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Siderophore-mediated iron acquisition in the staphylococci
Journal of Inorganic Biochemistry 104 (2010) 282–288
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Siderophore-mediated iron acquisition in the staphylococci Federico C. Beasley, David E. Heinrichs * Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada N6A 5C1
a r t i c l e
i n f o
Article history: Received 10 June 2009 Received in revised form 1 September 2009 Accepted 18 September 2009 Available online 26 September 2009 Keywords: Iron Siderophore Staphylococcus Iron acquisition ABC transporters
a b s t r a c t Iron is frequently a growth-limiting nutrient due to its propensity to interact with oxygen to form insoluble precipitates and, therefore, biological systems have evolved specialized uptake mechanisms to obtain this essential nutrient. Many pathogenic bacteria are capable of obtaining stringently sequestered iron from animal hosts by one or both of the following mechanisms: extraction of heme from host erythrocyte and serum hemoproteins, or through the use of high affinity, iron-scavenging molecules termed siderophores. This review summarizes our current knowledge of siderophore-mediated iron acquisition systems in the genus Staphylococcus. Ó 2009 Elsevier Inc. All rights reserved.
1. The staphylococci The staphylococci are facultatively aerobic Gram-positive bacteria. Most species are natural inhabitants of mammalian skin and mucous membranes [1]. A few are capable of causing disease, especially in an opportunistic fashion; the most studied pathogenic species is Staphylococcus aureus. Superficial colonization by staphylococci can result in furunculosis or impetigo [2], and systemic infiltration of tissues by S. aureus leads to syndromes including septicemia, endocarditis, and necrotizing pneumonia [3–5]. While S. aureus infections are especially frequent in hospital settings among patients suffering temporary or chronic compromise to the immune system, there exist some community-acquired strains that are particularly aggressive and can cause debilitating disease in healthy individuals [6]. For the purposes of identification, S. aureus is distinguished from other, less virulent staphylococci based on the species’ uniform expression of the enzyme coagulase; most other species are denoted as coagulase-negative (CoNS), though some contain strains that may be coagulase-positive [7]. CoNS infections are typically less severe, hospital acquired, and dependent on immunocompromise [8] or the opportunity to colonize implanted medical devices [9,10]. The best-characterized member of this group is Staphylococcus epidermidis, although Staphylococcus saprophyticus is noteworthy for being the second leading cause of uncomplicated urinary tract infection after Escherichia coli [11]. Pertinent to this review, another key distinction between S. aureus and CoNS is the genetic complement asso-
* Corresponding author. Tel.: +1 519 661 3984; fax: +1 519 661 3499. E-mail address: [email protected] (D.E. Heinrichs). 0162-0134/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2009.09.011
ciated with iron acquisition. Absent from CoNS (at least in genomes sequenced to date) but present in S. aureus are two key iron acquisition mechanisms (see Table 1). The first is the isd (iron-regulated surface determinant) gene set that mediates heme acquisition from mammalian heme-containing proteins, and the second is an Fe(III)-siderophore acquisition system that will be described in more detail below. It is feasible that these iron acquisition mechanisms contribute to the higher virulence potential of S. aureus versus its CoNS counterparts.
2. Iron With the exception of some lactobacilli and spirochaetes [12,13], iron is an essential nutrient for all bacteria. The Fe(III) centre of cytochromes is crucial to respiratory and photosynthetic ATP biosynthesis; Fe–S clusters are central to redox reactions mediated by proteins such as nitrogenase and hydrogenase; and iron is a cofactor for numerous other metalloproteins. Under physiological conditions, iron predominates in its ferric form, forming insoluble Fe(III) hydroxides [14]. In animal tissues and sera, iron is further sequestered by transferrin, so that the amount persisting free in solution is on the order of 10 24 M [15], well below the threshold required to sustain microbial life. Iron deprivation is a key sensory trigger for the expression of bacterial virulence factors, many of which are known to facilitate iron acquisition [16–19]. Bacteria use global, iron-responsive transcriptional regulators, such as the well-characterized Fur or the RirA (in Rhizobiales)-type regulatory proteins, to control gene expression (for reviews, see [20–23]). In brief, when cellular iron exceeds the level required for proper function of essential
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F.C. Beasley, D.E. Heinrichs / Journal of Inorganic Biochemistry 104 (2010) 282–288 Table 1 Known and putative genes involved in iron acquisition in S. aureus and S. epidermidis. Gene(s)
Locus number (S. aureus Newman or S. epidermidis RP62A)
Role of product(s)
References
S. aureus sbnABCDEFGHI sirABC isdB harA isdA isdC isdDEF
NWMN_0060-0068 NWMN_0059-0057 NWMN_1040 NWMN_2614 NWMN_1041 NWMN_1042 NWMN_1042-1045
NIS biosynthesis (staphyloferrin B) ABC transporter (staphyloferrin B) Hemoglobin and haptoglobin binding
[67] [68] [80,81]
Heme binding/transfer
[82–86] [83,87,88]
isdG isdI
NWMN_1047 NWMN_0111
Membrane proteins involved in heme transport Heme degradation
S. aureus and S. epidermidis sfaABC sfaD htsABC fhuD1, fhuD2 (aureus); fhuD (epidermidis) fhuCBG (aureus) fhuBC (epidermidis) sstABCD fur
[89]
NWMN_2081-2079 NWMN_2078 SERP1780-1778 SERP1781 NWMN_2078-2076 SERP1777-1775 NWMN_1931, NWMN_2185 SERP1777
NIS biosynthesis (staphyloferrin A)
[59,62]
ABC transporter (staphyloferrin A; heme?) Lipoprotein receptor (Fe3+ hydroxamates)
[59] [55,79]
NWMN_0616-0618 SERP1776-1775 NWMN_0702-0705 SERP0400-0403 NWMN_1406 SERP1061
ABC transporter (Fe3+ hydroxamates) ABC transporter (siderophore?) Transcriptional repressor of iron uptake
[72] [70] [90]
iron-containing metalloenzymes, association of an Fe(II) ion to each Fur monomer causes the dimeric protein to bind a roughly 19-bp DNA motif, called the Fur box, within the operator region of target genes. Typically, this binding blocks RNA polymerase, resulting in downregulation of gene transcription. Fur is known to indirectly activate some genes, via repression of small RNA silencing molecules [24], and direct upregulation of a target gene has also been reported [25].
3. Siderophore biosynthesis Numerous plants, fungi, saprophytic bacteria, and bacterial pathogens secrete small (typically less than 1 kDa) molecules called siderophores to serve as high affinity Fe(III) scavengers. Huge diversity has been documented in terms of the structure and iron-binding affinity of these compounds, although some broadly shared themes describe their physicochemical properties (for more comprehensive reviews, see [26–28]). Siderophores are highly electronegative and surround Fe(III) to form a hexacoordinated complex, although the stoichiometry of iron atoms to siderophore molecules may vary, depending on the number of donor atoms per siderophore molecule and its ability to use surrogate oxygen, nitrogen, or sulfur donors from solution to compensate for vacancies. In general, three types of functional groups constitute siderophore iron coordination motifs. First are the adjacent hydroxyl groups of catechol moieties, exemplified in the enterobactin structure (Fig. 1) [29]. Second are hydroxamate motifs, where the carbonyl and aminohydroxyl groups each occupy an iron coordination site, as in coprogen B [30] (Fig. 1). Third are carboxylate motifs. These may involve two sites provided by a-hydroxy-carboxylates derived from citrate, as in petrobactin [31], or from carboxylic acid moieties, as in rhizoferrin (Fig. 1) [32]. Numerous siderophores, such as the pyoverdins [33] or aerobactin [34] (Fig. 2), use combinations of the three iron coordinating groups. There are two structurally characterized siderophores produced by members of the staphylococci: staphyloferrin A and staphyloferrin B (Fig. 1). Siderophore assembly pathways fall into two broad classes: nonribosomal peptide synthesis (NRPS) and NRPS-independent siderophore (NIS) synthesis [35]. NRPS mechanisms have been characterized for numerous siderophores, including enterobactin [15], yersiniabactin [15,36], and vibriobactin [37], as well as impor-
tant antibiotics such as penicillin [38] and vancomycin [39]. NRPS siderophores have peptidic scaffolds, often incorporating nonproteinogenic amino acids and their derivatives, that are assembled stepwise without the benefit of a ribosomal template [26]. Less well characterized are the mechanisms for assembly of NIS siderophores. These are created from the condensation of alternating subunits of dicarboxylic acids (usually succinate, citrate, and a-ketoglutarate) with diamines, amino alcohols, and alcohols [35]. NIS synthetases have been sparingly investigated. For decades, the proposed assembly of aerobactin (Fig. 2) has represented the archetypal pathway of NIS biosynthesis [40,41], even though the biochemical roles of synthetases IucA and IucC have not been empirically characterized. Recent efforts have described the pathway involved in production of petrobactin [42,43], which is a rare example of a siderophore assembled via a hybrid NRPS/NIS strategy. Elucidation of the molecular structure of other siderophores and annotation of the genetic loci encoding their biosynthesis suggest rhizobactin [44], achromobactin [45], vibrioferrin [46], and alcaligin [47] are also assembled in an NRPS-independent fashion. NIS synthetases are not homologous to their modular counterparts in NRPS pathways. They primarily serve to catalyze formation of amide and ester bonds between an organic acid and a substrate bearing an amino or a hydroxyl group [35]. A model has been proposed to classify NIS synthetases based on specificity for a carboxylic acid [35,48]. Type A enzymes, represented by IucA, catalyze condensation of a prochiral carboxyl group of citrate to an amine or alcohol functional group of a second substrate, resulting in an amide or ester bond, respectively. Type B enzymes catalyze a similar reaction substituting the C5 carboxyl of a-ketoglutarate for the citrate carboxyl. Type C enzymes, represented by IucC, catalyze condensation of monoamide/monoester derivatives of citrate or succinate to carboxyl groups on molecules with an amine or an alcohol. Prior to condensation, NIS synthetases activate reacting carboxyl groups by adenylation with a nucleotide triphosphate. Acyl-adenylate intermediates are captured by an amine substrate leading to condensation to an amide [49]. For the most part, predictions of NIS synthetase type based on phylogenetic analysis have accurately described their role in functional enzyme assays [48], but this is based on limited data, and at least one exception has been uncovered and is described below. Additional modifications to precursors, such as decarboxylation, isomerization, or oxidation, are performed by separate enzymes typically encoded by genes clustering near to those encoding the synthetases.
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Fig. 1. Chemical structures of representative siderophores.
Fig. 2. Biosynthetic pathway for aerobactin.
4. Siderophore transport The cytoplasmic membrane in Gram-positive bacteria provides the main barrier to entry into the cytoplasm for small molecules such as Fe(III)-siderophore complexes. As such, Fe(III)-siderophores are brought into the cytoplasm through an active process involving ATP-binding cassette (ABC) transporters that use hydrolysis of ATP to provide the energy required for transport across the
membrane. ABC transporter systems are comprised of a substratebinding protein, a transmembrane permease and an ATPase. The ATPase interacts with the permease and couples active transport to ATP hydrolysis. For an excellent, comprehensive review of ABC-type transport systems, see [50]. Fe(III)-siderophore binding proteins in Gram-positive bacteria are lipoproteins tethered to the lipid bilayer via acylation to a conserved cysteine residue which resides at the end of the cleavable signal peptide, within
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the so-called ‘‘lipobox” domain (consensus: LXXC). The substratebinding proteins involved in Fe(III)-siderophore binding are part of a large family of proteins also known as the periplasmic binding protein (PBP) family (based on their location in the periplasm of Gram-negative bacteria). The Fe(III)-siderophore binding proteins fall into cluster 8 of this large family [51]. Binding proteins are characterized by two lobes, each made of mixed a/b structure, that surround the substrate-binding pocket; in cluster 8 proteins, the two lobes of the protein are connected by a long a-helical backbone that spans the width of the protein (Fig. 3), rather than by a more flexible series of short b-strands found in other binding proteins as exemplified by maltose binding protein. The resulting characteristic feature of the helical backbone type binding proteins is relatively restricted domain movement upon ligand binding and release [52–55]. Among the interactions between the binding protein and the membrane permease are key salt bridges that are formed between conserved glutamic acid residues on each of the lobes of the binding protein (Fig. 3) and three arginine residues that form a positively-charged pocket in each half of the permease [55,56]. 5. Staphyloferrin A biosynthesis Staphyloferrin A (Fig. 1) was first purified from culture supernatant of iron-starved Staphylococcus hyicus [57]. Staphyloferrin A (480 Da) is a highly hydrophilic molecule comprised of two molecules of citrate bridged by D-ornithine, thus having the structural identity N2,N5-di-(1-oxo-3-hydroxy-3,4-dicarboxylbutyl)-D-ornithine. The metabolite is produced by a wide variety of clinically relevant staphylococci, including S. aureus and S. epidermidis, and its production is stimulated by supplementation of the growth medium with ornithine [58]. To identify genes encoding for biosynthesis of this putative NIS siderophore, bioinformatic searches for IucA and IucC homologues uncovered the four-gene sfa locus (staphyloferrin A) in all sequenced staphylococcal genomes, including strains of S. aureus, S. epidermidis, S. saprophyticus, and Staphylococcus haemolyticus [59] (Fig. 4 and Table 1). The cluster comprises one three-gene operon, sfaABC, and a divergently transcribed sfaD gene. A consensus Fur box lies intergenically to sfaA and sfaD, suggesting transcriptional control by Fur which was confirmed in S. aureus using real-time PCR [59]. Production of staphy-
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loferrin A has been reported as constitutive among some CoNS isolates [60], suggesting a possible lack of Fur-dependent regulation of the locus in some species. Chromosomal deletion of the sfa locus in S. aureus abrogates production of a secreted metabolite with a mass and chemical structure corresponding to that of staphyloferrin A [59]. Surprisingly this mutation was not associated with an observable defect in bacterial growth in mammalian serum [59]. Only when staphyloferrin B production was also knocked out (sbn deletion – see below) in the same strain of S. aureus was there an observable staphyloferrin A-dependent growth defect in serum. The sfa mutant growth defect in an sbn deletion strain of S. aureus was restored by complementation with plasmid-borne sfa genes from S. aureus, as well as the homologous sfa gene locus from S. epidermidis, suggesting functional conservation of Sfa proteins among the staphylococci, despite protein sequence identities in the range of 50–80% between S. aureus and S. epidermidis. The observation that staphyloferrin A-deficient mutants of S. aureus, at least in strains producing another siderophore (staphyloferrin B, see below), do not suffer growth reduction, suggests that staphyloferrin A may not be a major contributor to iron extraction from transferrin, the primary iron substrate in serum. Extraction of transferrin iron may be dependent on other processes that affect glycoproteins, such as binding to a cell membrane receptor or proteolytic cleavage. Staphyloferrin A has been reported to leach iron from human transferrin, which is recognized by staphylococcal transferrin receptor proteins, as well as porcine transferrin, which is not recognized [61]. These studies may have used staphyloferrin A at concentrations exceeding what is produced in culture supernatants of S. aureus strains that are producing a second siderophore. The ability of staphyloferrin A to capture iron from serum glycoproteins merits further investigation. S. aureus proteins SfaB and SfaD have been characterized as NIS synthetases. When overexpressed and purified, they are capable of generating staphyloferrin A in vitro from citrate and D-, but not L-, ornithine [62]. The SfaC protein is homologous to amino acid racemases and likely racemases L-ornithine to D-ornithine to provide the substrate for staphyloferrin A synthesis. SfaD mediates the first committed step in synthesis, forming a d-citryl-D-ornithine intermediate, and SfaB condenses the second citric acid onto the intermediate (Fig. 5) [62]. While SfaB and SfaD cluster among B type NIS synthetases [35,62], their chemistry aligns them with the A-type (i.e. specificity for citrate) enzymes, forming a subgroup within the A-type enzymes. It will be interesting to follow the evolution of the NIS synthetase phylogeny as additional NIS synthetases are characterized biochemically. The SfaA protein, which shares similarity with membrane-embedded efflux proteins, is a likely candidate for involvement in staphyloferrin A efflux. Empirical corroboration of this function would provide valuable insight into the secretion of siderophores in Gram-positive bacteria.
6. Staphyloferrin A transport
Fig. 3. Crystal structure of the staphyloferrin A binding protein HtsA (PDB: 3EIW) [59], which is a member of the periplasmic binding protein superfamily. The Fe(III)siderophore binding subfamily is characterized by two mixed a/b domains (Nterminal domain of HtsA is shown in green and the C-terminal domain is shown in blue) separated by a long a-helical backbone (yellow) and conserved glutamic acid residues (represented in stick format) on the surface of the lobes. The glutamic acid residues form salt bridges with arginine residues in the membrane permease proteins to mediate productive protein:protein interactions and, therefore, transport.
The iron and Fur-regulated htsABC operon (heme transport system) lies adjacent to the sfa gene cluster in the chromosomes of S. aureus and CoNS (Fig. 4) [59]. An early study describes its role in heme transport [63], showing that inactivation of the permease components HtsB and HtsC shift the iron source preference of S. aureus from heme toward transferrin (and, implicitly, toward siderophore-mediated iron uptake). Although direct involvement of HtsABC in heme transport has not been demonstrated the Hts transporter is linked to staphyloferrin A uptake in S. aureus. Indeed, transport of staphyloferrin A by S. aureus is abolished in htsABC mutants, and the crystal structure of the unliganded HtsA substrate-binding protein reveals a positively-charged binding pocket, consistent with a role in binding of the anionic siderophore
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Fig. 4. Genetic loci in staphylococci involved in siderophore production. The sfa-hts locus is found in S. aureus and CoNS and is responsible for biosynthesis and transport of staphyloferrin A. The sbn-sir locus is found in S. aureus and a homologous cluster of genes is found in Ralstonia spp. The locus is responsible for synthesis and transport of staphyloferrin B.
Fig. 5. Biosynthetic pathway for staphyloferrin A, as determined in [62].
staphyloferrin A [59]. HtsA (Fig. 3) belongs to the cluster 8 subfamily of the periplasmic binding protein superfamily (described above). Strangely, despite the energy dependence of siderophore transport, the operon lacks a gene encoding an ATPase component and genetic evidence supports a role for the FhuC ATPase (discussed in more detail below) in staphyloferrin A transport [59]. 7. Staphyloferrin B biosynthesis A number of staphylococcal species have been shown to produce a second siderophore, termed staphyloferrin B. This is another highly hydrophilic NIS siderophore composed of L-2,3-diaminopropionic acid (DAPA), citrate, ethylenediamine, and a-ketoglutarate (Fig. 1), with a mass of 448 Da [64]. Purification and structural characterization of the metabolite was performed using culture supernatants of iron-starved S. hyicus [65]. HPLC analysis has identified it in supernatants of S. aureus and a wide range of CoNS species [58,64]. Interestingly, the molecule is also produced by the Gram-negative plant pathogen Ralstonia solanacearum and the metal-resistant soil bacterium Cupriavidus metallidurans [66]. In an attempt to uncover genes responsible for biosynthesis of siderophores in S. aureus, a search for homologues of IucA and IucC
in S. aureus uncovered a nine-gene operon designated sbn (siderophore biosynthesis). The sbn operon (Fig. 4 and Table 2) encodes three putative NIS synthetases: SbnC, SbnE, and SbnF [67]. Phylogenetic analysis groups them with type B, type A, and type C synthetases, respectively [35]. The sbn operon is highly similar in sequence and arrangement to the staphyloferrin B biosynthesis operon of R. solanacearum [66], suggesting that sbn genes encode the machinery required for staphyloferrin B biosynthesis, as opposed to the previous designation of staphylobactin for a structurally uncharacterized siderophore whose production was initially linked to the sbn operon [67]. A consensus Fur box lies within the sbn operon operator region, and b-galactosidase transcriptional fusions to sbn genes were used to demonstrate maximal expression of the operon under conditions of iron restriction [67]. Chromosomal inactivation of the sbn operon in S. aureus leads to impaired growth in serum, though culture densities eventually approach wild type levels with prolonged incubation, presumably through the production of staphyloferrin A [59]. An sbnE mutant strain proved attenuated in a mouse model of kidney infection [67], highlighting the importance of specialized iron uptake mechanisms in virulence S. aureus. The structure of staphyloferrin B by S. aureus could feasibly be assembled by the complement of predicted protein functions
F.C. Beasley, D.E. Heinrichs / Journal of Inorganic Biochemistry 104 (2010) 282–288 Table 2 Staphyloferrin biosynthesis and transport proteins in Staphylococcus aureus. Protein
Putative or characterized function
Staphyloferrin A SfaA Major facilitator superfamily protein; staphyloferrin A efflux SfaB NIS synthetase; staphyloferrin A synthesis SfaC Racemase; conversion of L-ornithine to D-ornithine SfaD NIS synthetase; staphyloferrin A synthesis HtsA Staphyloferrin A binding protein HtsB ABC transporter permease; staphyloferrin A transport HtsC ABC transporter permease; staphyloferrin A transport Staphyloferrin B SbnA O-acetyl-L-serine sulfylhydrylase; synthesis of L-2,3diaminpropionic acid SbnB Ornithine cyclodeaminase; synthesis of L-2,3-diaminopropionic acid SbnC Type B NIS synthetase; staphyloferrin B synthesis SbnD Major facilitator superfamily protein; staphyloferrin B efflux SbnE Type A NIS synthetase; staphyloferrin B synthesis SbnF Type C NIS synthetase; staphyloferrin B synthesis SbnG Aldolase SbnH Amino acid decarboxylase SbnI Unknown function SirA Staphyloferrin B receptor SirB Heterodimeric permease component; staphyloferrin B transporter SirC Heterodimeric permease component; staphyloferrin B transporter
encoded by the sbn operon [67]. No detailed characterization of Sbn proteins has as yet been published at the time of writing this review. However, the NIS synthetases (SbnC, SbnE and SbnF) could condense molecules of diaminopropionic acid (Dap), citrate, ethylenediamine, and 2-ketoglutaric acid. SbnA, a putative L-Oacetylserine sulfhydrylase, and SbnB, a putative ornithine cyclodeaminase, could generate Dap from a coupled reaction involving cyclization of the diamino acid ornithine to produce proline and ammonia, with the ammonia being used to convert O-acetyl-L-serine into Dap. SbnD, with similarity to antibiotic efflux proteins, is likely involved in secretion of siderophore into the extracellular milieu, while the roles of SbnG, SbnH and SbnI remain unclear at the time of writing this review. It is interesting to note that although staphyloferrin B was reported to be produced by some CoNS species [58,64], to date, the sbn gene cluster has not been identified in any of the available CoNS genome sequences (Table 1), nor was the sbn gene cluster detected in a screen of CoNS using low-stringency Southern blot hybridization analyses [67]. Searches of the rapidly expanding number of sequenced CoNS genomes (7 with BlastP functionality at the time of writing: S. capitis SK14, S. epidermidis RP62A, S. epidermidis ATCC12228, S. saprophyticus ATCC15305, S. haemolyticus JCSC1435, Staphylococcus carnosus TM300, and Staphylococcus hominis SK119) have not uncovered any obvious non-sfa NIS synthetase encoding genes.
8. Staphyloferrin B transport The sirABC transport operon (staphylococcal iron regulated) (Fig. 4) is divergently transcribed from the sbn operon in the chromosome of S. aureus. Similar to the sbn operon, sirABC is absent in all currently available CoNS genome sequences. Expression of this transport system is also regulated by iron and Fur [68,69]. As with the HtsABC transporter, no encoded ATP-binding component is found in the genomic vicinity. Uptake of S. aureus siderophore (staphyloferrin B) complexed with 55Fe was shown to be dependent on expression of the receptor protein SirA and the permease component SirB, and transport is an energy-dependent process [68]. Heterologous expression of sirABC in an S. epidermidis strain (which does not possess sbn genes and, thus, synthesize staphyloferrin B) rendered it capable of using the sbn siderophore as an iron
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source (F.C. Beasley, unpublished data). Two conclusions are derived following crossfeeding experiments between combinations of transporter and biosynthesis mutants of S. aureus [59]. First, as siderophore output is nil in a double sfa sbn biosynthesis mutant, staphyloferrin A and staphyloferrin B are likely the only siderophores produced by characterized strains of S. aureus. Second, as transport of these siderophores is dependent on HtsABC and SirABC, respectively, the two transporters specifically transport their cognate siderophore ligand, and it is unlikely that redundant transporters are encoded elsewhere in the genome. 9. Transport of exogenous siderophores by S. aureus To date, there are three ABC transporters demonstrated to be involved in Fe(III)-siderophore uptake in S. aureus. These are involved in the uptake of staphyloferrin A (HtsABC) and staphyloferrin B (SirABC) as described above, and a third which is required for uptake of Fe(III)-hydroxamate complexes (FhuCBG-D1-D2). A fourth iron-regulated ABC transporter (SstABCD) is encoded by S. aureus and the substrate for this transporter has not yet been identified [70]. Hydroxamate-type siderophores are considered exogenous metabolites, as studies have so far failed to detect hydroxamate siderophore production by S. aureus [71,72]. The transporter is comprised of permease components FhuBG, ATP-binding protein FhuC, and dual receptor proteins FhuD1 and FhuD2 which can compete for an interaction with FhuCBG. Component nomenclature is based on their homology to ferric hydroxamate uptake proteins of E. coli and Bacillus [73–76]. The fhuCBG operon and moncistronic genes fhuD1 and fhuD2 are located at widely segregated positions on the S. aureus chromosome; fhuCBG and single allele fhuD operons have also been annotated in CoNS genomes. Chromosomal inactivations of fhuB or fhuG result in an inability to use a range of hydroxamate siderophores, including the bacterial siderophore aerobactin (Fig. 2), the fungal siderophores ferrichrome and coprogen, and the clinically-utilized siderophore DesferalTM [72,77,78]. Compared to FhuD1, FhuD2 has a higher substrate range and affinity for hydroxamate siderophores (for example, DesferalTM: Kd FhuD2 = 5 10 8 M, Kd FhuD1 = 1 10 6 M) [55]. As observed for E. coli FhuD and other members of the Fe(III)-siderophore binding protein superfamily, substrate binding induces very little conformational change in FhuD1 and FhuD2 [55,79]. FhuD1, with reduced affinity for Fe-hydroxamates and inconsistent presence of fhuD1 gene within sequenced S. aureus genomes, appears to be redundant to FhuD2 and likely arose through a gene duplication event [79]. FhuC has been characterized as an ATPase required for Fe(III)hydroxamate uptake [78]. Interestingly, there is now significant genetic and microbiological evidence demonstrating that FhuC is also required for staphyloferrin A and staphyloferrin B transport [78]. 10. Concluding remarks The ever-expanding repertoire of bacterial genomic sequences has resulted in the identification of a large number of genes predicted to be involved in siderophore biosynthesis and transport. Concomitantly, our knowledge of NRPS-independent siderophore biosynthesis and transport has rapidly expanded in recent years, resulting in significant leaps forward in our understanding of how these siderophores are built. A detailed understanding of the biochemistry and biology of NRPS-independent siderophores in the staphylococci will provide important insight into the biology of these medically relevant bacteria, and may be invaluable to uncover methodologies to control staphylococcal infections through
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Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio
Siderophore-mediated iron acquisition in the staphylococci Federico C. Beasley, David E. Heinrichs * Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada N6A 5C1
a r t i c l e
i n f o
Article history: Received 10 June 2009 Received in revised form 1 September 2009 Accepted 18 September 2009 Available online 26 September 2009 Keywords: Iron Siderophore Staphylococcus Iron acquisition ABC transporters
a b s t r a c t Iron is frequently a growth-limiting nutrient due to its propensity to interact with oxygen to form insoluble precipitates and, therefore, biological systems have evolved specialized uptake mechanisms to obtain this essential nutrient. Many pathogenic bacteria are capable of obtaining stringently sequestered iron from animal hosts by one or both of the following mechanisms: extraction of heme from host erythrocyte and serum hemoproteins, or through the use of high affinity, iron-scavenging molecules termed siderophores. This review summarizes our current knowledge of siderophore-mediated iron acquisition systems in the genus Staphylococcus. Ó 2009 Elsevier Inc. All rights reserved.
1. The staphylococci The staphylococci are facultatively aerobic Gram-positive bacteria. Most species are natural inhabitants of mammalian skin and mucous membranes [1]. A few are capable of causing disease, especially in an opportunistic fashion; the most studied pathogenic species is Staphylococcus aureus. Superficial colonization by staphylococci can result in furunculosis or impetigo [2], and systemic infiltration of tissues by S. aureus leads to syndromes including septicemia, endocarditis, and necrotizing pneumonia [3–5]. While S. aureus infections are especially frequent in hospital settings among patients suffering temporary or chronic compromise to the immune system, there exist some community-acquired strains that are particularly aggressive and can cause debilitating disease in healthy individuals [6]. For the purposes of identification, S. aureus is distinguished from other, less virulent staphylococci based on the species’ uniform expression of the enzyme coagulase; most other species are denoted as coagulase-negative (CoNS), though some contain strains that may be coagulase-positive [7]. CoNS infections are typically less severe, hospital acquired, and dependent on immunocompromise [8] or the opportunity to colonize implanted medical devices [9,10]. The best-characterized member of this group is Staphylococcus epidermidis, although Staphylococcus saprophyticus is noteworthy for being the second leading cause of uncomplicated urinary tract infection after Escherichia coli [11]. Pertinent to this review, another key distinction between S. aureus and CoNS is the genetic complement asso-
* Corresponding author. Tel.: +1 519 661 3984; fax: +1 519 661 3499. E-mail address: [email protected] (D.E. Heinrichs). 0162-0134/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2009.09.011
ciated with iron acquisition. Absent from CoNS (at least in genomes sequenced to date) but present in S. aureus are two key iron acquisition mechanisms (see Table 1). The first is the isd (iron-regulated surface determinant) gene set that mediates heme acquisition from mammalian heme-containing proteins, and the second is an Fe(III)-siderophore acquisition system that will be described in more detail below. It is feasible that these iron acquisition mechanisms contribute to the higher virulence potential of S. aureus versus its CoNS counterparts.
2. Iron With the exception of some lactobacilli and spirochaetes [12,13], iron is an essential nutrient for all bacteria. The Fe(III) centre of cytochromes is crucial to respiratory and photosynthetic ATP biosynthesis; Fe–S clusters are central to redox reactions mediated by proteins such as nitrogenase and hydrogenase; and iron is a cofactor for numerous other metalloproteins. Under physiological conditions, iron predominates in its ferric form, forming insoluble Fe(III) hydroxides [14]. In animal tissues and sera, iron is further sequestered by transferrin, so that the amount persisting free in solution is on the order of 10 24 M [15], well below the threshold required to sustain microbial life. Iron deprivation is a key sensory trigger for the expression of bacterial virulence factors, many of which are known to facilitate iron acquisition [16–19]. Bacteria use global, iron-responsive transcriptional regulators, such as the well-characterized Fur or the RirA (in Rhizobiales)-type regulatory proteins, to control gene expression (for reviews, see [20–23]). In brief, when cellular iron exceeds the level required for proper function of essential
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F.C. Beasley, D.E. Heinrichs / Journal of Inorganic Biochemistry 104 (2010) 282–288 Table 1 Known and putative genes involved in iron acquisition in S. aureus and S. epidermidis. Gene(s)
Locus number (S. aureus Newman or S. epidermidis RP62A)
Role of product(s)
References
S. aureus sbnABCDEFGHI sirABC isdB harA isdA isdC isdDEF
NWMN_0060-0068 NWMN_0059-0057 NWMN_1040 NWMN_2614 NWMN_1041 NWMN_1042 NWMN_1042-1045
NIS biosynthesis (staphyloferrin B) ABC transporter (staphyloferrin B) Hemoglobin and haptoglobin binding
[67] [68] [80,81]
Heme binding/transfer
[82–86] [83,87,88]
isdG isdI
NWMN_1047 NWMN_0111
Membrane proteins involved in heme transport Heme degradation
S. aureus and S. epidermidis sfaABC sfaD htsABC fhuD1, fhuD2 (aureus); fhuD (epidermidis) fhuCBG (aureus) fhuBC (epidermidis) sstABCD fur
[89]
NWMN_2081-2079 NWMN_2078 SERP1780-1778 SERP1781 NWMN_2078-2076 SERP1777-1775 NWMN_1931, NWMN_2185 SERP1777
NIS biosynthesis (staphyloferrin A)
[59,62]
ABC transporter (staphyloferrin A; heme?) Lipoprotein receptor (Fe3+ hydroxamates)
[59] [55,79]
NWMN_0616-0618 SERP1776-1775 NWMN_0702-0705 SERP0400-0403 NWMN_1406 SERP1061
ABC transporter (Fe3+ hydroxamates) ABC transporter (siderophore?) Transcriptional repressor of iron uptake
[72] [70] [90]
iron-containing metalloenzymes, association of an Fe(II) ion to each Fur monomer causes the dimeric protein to bind a roughly 19-bp DNA motif, called the Fur box, within the operator region of target genes. Typically, this binding blocks RNA polymerase, resulting in downregulation of gene transcription. Fur is known to indirectly activate some genes, via repression of small RNA silencing molecules [24], and direct upregulation of a target gene has also been reported [25].
3. Siderophore biosynthesis Numerous plants, fungi, saprophytic bacteria, and bacterial pathogens secrete small (typically less than 1 kDa) molecules called siderophores to serve as high affinity Fe(III) scavengers. Huge diversity has been documented in terms of the structure and iron-binding affinity of these compounds, although some broadly shared themes describe their physicochemical properties (for more comprehensive reviews, see [26–28]). Siderophores are highly electronegative and surround Fe(III) to form a hexacoordinated complex, although the stoichiometry of iron atoms to siderophore molecules may vary, depending on the number of donor atoms per siderophore molecule and its ability to use surrogate oxygen, nitrogen, or sulfur donors from solution to compensate for vacancies. In general, three types of functional groups constitute siderophore iron coordination motifs. First are the adjacent hydroxyl groups of catechol moieties, exemplified in the enterobactin structure (Fig. 1) [29]. Second are hydroxamate motifs, where the carbonyl and aminohydroxyl groups each occupy an iron coordination site, as in coprogen B [30] (Fig. 1). Third are carboxylate motifs. These may involve two sites provided by a-hydroxy-carboxylates derived from citrate, as in petrobactin [31], or from carboxylic acid moieties, as in rhizoferrin (Fig. 1) [32]. Numerous siderophores, such as the pyoverdins [33] or aerobactin [34] (Fig. 2), use combinations of the three iron coordinating groups. There are two structurally characterized siderophores produced by members of the staphylococci: staphyloferrin A and staphyloferrin B (Fig. 1). Siderophore assembly pathways fall into two broad classes: nonribosomal peptide synthesis (NRPS) and NRPS-independent siderophore (NIS) synthesis [35]. NRPS mechanisms have been characterized for numerous siderophores, including enterobactin [15], yersiniabactin [15,36], and vibriobactin [37], as well as impor-
tant antibiotics such as penicillin [38] and vancomycin [39]. NRPS siderophores have peptidic scaffolds, often incorporating nonproteinogenic amino acids and their derivatives, that are assembled stepwise without the benefit of a ribosomal template [26]. Less well characterized are the mechanisms for assembly of NIS siderophores. These are created from the condensation of alternating subunits of dicarboxylic acids (usually succinate, citrate, and a-ketoglutarate) with diamines, amino alcohols, and alcohols [35]. NIS synthetases have been sparingly investigated. For decades, the proposed assembly of aerobactin (Fig. 2) has represented the archetypal pathway of NIS biosynthesis [40,41], even though the biochemical roles of synthetases IucA and IucC have not been empirically characterized. Recent efforts have described the pathway involved in production of petrobactin [42,43], which is a rare example of a siderophore assembled via a hybrid NRPS/NIS strategy. Elucidation of the molecular structure of other siderophores and annotation of the genetic loci encoding their biosynthesis suggest rhizobactin [44], achromobactin [45], vibrioferrin [46], and alcaligin [47] are also assembled in an NRPS-independent fashion. NIS synthetases are not homologous to their modular counterparts in NRPS pathways. They primarily serve to catalyze formation of amide and ester bonds between an organic acid and a substrate bearing an amino or a hydroxyl group [35]. A model has been proposed to classify NIS synthetases based on specificity for a carboxylic acid [35,48]. Type A enzymes, represented by IucA, catalyze condensation of a prochiral carboxyl group of citrate to an amine or alcohol functional group of a second substrate, resulting in an amide or ester bond, respectively. Type B enzymes catalyze a similar reaction substituting the C5 carboxyl of a-ketoglutarate for the citrate carboxyl. Type C enzymes, represented by IucC, catalyze condensation of monoamide/monoester derivatives of citrate or succinate to carboxyl groups on molecules with an amine or an alcohol. Prior to condensation, NIS synthetases activate reacting carboxyl groups by adenylation with a nucleotide triphosphate. Acyl-adenylate intermediates are captured by an amine substrate leading to condensation to an amide [49]. For the most part, predictions of NIS synthetase type based on phylogenetic analysis have accurately described their role in functional enzyme assays [48], but this is based on limited data, and at least one exception has been uncovered and is described below. Additional modifications to precursors, such as decarboxylation, isomerization, or oxidation, are performed by separate enzymes typically encoded by genes clustering near to those encoding the synthetases.
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Fig. 1. Chemical structures of representative siderophores.
Fig. 2. Biosynthetic pathway for aerobactin.
4. Siderophore transport The cytoplasmic membrane in Gram-positive bacteria provides the main barrier to entry into the cytoplasm for small molecules such as Fe(III)-siderophore complexes. As such, Fe(III)-siderophores are brought into the cytoplasm through an active process involving ATP-binding cassette (ABC) transporters that use hydrolysis of ATP to provide the energy required for transport across the
membrane. ABC transporter systems are comprised of a substratebinding protein, a transmembrane permease and an ATPase. The ATPase interacts with the permease and couples active transport to ATP hydrolysis. For an excellent, comprehensive review of ABC-type transport systems, see [50]. Fe(III)-siderophore binding proteins in Gram-positive bacteria are lipoproteins tethered to the lipid bilayer via acylation to a conserved cysteine residue which resides at the end of the cleavable signal peptide, within
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the so-called ‘‘lipobox” domain (consensus: LXXC). The substratebinding proteins involved in Fe(III)-siderophore binding are part of a large family of proteins also known as the periplasmic binding protein (PBP) family (based on their location in the periplasm of Gram-negative bacteria). The Fe(III)-siderophore binding proteins fall into cluster 8 of this large family [51]. Binding proteins are characterized by two lobes, each made of mixed a/b structure, that surround the substrate-binding pocket; in cluster 8 proteins, the two lobes of the protein are connected by a long a-helical backbone that spans the width of the protein (Fig. 3), rather than by a more flexible series of short b-strands found in other binding proteins as exemplified by maltose binding protein. The resulting characteristic feature of the helical backbone type binding proteins is relatively restricted domain movement upon ligand binding and release [52–55]. Among the interactions between the binding protein and the membrane permease are key salt bridges that are formed between conserved glutamic acid residues on each of the lobes of the binding protein (Fig. 3) and three arginine residues that form a positively-charged pocket in each half of the permease [55,56]. 5. Staphyloferrin A biosynthesis Staphyloferrin A (Fig. 1) was first purified from culture supernatant of iron-starved Staphylococcus hyicus [57]. Staphyloferrin A (480 Da) is a highly hydrophilic molecule comprised of two molecules of citrate bridged by D-ornithine, thus having the structural identity N2,N5-di-(1-oxo-3-hydroxy-3,4-dicarboxylbutyl)-D-ornithine. The metabolite is produced by a wide variety of clinically relevant staphylococci, including S. aureus and S. epidermidis, and its production is stimulated by supplementation of the growth medium with ornithine [58]. To identify genes encoding for biosynthesis of this putative NIS siderophore, bioinformatic searches for IucA and IucC homologues uncovered the four-gene sfa locus (staphyloferrin A) in all sequenced staphylococcal genomes, including strains of S. aureus, S. epidermidis, S. saprophyticus, and Staphylococcus haemolyticus [59] (Fig. 4 and Table 1). The cluster comprises one three-gene operon, sfaABC, and a divergently transcribed sfaD gene. A consensus Fur box lies intergenically to sfaA and sfaD, suggesting transcriptional control by Fur which was confirmed in S. aureus using real-time PCR [59]. Production of staphy-
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loferrin A has been reported as constitutive among some CoNS isolates [60], suggesting a possible lack of Fur-dependent regulation of the locus in some species. Chromosomal deletion of the sfa locus in S. aureus abrogates production of a secreted metabolite with a mass and chemical structure corresponding to that of staphyloferrin A [59]. Surprisingly this mutation was not associated with an observable defect in bacterial growth in mammalian serum [59]. Only when staphyloferrin B production was also knocked out (sbn deletion – see below) in the same strain of S. aureus was there an observable staphyloferrin A-dependent growth defect in serum. The sfa mutant growth defect in an sbn deletion strain of S. aureus was restored by complementation with plasmid-borne sfa genes from S. aureus, as well as the homologous sfa gene locus from S. epidermidis, suggesting functional conservation of Sfa proteins among the staphylococci, despite protein sequence identities in the range of 50–80% between S. aureus and S. epidermidis. The observation that staphyloferrin A-deficient mutants of S. aureus, at least in strains producing another siderophore (staphyloferrin B, see below), do not suffer growth reduction, suggests that staphyloferrin A may not be a major contributor to iron extraction from transferrin, the primary iron substrate in serum. Extraction of transferrin iron may be dependent on other processes that affect glycoproteins, such as binding to a cell membrane receptor or proteolytic cleavage. Staphyloferrin A has been reported to leach iron from human transferrin, which is recognized by staphylococcal transferrin receptor proteins, as well as porcine transferrin, which is not recognized [61]. These studies may have used staphyloferrin A at concentrations exceeding what is produced in culture supernatants of S. aureus strains that are producing a second siderophore. The ability of staphyloferrin A to capture iron from serum glycoproteins merits further investigation. S. aureus proteins SfaB and SfaD have been characterized as NIS synthetases. When overexpressed and purified, they are capable of generating staphyloferrin A in vitro from citrate and D-, but not L-, ornithine [62]. The SfaC protein is homologous to amino acid racemases and likely racemases L-ornithine to D-ornithine to provide the substrate for staphyloferrin A synthesis. SfaD mediates the first committed step in synthesis, forming a d-citryl-D-ornithine intermediate, and SfaB condenses the second citric acid onto the intermediate (Fig. 5) [62]. While SfaB and SfaD cluster among B type NIS synthetases [35,62], their chemistry aligns them with the A-type (i.e. specificity for citrate) enzymes, forming a subgroup within the A-type enzymes. It will be interesting to follow the evolution of the NIS synthetase phylogeny as additional NIS synthetases are characterized biochemically. The SfaA protein, which shares similarity with membrane-embedded efflux proteins, is a likely candidate for involvement in staphyloferrin A efflux. Empirical corroboration of this function would provide valuable insight into the secretion of siderophores in Gram-positive bacteria.
6. Staphyloferrin A transport
Fig. 3. Crystal structure of the staphyloferrin A binding protein HtsA (PDB: 3EIW) [59], which is a member of the periplasmic binding protein superfamily. The Fe(III)siderophore binding subfamily is characterized by two mixed a/b domains (Nterminal domain of HtsA is shown in green and the C-terminal domain is shown in blue) separated by a long a-helical backbone (yellow) and conserved glutamic acid residues (represented in stick format) on the surface of the lobes. The glutamic acid residues form salt bridges with arginine residues in the membrane permease proteins to mediate productive protein:protein interactions and, therefore, transport.
The iron and Fur-regulated htsABC operon (heme transport system) lies adjacent to the sfa gene cluster in the chromosomes of S. aureus and CoNS (Fig. 4) [59]. An early study describes its role in heme transport [63], showing that inactivation of the permease components HtsB and HtsC shift the iron source preference of S. aureus from heme toward transferrin (and, implicitly, toward siderophore-mediated iron uptake). Although direct involvement of HtsABC in heme transport has not been demonstrated the Hts transporter is linked to staphyloferrin A uptake in S. aureus. Indeed, transport of staphyloferrin A by S. aureus is abolished in htsABC mutants, and the crystal structure of the unliganded HtsA substrate-binding protein reveals a positively-charged binding pocket, consistent with a role in binding of the anionic siderophore
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Fig. 4. Genetic loci in staphylococci involved in siderophore production. The sfa-hts locus is found in S. aureus and CoNS and is responsible for biosynthesis and transport of staphyloferrin A. The sbn-sir locus is found in S. aureus and a homologous cluster of genes is found in Ralstonia spp. The locus is responsible for synthesis and transport of staphyloferrin B.
Fig. 5. Biosynthetic pathway for staphyloferrin A, as determined in [62].
staphyloferrin A [59]. HtsA (Fig. 3) belongs to the cluster 8 subfamily of the periplasmic binding protein superfamily (described above). Strangely, despite the energy dependence of siderophore transport, the operon lacks a gene encoding an ATPase component and genetic evidence supports a role for the FhuC ATPase (discussed in more detail below) in staphyloferrin A transport [59]. 7. Staphyloferrin B biosynthesis A number of staphylococcal species have been shown to produce a second siderophore, termed staphyloferrin B. This is another highly hydrophilic NIS siderophore composed of L-2,3-diaminopropionic acid (DAPA), citrate, ethylenediamine, and a-ketoglutarate (Fig. 1), with a mass of 448 Da [64]. Purification and structural characterization of the metabolite was performed using culture supernatants of iron-starved S. hyicus [65]. HPLC analysis has identified it in supernatants of S. aureus and a wide range of CoNS species [58,64]. Interestingly, the molecule is also produced by the Gram-negative plant pathogen Ralstonia solanacearum and the metal-resistant soil bacterium Cupriavidus metallidurans [66]. In an attempt to uncover genes responsible for biosynthesis of siderophores in S. aureus, a search for homologues of IucA and IucC
in S. aureus uncovered a nine-gene operon designated sbn (siderophore biosynthesis). The sbn operon (Fig. 4 and Table 2) encodes three putative NIS synthetases: SbnC, SbnE, and SbnF [67]. Phylogenetic analysis groups them with type B, type A, and type C synthetases, respectively [35]. The sbn operon is highly similar in sequence and arrangement to the staphyloferrin B biosynthesis operon of R. solanacearum [66], suggesting that sbn genes encode the machinery required for staphyloferrin B biosynthesis, as opposed to the previous designation of staphylobactin for a structurally uncharacterized siderophore whose production was initially linked to the sbn operon [67]. A consensus Fur box lies within the sbn operon operator region, and b-galactosidase transcriptional fusions to sbn genes were used to demonstrate maximal expression of the operon under conditions of iron restriction [67]. Chromosomal inactivation of the sbn operon in S. aureus leads to impaired growth in serum, though culture densities eventually approach wild type levels with prolonged incubation, presumably through the production of staphyloferrin A [59]. An sbnE mutant strain proved attenuated in a mouse model of kidney infection [67], highlighting the importance of specialized iron uptake mechanisms in virulence S. aureus. The structure of staphyloferrin B by S. aureus could feasibly be assembled by the complement of predicted protein functions
F.C. Beasley, D.E. Heinrichs / Journal of Inorganic Biochemistry 104 (2010) 282–288 Table 2 Staphyloferrin biosynthesis and transport proteins in Staphylococcus aureus. Protein
Putative or characterized function
Staphyloferrin A SfaA Major facilitator superfamily protein; staphyloferrin A efflux SfaB NIS synthetase; staphyloferrin A synthesis SfaC Racemase; conversion of L-ornithine to D-ornithine SfaD NIS synthetase; staphyloferrin A synthesis HtsA Staphyloferrin A binding protein HtsB ABC transporter permease; staphyloferrin A transport HtsC ABC transporter permease; staphyloferrin A transport Staphyloferrin B SbnA O-acetyl-L-serine sulfylhydrylase; synthesis of L-2,3diaminpropionic acid SbnB Ornithine cyclodeaminase; synthesis of L-2,3-diaminopropionic acid SbnC Type B NIS synthetase; staphyloferrin B synthesis SbnD Major facilitator superfamily protein; staphyloferrin B efflux SbnE Type A NIS synthetase; staphyloferrin B synthesis SbnF Type C NIS synthetase; staphyloferrin B synthesis SbnG Aldolase SbnH Amino acid decarboxylase SbnI Unknown function SirA Staphyloferrin B receptor SirB Heterodimeric permease component; staphyloferrin B transporter SirC Heterodimeric permease component; staphyloferrin B transporter
encoded by the sbn operon [67]. No detailed characterization of Sbn proteins has as yet been published at the time of writing this review. However, the NIS synthetases (SbnC, SbnE and SbnF) could condense molecules of diaminopropionic acid (Dap), citrate, ethylenediamine, and 2-ketoglutaric acid. SbnA, a putative L-Oacetylserine sulfhydrylase, and SbnB, a putative ornithine cyclodeaminase, could generate Dap from a coupled reaction involving cyclization of the diamino acid ornithine to produce proline and ammonia, with the ammonia being used to convert O-acetyl-L-serine into Dap. SbnD, with similarity to antibiotic efflux proteins, is likely involved in secretion of siderophore into the extracellular milieu, while the roles of SbnG, SbnH and SbnI remain unclear at the time of writing this review. It is interesting to note that although staphyloferrin B was reported to be produced by some CoNS species [58,64], to date, the sbn gene cluster has not been identified in any of the available CoNS genome sequences (Table 1), nor was the sbn gene cluster detected in a screen of CoNS using low-stringency Southern blot hybridization analyses [67]. Searches of the rapidly expanding number of sequenced CoNS genomes (7 with BlastP functionality at the time of writing: S. capitis SK14, S. epidermidis RP62A, S. epidermidis ATCC12228, S. saprophyticus ATCC15305, S. haemolyticus JCSC1435, Staphylococcus carnosus TM300, and Staphylococcus hominis SK119) have not uncovered any obvious non-sfa NIS synthetase encoding genes.
8. Staphyloferrin B transport The sirABC transport operon (staphylococcal iron regulated) (Fig. 4) is divergently transcribed from the sbn operon in the chromosome of S. aureus. Similar to the sbn operon, sirABC is absent in all currently available CoNS genome sequences. Expression of this transport system is also regulated by iron and Fur [68,69]. As with the HtsABC transporter, no encoded ATP-binding component is found in the genomic vicinity. Uptake of S. aureus siderophore (staphyloferrin B) complexed with 55Fe was shown to be dependent on expression of the receptor protein SirA and the permease component SirB, and transport is an energy-dependent process [68]. Heterologous expression of sirABC in an S. epidermidis strain (which does not possess sbn genes and, thus, synthesize staphyloferrin B) rendered it capable of using the sbn siderophore as an iron
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source (F.C. Beasley, unpublished data). Two conclusions are derived following crossfeeding experiments between combinations of transporter and biosynthesis mutants of S. aureus [59]. First, as siderophore output is nil in a double sfa sbn biosynthesis mutant, staphyloferrin A and staphyloferrin B are likely the only siderophores produced by characterized strains of S. aureus. Second, as transport of these siderophores is dependent on HtsABC and SirABC, respectively, the two transporters specifically transport their cognate siderophore ligand, and it is unlikely that redundant transporters are encoded elsewhere in the genome. 9. Transport of exogenous siderophores by S. aureus To date, there are three ABC transporters demonstrated to be involved in Fe(III)-siderophore uptake in S. aureus. These are involved in the uptake of staphyloferrin A (HtsABC) and staphyloferrin B (SirABC) as described above, and a third which is required for uptake of Fe(III)-hydroxamate complexes (FhuCBG-D1-D2). A fourth iron-regulated ABC transporter (SstABCD) is encoded by S. aureus and the substrate for this transporter has not yet been identified [70]. Hydroxamate-type siderophores are considered exogenous metabolites, as studies have so far failed to detect hydroxamate siderophore production by S. aureus [71,72]. The transporter is comprised of permease components FhuBG, ATP-binding protein FhuC, and dual receptor proteins FhuD1 and FhuD2 which can compete for an interaction with FhuCBG. Component nomenclature is based on their homology to ferric hydroxamate uptake proteins of E. coli and Bacillus [73–76]. The fhuCBG operon and moncistronic genes fhuD1 and fhuD2 are located at widely segregated positions on the S. aureus chromosome; fhuCBG and single allele fhuD operons have also been annotated in CoNS genomes. Chromosomal inactivations of fhuB or fhuG result in an inability to use a range of hydroxamate siderophores, including the bacterial siderophore aerobactin (Fig. 2), the fungal siderophores ferrichrome and coprogen, and the clinically-utilized siderophore DesferalTM [72,77,78]. Compared to FhuD1, FhuD2 has a higher substrate range and affinity for hydroxamate siderophores (for example, DesferalTM: Kd FhuD2 = 5 10 8 M, Kd FhuD1 = 1 10 6 M) [55]. As observed for E. coli FhuD and other members of the Fe(III)-siderophore binding protein superfamily, substrate binding induces very little conformational change in FhuD1 and FhuD2 [55,79]. FhuD1, with reduced affinity for Fe-hydroxamates and inconsistent presence of fhuD1 gene within sequenced S. aureus genomes, appears to be redundant to FhuD2 and likely arose through a gene duplication event [79]. FhuC has been characterized as an ATPase required for Fe(III)hydroxamate uptake [78]. Interestingly, there is now significant genetic and microbiological evidence demonstrating that FhuC is also required for staphyloferrin A and staphyloferrin B transport [78]. 10. Concluding remarks The ever-expanding repertoire of bacterial genomic sequences has resulted in the identification of a large number of genes predicted to be involved in siderophore biosynthesis and transport. Concomitantly, our knowledge of NRPS-independent siderophore biosynthesis and transport has rapidly expanded in recent years, resulting in significant leaps forward in our understanding of how these siderophores are built. A detailed understanding of the biochemistry and biology of NRPS-independent siderophores in the staphylococci will provide important insight into the biology of these medically relevant bacteria, and may be invaluable to uncover methodologies to control staphylococcal infections through
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