism, transport, cell envelope and regulation, protein synthesis and fate, and the biosynthesis of amino acids.88 The repressed metal-related genes include the 10-gene sia operon, Per-regulated metal transporter pmtA (encoding a P-type ATPase and mediating metal export89 and peroxide resistance) and the P-type ATPase family heavy metal transporter copA.88 However no changes in mts and ahpC transcripts were observed in this transcriptome study, demonstrating some strain-dependent variation of the regulon.
Conclusions and perspectives Streptococcus is a genus of pathological Gram-positive bacteria and streptococci have been closely associated with millions of cases of meningitis, bacterial pneumonia, endocarditis, erysipelas and necrotizing fasciitis. As iron is essential for almost all living organisms, investigation of the iron acquisition and regulation systems is important to understand the pathological roles of the Streptococcus species. The relevant iron acquisition proteins may be the future targets of antibiotics or other antibacterial treatments. The available information about iron uptake and regulation of the Streptococcus species has been discussed in this concise review. Ferrous iron is transported through the membrane by MtsABC. However, what is the ferrous iron origin, from free ferrous iron pool in the host, from host ferrous-storage proteins, or from the reduction of ferric iron sources, such as ferric-storage proteins, heme-containing proteins and ferric-siderophores? What kind of reductase is responsible for the reduction of ferric into ferrous iron in the extracellular space? The ferric iron molecules may be uptaken through the Shr/Shp/ SiaABC system (heme) or FtsABCD transporter (ferric-siderophore). However it is not yet clear how iron is removed from the various carriers (heme and siderophore) once inside the cell. Iron may be the active cofactor in quite a number of enzymes, such as hydrogenase and SOD. Which kinds of iron chaperone proteins are involved in the maturation of these iron-containing enzymes in the Streptococcus species? The iron trafficking system in the Streptococcus species has not been studied systematically with metallomic or metalloproteomic methods. Advanced nuclear analytical techniques, such as neutron activation analysis, X-ray emission/fluorescence spectroscopy, isotope dilution and tracing, X-ray absorption, neutron scattering, electron paramagnetic resonance and nuclear magnetic resonance, are highly sensitive, accurate and non-destructive and play increasingly important roles in the studies of metallomics and metalloproteomics.90,91 The application of these techniques will help clarify in several ways the iron-trafficking proteins in the Streptococcus species in vivo: the cellular localization of iron inside the bacterium; the structure of iron-containing metalloenzymes; and so on. All these questions regarding the iron trafficking system in the Streptococcus species need further work to fully understand.
Acknowledgements This work was supported by National Natural Science Foundation of China (20801061, 31000373 and 21371182), the Fundamental
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Metallomics
Research Funds for the Central Universities (10lgpy19 and 21611201), Pearl River Rising Star of Science and Technology of Guangzhou City (2011J2200079 and 2011J2200003) and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Jinan University.
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84 R. Ge, Y. Zhang, X. Sun, R. M. Watt, Q. Y. He, J. D. Huang, D. E. Wilcox and H. Sun, J. Am. Chem. Soc., 2006, 128, 11330–11331. 85 N. S. Jakubovics, A. W. Smith and H. F. Jenkinson, Mol. Microbiol., 2000, 38, 140–153. 86 C. S. Bates, C. Toukoki, M. N. Neely and Z. Eichenbaum, Infect. Immun., 2005, 73, 5743–5753. 87 S. B. Beres, E. W. Richter, M. J. Nagiec, P. Sumby, S. F. Porcella, F. R. DeLeo and J. M. Musser, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 7059–7064. 88 C. Toukoki, K. M. Gold, K. S. McIver and Z. Eichenbaum, Mol. Microbiol., 2010, 76, 971–989. 89 A. Brenot, B. F. Weston and M. G. Caparon, Mol. Microbiol., 2007, 63, 1185–1196. 90 R. Ge, I. K. Chu and H. Sun, in Nuclear Analytical Techniques for Metallomics and Metalloproteomics, ed. C. Chen, Z. Chai and Y. Gao, Royal Society of Chemistry, London, 2011, pp. 265–298. 91 R. Ge, X. Sun and Q. Y. He, Curr. Drug Metab., 2011, 12, 287–299.
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Iron acquisition and regulation systems in Streptococcus species Ruiguang Ge*a and Xuesong Sun*b Gram-positive Streptococcus species are responsible for millions of cases of meningitis, bacterial pneumonia, endocarditis, erysipelas and necrotizing fasciitis. Iron is essential for the growth and survival of Streptococcus in the host environment. Streptococcus species have developed various mechanisms to uptake iron from an environment with limited available iron. Streptococcus can directly extract iron
Received 13th January 2014, Accepted 13th March 2014
from host iron-containing proteins such as ferritin, transferrin, lactoferrin and hemoproteins, or indirectly
DOI: 10.1039/c4mt00011k
by relying on the employment of specialized secreted hemophores (heme chelators) and small siderophore molecules (high affinity ferric chelators). This review presents the most recent discoveries in
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the iron acquisition system of Streptococcus species – the transporters as well as the regulators.
Introduction Almost all organisms need iron for a wide range of cellular pathways, including electron transport, oxygen activation, peroxide reduction, amino acid and nucleoside synthesis, and photosynthesis. An environmental concentration of 10 8 M iron is commonly required for the growth of most bacteria.1,2 However in aqueous neutral and aerobic environments, Fe3+ forms stable ferric oxide hydrate complexes (Fe2O3nH2O), leading to a free a
Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China. E-mail: [email protected]; Fax: +86-20-39332973; Tel: +86-20-39332973 b Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institutes of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China. E-mail: [email protected]; Fax: +86-20-85226165; Tel: +86-20-85226165
Ruiguang Ge
Ruiguang Ge received his PhD in 2006 with Profs. Hongzhe Sun and Jian-Dong Huang from The University of Hong Kong. After postdoctoral training at Washington University in St. Louis with Prof. Dan Goldberg, he joined the Faculty of Sun Yat-Sen University where he is currently an associate professor of Biochemistry and Molecular Biology. His research focuses on metalloproteins and metallodrugs.
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Fe3+ concentration of around 10 10 M.3 The availability of iron to bacterial pathogens in the host is further restricted due to the iron-binding proteins.4 In serum the free iron concentration is calculated to be around 10 24 M due to sequestration by transferrins and lactoferrins.5 Therefore bacterial pathogens have to develop various ways to overcome the limited availability of iron in the host environment. One way is to replace the iron in cellular pathways with abundantly available metal ions, such as copper, as in the case of Borrelia burgdorferi.6 However, this strategy is probably only feasible for pathogenic bacteria which do not require many of the biosynthetic enzymes that use iron as an essential cofactor. Instead, most of the bacterial pathogens have developed direct or indirect high-affinity iron uptake systems. Direct mechanisms comprise the uptake of iron either from host iron-containing proteins (ferritin, transferrin or lactoferrin) or from heme-containing proteins (hemoglobin, haptoglobin or hemopexin). Indirect strategies use secreted hemophores to
Xuesong Sun is currently a full professor in College of Life Science and Technology of Jinan University. She received her PhD in 2006 with Profs. Qing-Yu He and Hongzhe Sun from The University of Hong Kong. After postdoctoral training at The University of Hong Kong, she joined Jinan University as an associate professor in 2008. Her research interests include metalloproteins and metalloproteomics. Xuesong Sun
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Table 1
Metallomics
Iron uptake systems of the Streptococcus species
Organism
Transporter
Substrate
Ref.
S. pyogenes
Fhu/FtsABCD/SiuADBG MtsABC SiaABC/HtsABC Shr Shp
Heme/siderophore Fe2+ Heme Heme Heme
59 and 60 16, 18 and 19 29–31 38 31, 36, 40, 44 and 45
S. pneumoniae
PiuBCDA/Pit1BCDA/FhuDBGC PiaABCD/Pit2ABCD PitBCDA
Siderophore/heme Siderophore Siderophore
65–67 and 69 67 65
S. equi
EqbHIJ Shr/Shp/HtsA
Siderophore Heme
70 47
S. iniae
MtsABC FtsABCD
Heme Heme
25 50
S. agalactiae
MtsABC FhuCDBG PefAB PefRCD
Fe2+/Mn2+ Siderophore Heme Heme
17 62 53 53
acquire heme or small siderophore molecules (normally o1 kDa) as high affinity ferric chelators.7 The uptake of iron from either direct or indirect sources requires specific receptors, such as ATP-binding protein cassette (ABC) transporters on the cell surface to facilitate the transmembrane delivery.8 Both Gram-positive and Gram-negative bacteria have evolved similar strategies but different tactics in the iron acquisition, possibly due to the different cellular architecture.9 For example, Gram-negative bacteria use siderophore-binding outer membrane receptors, such as E. coli FepA to transport ferric-siderophore into the periplasmic space as driven by the TonB–ExbB–ExbD energy-transducing system.10 Afterwards, the ferric-siderophores are imported into the cytosol via the inner membrane by ABC transporters. In contrast, the Gram-positive bacteria can directly uptake ferric-siderophores from the environments through the ABC transporters. Streptococcus is a genus of Gram-positive pathological bacteria. For example, S. pyogenes is responsible for streptococcal toxic shock, rheumatic fever, rheumatic heart disease, pharyngitis and bacteremia.11 S. pneumoniae is the most common cause of bacterial pneumonia and a frequent cause of septicemia and meningitis.12 S. agalactiae is the leading cause of invasive infection among newborns possibly via aspiration of the vaginal content or the amniotic fluid during delivery.13 S. iniae causes serious infections such as meningoencephalitis and generalized septicemia with considerable incidence of disease and mortality in cultured fish worldwide.14 The iron trafficking mechanisms in these Gram-positive Streptococcus species have been a hot topic for some time.15 The present review focuses on the most recent reports and discoveries in the field of streptococcal iron acquisition and regulation systems.
conditions, and is taken up nonspecifically by the ubiquitous divalent metal ABC transporter. However, in most microbial habitats, Fe2+ is oxidized to Fe3+ enzymatically or through reactions with molecular oxygen. As listed in Table 1, MtsABC (divalent metal transporter of Streptococcus) is the only one proposed to be a ferrous iron transporter in two Streptococcus species.16–19 MtsA is a lipoprotein tethered to the outside of cell membrane with an N-terminal lipid anchor and is functionally similar to the periplasmic ferric-ion binding protein (Fbp) in Gram-negative bacteria (Fig. 1).20,21 With the ATP-binding protein in MtsABC providing energy, MtsA interacts with the integral membrane component for the transmembrane iron uptake. MtsA was supposed to primarily bind to Fe2+ under physiologically relevant conditions.16,22 Considering that the
Ferrous iron transporter
Fig. 1 A schematic overview of the major iron, heme and siderophore acquisition systems in S. pyogenes. Ferric iron is reduced to the ferrous form and is transported by the MtsABC transporter; heme is delivered into cells via the Shr/Shp/SiaABC system; and Fch iron is transported through the FtsABCD transporter. Intracellular iron is temporarily stored in Dpr (ferrous or ferric iron) or AhpC (heme).
The cellular uptake of iron is restricted to its physiologically relevant species, Fe2+ (ferrous iron) and Fe3+ (ferric iron). Fe2+ is soluble in aqueous solution under anaerobic or reducing
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S. pyogenes genome encodes a ferredoxin23 which may reduce environmental Fe3+ to Fe2+, it is possible that MtsA may work as a specific receptor targeting Fe2+ for iron acquisition. This was further supported by a study with the S. agalactiae MtsA homologue that MtsA was coordinately regulated by both manganese and ferrous ions.17 The order of MtsA affinity for metal ions is as follows: Fe2+ ((2.23 0.16) 105 M 1) 4 Fe3+ ((8.36 1.05) 104 M 1) 4 Cu2+ ((5.97 0.22) 104 M 1) 4 Mn2+ ((1.84 0.11) 104 M 1) 4 Zn2+ ((1.30 0.03) 104 M 1).16 Bicarbonate was required as a synergistic anion for the stable iron binding in MtsA,16 similar to the iron binding characteristics of human transferrin in which bicarbonate is needed for Fe3+ coordination.24 This is different from the active site of FbpA where the synergistic anion is monodentate and labile, e.g. phosphate at neutral pH and carbonate at higher pH values.21 The ferric binding site is composed of four amino acid side-chains: aspartate, histidine and two tyrosines in human transferrin, and glutamate, histidine and two tyrosines in FbpA.21 However in the MtsA crystal structure we determined, Fe was bound to the side chains of His68, His140, Glu206 and Asp281, without any hint of the presence of bicarbonate in the metal binding.22 This suggests that the anion may be displaced during the final stages of metal binding during the crystallization. Although the MtsAs in S. pyogenes and S. agalactiae were supposed to be responsible for ferrous transport, the S. iniae homologue was found to be involved in heme utilization.25 This suggests that MtsAs may have differential roles in the iron uptake, which is strain dependent.
Heme transporter Heme is the most abundant source of iron in the body. One method of heme uptake is via ABC transporters.26 The heme is first bound by a heme-binding protein (Hbp), and then is transferred to the permease for passage through the cell membrane. During the process, ATPase provides the energy through the hydrolysis of ATP. In some hemoproteins, iron is hexa-coordinated with four ligands from protoporphyrin IX and two axial ligands from the side chains of His, Lys, Tyr, Met or Cys, resulting in the low spin iron states.27,28 In S. pyogenes, a 10-gene operon named sia (Streptococcus iron acquisition) or hts (heme transport S. pyogenes) locus mediates the iron uptake from hemoproteins.29–31 The suppression of sia resulted in reduced iron uptake and decreased hemoglobin binding. The sia locus encodes a hemoprotein receptor Shr, a heme binding protein Shp and an ABC transporter SiaABC. Shr and Shp are two NEAT (near-iron transporter) type receptors and are anchored to the cell membrane, contributing to bacterial adherence and virulence.32 Each NEAT domain has around 120 amino acids and adopts an eight-b-strand sandwich structure.33 Shr has a unique functional N-terminal domain (NTD) that interacts with methemoglobin and two distinctive heme-binding NEAT domains separated by a series of LRRs (leucine-rich repeats). Besides heme-binding, NEAT2 also binds extracellular matrix components34,35 and exhibits novel heme iron-reduction ability.34 Shr and Shp do not have the axial ligand (mostly tyrosine) to
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acquire and transfer heme, commonly found in the ligandbinding pocket of other NEAT domain-containing proteins. The NEAT domain in Shp uses bis-methionyl (Met66/Met153) for heme ligation,36 while the binding ligands in NEAT1 and NEAT2 in Shr remain unknown. Shr extracts heme from host hemoproteins and delivers it to a NEAT module in the adjacent Shp on the surface.37,38 The transfer is through a biphasic process and only partial with around 63% of Shr heme transferred to Shp.37 Further study with stopped-flow suggested that holoNEAT1 quickly delivered heme to apoShp, while holoNEAT2 is transferred only slowly and negligibly to apoShp.39 The heme in the NEATs of Shr can be transported in between from NEAT1 to NEAT2 by a fast and reversible process,39 suggesting that NEAT1 is responsible for quick heme delivery to Shp and NEAT2 serves as a temporary storage for heme on the bacterial surface (Fig. 2). Heme is then further transferred to SiaABC proteins for the subsequent transport across the membrane. Shp cannot directly extract heme from hemoglobin as the heme transfer from hemoglobin to apo-Shp is similar to the dissociation rate constants of heme from hemoglobin.38 The crystal structure of the heme-binding domain of Shp exhibited an immunoglobulin-like b-sandwich fold (Fig. 3). This domain is located in the N-terminus.36 The N-terminus is functionally active by itself, although inclusion of the C-terminal domain speeds up the process to around 10-folds. Besides the biaxial ligands of Met66 and Met153 in Shp, hydrogen bonds between N2 of heme O and Ne of Arg155 are formed to stabilize the structure. In the SiaABC transporter, SiaA is a heme-binding lipoprotein, and is associated with SiaB and SiaC, the membrane permease and ATPase, respectively. ESR studies revealed that SiaA has a bisligated heme center,40 with the biaxial ligands to be methionine (Met79) and histidine (His229).41,42 The heme iron in SiaA is identified to be six-coordinate and low-spin through characterization by Raman, magnetic circular dichroism and
Fig. 2 Scheme for the heme acquisition by the Shr/Shp/SiaABC system in S. pyogenes. Shr obtains heme upon binding of hemoglobin to the NTD domain. The heme in the NEAT1 of Shr is rapidly transferred to Shp in the presence of high amounts of environmental heme. The NEAT2 is a temporary storage place for heme. When environmental heme availability is limited, heme from NEAT2 is transferred to NEAT1 for the subsequent transfer to Shp. The heme in Shp is further transported to SiaA protein of the SiaABC transporter for the subsequent transmembrane transport. The zoomed-in area shows the intermediate state of heme transfer from Shp to SiaA, with the axial heme ligands highlighted.
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Once internalized, heme may bind to AhpC in a 1 : 1 ratio with a Kd of 0.5 mM.54 AhpC has roles in the intracellular fate of heme due to the following findings: (1) AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo; (2) the bacterium with AhpC inactivated displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase; and (3) heme is protected from chemical degradation in vitro upon AhpC binding.54
Siderophore transporter Fig. 3 Bis-methionyl coordination in Shp and heme stacking in the crystal interface (PDB ID: 2Q7A). Heme packing is shown between the bound and the exogenous hemes. Hydrogen bonds formed between Arg155 and a propionate on the bound heme molecules.
nuclear magnetic resonance.41 The biaxial ligands Met79 and His229 of SiaA have differential roles with His229 as the key residue for heme binding,42 further supported by competitive binding experiments with exogenous imidazole, cyanide and CO.43 Shp can rapidly transfer heme to SiaA in a concerted two-step process: holoShp forms a complex with apoSiaA prior to heme transfer.40,44,45 Further study implies that the axial residues of Met79 and His229 of SiaA displace the Met66 and Met153 residues of Shp, respectively, during the heme transfer reaction.46 The axial ligand replacement during the heme transfer between holoShp and apoSiaA displays a first-order kinetic phase, indicating that the displacement of the first Met ligand is rate limiting and the two new coordination bonds in SiaA form roughly at the same time.40,45 The heme transportation system in Streptococcus species other than S. pyogenes is still far from clear. The Shr/Shp/ HtsABC(SiaABC) system was found to be important for heme acquisition in S. equi,47,48 albeit detailed mechanisms have not been established. Two membrane proteins from S. pneumonia were identified to be potential heme acquisition proteins through a proteomic work on the proteins purified from heme-affinity chromatography and competitive binding experiments with heme and iron.49 MtsABC25 and FtsABCD50 transporters were supposed to be involved in heme utilization in S. iniae. The internalized heme was important to the activation of the respiratory chain in S. agalactiae in the presence of exogenous menaquinone or quinone.51,52 S. agalactiae cells with an activated respiratory chain displayed improved survival in an aerobic environment and greater virulence in a murine septicemia model.51 Although the heme acquisition system is unknown for S. agalactiae, two efflux operons (pefAB and pefRCD)53 and one intracellular heme binding protein (alkyl hydroperoxide reductase AhpC)54 were identified to be important for heme and protoporphyrin IX homeostasis. The bacterium with both pefAB and pefRCD inactivated displayed accrued sensitivity to heme and protoporphyrin IX.53 The porphyrins accumulate intracellularly.
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Siderophores are small organic molecules with variable Fe3+ affinities (ranging over 30 orders of magnitude).55–58 Siderophores are classified into catecholates (e.g. enterobactin), hydroxamates (e.g. ferrichrome (Fch)) and hydroxycarboxylate (e.g. staphyloferrin A) based on their structures. The iron utilization as mediated by siderophores consists of a series of steps: siderophore synthesis, apo-siderophore secretion and the subsequent transmembrane transportation through holo-siderophore specific transporters or through ferrous transporters such as MtsABC. S. pyogenes genome encodes three ABC transporters, HtsABC/SiaABC, MtsABC, and FtsABCD (ferrichrome transporter system)/SiuADBG (Streptococcal iron uptake).59,60 In a study conducted by Montanez and coworkers, SiuADBG was named to emphasize its role in iron uptake and was proposed to be involved in heme utilization by S. pyogenes.60 In a later report by Lei and coworkers, the ABC transporter was renamed as FtsABCD,59 as this transporter was found to be important for Fe3+–Fch uptake. During Fch utilization, Fch is captured by the lipoprotein FtsB in 1 : 1 stoichiometry and then transferred to the permease FtsCD for subsequent transmembrane delivery. FtsA is an ATPase which provides energy for the Fch transfer. Fch binding in FtsB occurs in two steps with the C-terminal Trp204 and the N-terminal Tyr137 to be two essential residues.61 One hydroxamate-type siderophore transporter FhuCDBG was identified in S. agalactiae, containing one putative ATPase (FhuC, at the inner side of the cytoplasmic membrane), a cell surface binding lipoprotein (FhuD) and two transmembrane proteins (FhuB and FhuG, at the outer and inner cytoplasmic membranes respectively).62 FhuD binds hydroxamate-type siderophores with high affinities (e.g. for Fe3+–desferroxamine interaction, Kd = 50 nM).62 The role of FhuD in the acquisition of siderophore iron was also established through Fe accumulation assays. In S. pneumoniae, three ABC transporters are involved in siderophore acquisition, i.e., piuBCDA/pit1BCDA/fhuDBGC,63,64 piaABCD/pit2ABCD56,57 and pitBCDA.65 In S. pneumoniae, all the four fhu genes have the same transcription direction with the homologues in S. agalactiae but are arranged as fhuDBGC.66 This siderophore system was first described as pit2 (pneumococcal iron transporter) and pia (pneumococcal iron acquisition).65,67 FhuD of S. pneumoniae is more closely related to the fhuD homologue of E. coli than to other Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus.66 Therefore the fhu operon of S. pneumoniae is probably acquired by gene transfer as evidenced by the presence of a 27 kb region with characteristics of a
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Gram-negative bacterial pathogenicity island,67 a hint that the acquisition played a significant role in the evolution of this pathological bacterium. The membrane-anchored substratebinding protein PiaA is composed of an N-terminal and a C-terminal domain bridged by an a-helix.68 Fch is bound at the inter-domain cleft and stabilized by a number of amino acids: (1) two hydrogen bonds between Arg231 and carbonyl oxygen atoms of two hydroxamic acid moieties from Fch; (2) one hydrogen bond between the hydroxyl group of Tyr225 and the carbonyl oxygen atom of the third hydroxamic acid moiety; (3) three hydrogen bonds between the backbone of Fch and the side chains of Trp158 and Asn83; (4) hydrophobic interactions between the methylene carbon atoms of the hydroxyornithine moieties with the hydrophobic barrel formed by Met213, Trp223, Tyr225 and Phe255 from the C-terminal and Trp63, Tyr84 and Trp158 from the N-terminal domain.68 Besides siderophore uptake, piu and pia were also identified as responsible for hemoglobin utilization of S. pneumoniae,69 indicating that they may have variable substrates. In S. equi, a siderophore named equibactin is produced by a non-ribosomal peptide synthetase system within the locus eqbA-N.70 The production of equibactin was abolished in the absence of eqbE, eqbG and eqbM and was reduced in the absence of eqbN. Therefore the equibactin locus containing all of the genes unique to S. equi is required for the biosynthesis. EqbK and EqbL share 29% amino acid sequence identity with YbtP and YbtiQ of Y. pestis which are involved in siderophoremediated iron import and are structurally similar to the ABC transporters associated with iron acquisition.71 The ABC transporters encoded by the genes of eqbH, eqbI and eqbJ are required for the associated iron import.
Iron regulation Bacteria need to achieve intracellular iron homeostasis to fulfill their metabolic functions and to prevent the toxic effects attributed to the oxygen radicals produced by excessive iron (Fenton reaction: H2O2 + Fe2+ - OH + OH + Fe3+).72 Metaldependent transcription regulators, Fur or DtxR, are commonly found in bacteria to maintain iron homeostasis.73 Although the sequence homology between Fur and DtxR is modest, crystallographic data indicated substantial structural similarities,74,75 all sharing an N-terminal DNA binding domain and a dimerization domain with two metal binding sites per subunit. In S. pyogenes, PerR is a Fur type peroxide stress response regulator (Fig. 4).76–78 Oxidative conditions stimulate the oxidation of selected histidine residues of PerR and the subsequent release of the regulator from the DNA.77 One transcriptome study of the wild type and perR mutant indicates that PerRdependent genes were almost exclusively down-regulated and are involved in purine and deoxyribonucleotide biosynthesis, iron and heme homeostasis and amino acid/peptide transport.79 The regulated iron and heme homeostasis genes include the mts ferrous iron transporter, fhu/fts heme/Fch transport system, shr/shp/sia heme acquisition system,79 and the only
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Fig. 4 A schematic overview of the iron regulation system in S. pyogenes. PerR and MtsR coordinate together to control the iron uptake system in S. pyogenes.
ferritin-like iron storage protein Dpr/MrgA.80,81 Dpr expression was elevated upon treatment with millimolar concentrations of iron. Further study indicated that PerR binds the promoter region of dpr and increased iron concentrations and decreased PerR binding to the promoter region of dpr,80 thus resulting in an elevated expression of Dpr. DNase I footprinting revealed that S. pyogenes PerR binds to a single site upstream of the promoter for the gene encoding AhpC,81 the S. agalactiae homologue involved in intracellular heme homeostasis.54 However the regulation of AhpC was revealed to be independent of PerR.81 A structural study of the S. pyogenes PerR revealed that PerR exists as a homodimer with two metal-binding sites per subunit: one structural zinc site and one regulatory metal site.82 The regulatory site has an N-terminal HXH metal-binding motif83,84 fully conserved among PerRs from Streptococcus species and unique from other Fur family proteins. The integrity of this site is critical to bacterial pathology.82 A second S. pyogenes metal-dependent transcription regulator protein, MtsR, belongs to the DtxR family of metallo-repressors, and is located upstream and divergently transcribed from the mts gene cluster.85 PerR and MtsR coordinate the expression of genes involved in metal uptake and homeostasis as well as genes essential for the production of infection and pathology. MtsR directly represses the sia and mts transporters30,86 and the heme binding protein ahpC87 under conditions of high metal levels. MtsR has a more robust control of mtsA expression than of siaA, possibly attributed to its higher binding affinity for the mts promoter region.30,86 An mtsR mutant derivative of serotype M49 strain NZ131 has an elevated level of intracellular iron and is more sensitive to oxidative stress and less virulent in zebrafish infection models.86 A transcriptome analysis of the wild type and the mtsR inactivated mutant S. pyogenes revealed 44 up-regulated genes and 20 down-regulated genes,88 indicating that MtsR has an extensive regulatory role in S. pyogenes. The activated genes contribute to nucleotide metabolism, transport,
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cell envelope and regulation. The repressed genes are involved in nucleotide metabolism, transport, cell envelope and regulation, protein synthesis and fate, and the biosynthesis of amino acids.88 The repressed metal-related genes include the 10-gene sia operon, Per-regulated metal transporter pmtA (encoding a P-type ATPase and mediating metal export89 and peroxide resistance) and the P-type ATPase family heavy metal transporter copA.88 However no changes in mts and ahpC transcripts were observed in this transcriptome study, demonstrating some strain-dependent variation of the regulon.
Conclusions and perspectives Streptococcus is a genus of pathological Gram-positive bacteria and streptococci have been closely associated with millions of cases of meningitis, bacterial pneumonia, endocarditis, erysipelas and necrotizing fasciitis. As iron is essential for almost all living organisms, investigation of the iron acquisition and regulation systems is important to understand the pathological roles of the Streptococcus species. The relevant iron acquisition proteins may be the future targets of antibiotics or other antibacterial treatments. The available information about iron uptake and regulation of the Streptococcus species has been discussed in this concise review. Ferrous iron is transported through the membrane by MtsABC. However, what is the ferrous iron origin, from free ferrous iron pool in the host, from host ferrous-storage proteins, or from the reduction of ferric iron sources, such as ferric-storage proteins, heme-containing proteins and ferric-siderophores? What kind of reductase is responsible for the reduction of ferric into ferrous iron in the extracellular space? The ferric iron molecules may be uptaken through the Shr/Shp/ SiaABC system (heme) or FtsABCD transporter (ferric-siderophore). However it is not yet clear how iron is removed from the various carriers (heme and siderophore) once inside the cell. Iron may be the active cofactor in quite a number of enzymes, such as hydrogenase and SOD. Which kinds of iron chaperone proteins are involved in the maturation of these iron-containing enzymes in the Streptococcus species? The iron trafficking system in the Streptococcus species has not been studied systematically with metallomic or metalloproteomic methods. Advanced nuclear analytical techniques, such as neutron activation analysis, X-ray emission/fluorescence spectroscopy, isotope dilution and tracing, X-ray absorption, neutron scattering, electron paramagnetic resonance and nuclear magnetic resonance, are highly sensitive, accurate and non-destructive and play increasingly important roles in the studies of metallomics and metalloproteomics.90,91 The application of these techniques will help clarify in several ways the iron-trafficking proteins in the Streptococcus species in vivo: the cellular localization of iron inside the bacterium; the structure of iron-containing metalloenzymes; and so on. All these questions regarding the iron trafficking system in the Streptococcus species need further work to fully understand.
Acknowledgements This work was supported by National Natural Science Foundation of China (20801061, 31000373 and 21371182), the Fundamental
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Research Funds for the Central Universities (10lgpy19 and 21611201), Pearl River Rising Star of Science and Technology of Guangzhou City (2011J2200079 and 2011J2200003) and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Jinan University.
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