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J Bacteriol. Mar 2006; 188(6): 2048–2055.
PMCID: PMC1428144

Requirement of Staphylococcus aureus ATP-Binding Cassette-ATPase FhuC for Iron-Restricted Growth and Evidence that It Functions with More than One Iron Transporter

Abstract

In Staphylococcus aureus, fhuCBG encodes an ATP-binding cassette (ABC) transporter that is required for the transport of iron(III)-hydroxamates; mutation of either fhuB or fhuG eliminates transport. In this paper, we describe construction and characterization of an S. aureus fhuCBG deletion strain. The ΔfhuCBG::ermC mutation not only resulted in a strain that was incapable of growth on iron(III)-hydroxamates as a sole source of iron but also resulted in a strain which had a profound growth defect in iron-restricted laboratory media. The growth defect was not a result of the inability to transport iron(III)-hydroxamates since S. aureus fhuG::Tn917 and S. aureus fhuD1::Km fhuD2::Tet mutants, which are also unable to transport iron(III)-hydroxamates, do not have similar iron-restricted growth defects. Complementation experiments demonstrated that the growth defect of the ΔfhuCBG::ermC mutant was the result of the inability to express FhuC and that this was the result of an inability to transport iron complexed to the S. aureus siderophore staphylobactin. Transport of iron(III)-staphylobactin is dependent upon SirA (binding protein), SirB (permease), and SirC (permease). S. aureus expressing FhuC with a Walker A K42N mutation could not utilize iron(III)-hydroxamates or iron(III)-staphylobactin as a sole source of iron, supporting the conclusion that FhuC, as expected, functions with FhuB, FhuG, and FhuD1 or FhuD2 to transport iron(III)-hydroxamates and is the “genetically unlinked” ABC-ATPase that functions with SirA, SirB, and SirC to transport iron(III)-staphylobactin. Finally, we demonstrated that the ΔfhuCBG::ermC strain had decreased virulence in a murine kidney abscess model.

Iron is an essential micronutrient for virtually all microorganisms owing to its wide range of redox potentials. Either alone or incorporated into heme or iron-sulfur clusters, iron serves as the catalytic center of enzymes involved in critical cellular processes such as DNA synthesis and electron transport. However, despite the fact that iron is plentiful on Earth, the amount of free iron in biological systems is low due to its tendency to form insoluble oxyhydroxides under aerobic conditions at a neutral pH. The amount of iron available in the host environment is further reduced by sequestration into proteins such as transferrin and lactoferrin; virtually no free iron exists in living organisms (7).

In order to overcome iron limitation, most successful pathogens employ several strategies to acquire the quantities of iron necessary to cause infection (49). One such strategy is production of low-molecular-weight iron-chelating molecules termed siderophores which, together with their cognate cell surface binding proteins and transporters, provide an efficient system for iron acquisition (for a recent review, see reference 25). Indeed, there is considerable evidence demonstrating the importance of siderophore-mediated iron acquisition systems for the virulence of many disease-causing bacteria, including gram-negative bacteria such as Escherichia coli (50) and Yersinia enterocolitica (22) and gram-positive bacteria such as Staphylococcus aureus (14) and Bacillus anthracis (9).

S. aureus is an important human pathogen in both community and hospital settings; it is a leading cause of nosocomially acquired infection. This organism is capable of causing infections ranging from minor (e.g., impetigo and food poisoning) to more severe (e.g., bacteremia, necrotizing pneumonia, endocarditis, and osteomyelitis) (13). Moreover, S. aureus is a major threat to the health care system since it is becoming increasingly resistant to antibiotics. Indeed, strains resistant to methicillin and, more recently, to vancomycin exist (1, 2), and the problem is further complicated by the recent emergence of methicillin-resistant S. aureus carriage and disease in communities (20, 34).

S. aureus possesses multiple systems for the uptake of iron(III) siderophores in order to satisfy its requirement for iron. The best-characterized system in S. aureus is the ferric hydroxamate uptake (Fhu) system, which is encoded by up to five genes. fhuC, fhuB, and fhuG are in an operon that encodes an ATP-binding cassette (ABC) transporter; FhuC is a predicted ATPase; and FhuB and FhuG are membrane-spanning proteins (41). The genetically unlinked fhuD1 and fhuD2 genes encode iron(III)-hydroxamate-binding lipoproteins (40). Previous work in our laboratory has demonstrated that fhuG is required for transport of iron(III)-hydroxamates (41) and has also elucidated the relative contributions of FhuD1 and FhuD2 to the binding and utilization of these siderophores through extensive biochemical characterization of these proteins (42, 43). Interestingly, although S. aureus possesses this high-affinity uptake system for hydroxamate siderophores, it does not itself produce a hydroxamate siderophore (12, 41), implying that the Fhu system has a role in the transport of xenosiderophores. The potential in vivo relevance of the Fhu system has not been studied yet.

Additional studies in our laboratory resulted in identification of a nine-gene operon (sbnA to sbnI) involved in the production of staphylobactin, a nonhydroxamate S. aureus siderophore (14). The important contribution of this operon to the growth of S. aureus under iron limitation conditions, both in vitro and in vivo, was demonstrated. Transport of iron(III)-staphylobactin into the S. aureus cell is mediated by the sirABC operon (15), which is transcribed divergently from the sbn operon and encodes a lipoprotein (SirA) and two predicted membrane-spanning proteins (SirBC) (15, 23). Conspicuously absent from the sirABC operon is a gene encoding the ATPase component of a classical ABC-type transporter.

Here, we describe the creation and characterization of an fhuCBG deletion mutant and provide evidence that fhuC is involved in iron acquisition systems separate from its role in the transport of iron(III)-hydroxamates. In this study we demonstrated that an fhuCBG operon deletion mutant, in contrast to fhuG, fhuD1, and fhuD2 mutants, had a marked growth defect under iron-restricted growth conditions and that the growth-deficient phenotype could be restored by introducing fhuC alone in trans but not by introducing fhuC that included a Walker A K42N mutation. We further demonstrated that the growth deficiency was due to an inability to transport iron(III)-staphylobactin and that iron(III)-staphylobactin transport was restored in the mutant in which fhuC was present in trans, indicating that fhuC encodes the “missing” ATPase required for staphylobactin uptake via the SirABC transporter. Finally, we found that the fhuCBG mutant was less virulent in a mouse kidney abscess model.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

Bacterial strains and plasmids used in this study are described in Table Table1.1. E. coli was grown in Luria-Bertani broth (Difco). For experiments that did not directly involve analysis of iron uptake, S. aureus was grown in tryptic soy broth (Difco). Tris-minimal succinate (TMS) was prepared as described previously (43) and used as an iron-limited minimal medium. To further restrict the level of free iron in TMS, the iron-chelating compounds 2,2′-dipyridyl and ethylene diamine-di(o-hydroxyphenol acetic acid) were added as indicated below. When necessary, ampicillin (100 μg/ml) and kanamycin (30 μg/ml) were incorporated into the medium for growth of E. coli strains. For S. aureus, chloramphenicol (5 μg/ml), kanamycin (50 μg/ml), neomycin (50 μg/ml), erythromycin (3 μg/ml), and lincomycin (20 μg/ml) were incorporated into growth media as required. Solid media were obtained by addition of 1.5% (wt/vol) Bacto agar (Difco). All bacterial growth was performed at 37°C unless indicated otherwise. Iron-free water for preparation of growth media and solutions was obtained by passage through a Milli-Q water filtration system (Millipore Corp.).

TABLE 1.
Bacterial strains and plasmids used in this study

Recombinant DNA methodology.

Standard DNA manipulations were performed essentially as described by Sambrook et al. (39). Restriction endonucleases and DNA-modifying enzymes were purchased from Roche Diagnostics (Laval, Quebec, Canada), New England Biolabs (Mississauga, Ontario, Canada), Life Technologies Inc. (Burlington, Ontario, Canada), and MBI Fermentas (Flamborough, Ontario, Canada). Plasmid DNA was purified using QIAprep plasmid spin columns (QIAgen Inc., Santa Clarita, CA) as described by the manufacturer. For plasmid isolation from S. aureus, lysostaphin (50 μg/ml) was added to buffer P1. Chromosomal DNA from S. aureus was isolated using the InstaGene matrix (Bio-Rad) as described by the manufacturer. PCRs were performed using either PwoI or Taq DNA polymerase (Roche Diagnostics).

Construction of a ΔfhuCBG::ermC mutant.

To create a deletion in the fhuCBG operon in the chromosome of RN6390, two regions of DNA flanking the operon were amplified from the RN6390 chromosome by PCR. Primers fhuC upstream sense (5′-TTGAATTCAATACCTCGATGTAAGCACG-3′) and fhuC upstream antisense (5′-TTGGATCCACGATTCATAATTTCCCTAC-3′) were used to amplify a 709-bp fragment upstream of fhuC that included the first 9 bp of the fhuC open reading frame, and primers fhuG downstream sense (5′-TTGGATCCAACGAAAAATGTATAGTGTC-3′) and fhuG downstream antisense (5′-TTTCTAGACGGCAAGCTTATGAACAAAC-3′) were used to amplify a 718-bp fragment downstream of fhuG that included the final 13 bp of the fhuG open reading frame.

The fhuC upstream fragment was digested with EcoRI and BamHI (recognition sites are underlined in the primer sequences above), the fhuG downstream fragment was digested with BamHI and XbaI (recognition sites are underlined in the primer sequences above), and the two fragments were cloned together into pUC19 digested with EcoRI and XbaI. The resulting construct was digested with BamHI, and a 1.6-kb BamHI fragment from pDG646, carrying the ermC gene, was inserted. The resulting plasmid construct was digested with EcoRI and XbaI, and a 3,027-bp fragment harboring ermC between the fhuC upstream and fhuG downstream fragments was ligated into pAUL-A Km digested with EcoRI and XbaI to create plasmid pΔfhuCBG.

Plasmid pΔfhuCBG was introduced into S. aureus RN4220 by electroporation, and colonies resistant to kanamycin and neomycin were selected after growth at 30°C. Kanamycin-resistant clones were subjected to a temperature shift to 42°C to select for plasmid integration into the chromosome. Bacteria resistant to erythromycin and lincomycin but sensitive to kanamycin and neomycin were selected. The ΔfhuCBG::ermC mutation was confirmed by PCR, and the mutation was subsequently transduced into the RN6390 and Newman backgrounds to create strains H1071 and H1074, respectively, using procedures described previously (33, 41).

Construction of complementing plasmids.

In order to complement the ΔfhuCBG::ermC mutation, pMTS20 (41) was digested with BamHI, and the resulting 3.7-kb fragment harboring the fhuCBG operon with approximately 400 bp of upstream DNA (encompassing the Fur box and promoter sequences) was ligated into the BamHI site of pLI50 to create pFhuCBG. To create pFhuC, primers fhuCBG2 (5′-TTTGGATCCACAAGTTTCAAAAGCAAAGC-3′) and fhuC antisense (5′-TTGGATCCATTTGTCATGTTAATTGTCC-3′) were used to amplify a 1.2-kb region containing the fhuC coding region plus the same 400-bp upstream region that was in pFhuCBG, and the resulting PCR product was cloned into the BamHI site of pLI50.

Construction of FhuC K42N.

Plasmid pFhuC DNA was isolated using QIAprep plasmid spin columns (QIAGEN Inc., Santa Clarita, CA) as described by the manufacturer. A K42N mutation was introduced into the fhuC gene carried on plasmid pFhuC or pFhuCBG by using a QuikChange site-directed mutagenesis kit and the procedure recommended by Stratagene. The nucleotide sequence of the sense primer used for generation of the mutation was 5′-CGGCTGCGGGAACTCTACTTTGCTA-3′; the sequence of the antisense primer was the reverse and complement of the sequence of the sense primer. The mutation was confirmed by automated DNA sequencing.

Siderophores.

Ferrichrome was purchased from Sigma (Mississauga, Ontario, Canada), desferrioxamine B, used as Desferal (Novartis), was obtained from the London Health Sciences Centre (London, Ontario, Canada), and enterobactin was purchased from EMC Microcollections GmbH (Tübingen, Germany). Staphylobactin was prepared as previously described (15).

Bioassays.

Siderophore plate bioassays were performed essentially as described previously (41). Briefly, 104 cells/ml was added to molten TMS agar containing 25 μM ethylene diamine-di(o-hydroxyphenol acetic acid) as an iron-chelating agent. Ten-microliter portions of the iron sources to be tested (Desferal [50 μM], ferrichrome [50 μM], enterobactin [500 μM], staphylobactin [50 μM Desferal equivalents], hemin [250 μg/ml], hemoglobin [2 mg/ml], FeCl3 [50 mM], and ferric citrate [5 mM]) were added to sterile 6-mm-diameter paper disks and placed on the surfaces of the plates. In most cases growth promotion, as measured by the diameter of the growth halo around each disk, was determined after 48 h of incubation; the exceptions were the diameters of the haloes obtained with heme and hemoglobin, which were measured after 72 h.

Determination of MIC of 2,2′-dipyridyl for S. aureus strains.

Strains to be tested were pregrown in TMS, and cells were washed in fresh TMS prior to the assay. 2,2′-Dipyridyl was added to TMS, and the preparations were serially diluted to obtain concentrations ranging from 1 mM to 32 μM. Following serial dilution, 5 × 104 CFU was added to each 5-ml culture, and growth was recorded after 24 h of incubation.

Bacterial growth curves.

Strains were pregrown overnight in TMS. Cells were washed with TMS, and 5 × 106 CFU was added to 50 ml of fresh TMS or TMS supplemented with 50 μM 2,2′-dipyridyl in acid-washed flasks. When necessary, 50 μM FeCl3 was added to the medium to create iron-replete conditions. Bacterial growth was monitored by measuring the optical density at 600 nm until the stationary phase was reached.

55Fe transport assays.

Siderophore uptake was measured as previously described (15), with the following modifications: all strains except RN6390 ΔfhuCBG::ermC (H1071) were grown overnight in TMS containing 50 μM 2,2′-dipyridyl and appropriate antibiotics, and RN6390 ΔfhuCBG::ermC (H1071) was grown in TMS alone. Cells were washed three times in TMS, and the optical density at 600 nm was normalized to 2.0. Siderophores (~200 μM Desferal and ~200 μM staphylobactin) were mixed with 55FeCl3 (75 μM) in the presence of 4 μM nitrilotriacetic acid, and the mixtures were equilibrated at room temperature for 30 min. Iron uptake was initiated by adding 10 μl of each 55Fe-siderophore mixture to 1 ml of cells. At various times, 200 μl of cells was removed and washed twice with 100 mM LiCl over a 0.45-μm-pore-size membrane (Pall Gelman). Dried membranes were counted in CytoScint fluid using the tritium channel of a Beckman LS-6500 scintillation system. In some experiments, bacteria were exposed to 10 mM potassium cyanide for 15 min at room temperature before uptake with the 55Fe-siderophore mixtures was initiated. The data were expressed in picomoles of 55Fe transported normalized to the optical densities of the cultures.

Mouse kidney abscess model.

Female Swiss-Webster mice (25 g each) were purchased from Charles River Laboratories Canada Inc. and were housed in microisolator cages. S. aureus strains Newman and Newman ΔfhuCBG::ermC (H1074) were grown overnight in TSB and washed three times with sterile saline, and suspensions containing 1 × 108 CFU/ml in sterile saline were prepared. One-hundred-microliter portions of the cell suspensions were administered intravenously via the tail vein. The number of viable bacteria injected was confirmed by plating serial dilutions of the inocula on TSB agar. In a blinded fashion, University of Western Ontario Animal Care and Veterinary Services personnel scored mice throughout the experiment for alertness, activity, and coat condition. In each of the three categories, a score of 0 was normal, a score of 1 was slightly abnormal, and a score of 2 was very abnormal. On day 7, the mice were euthanized, and the kidneys were aseptically removed. Again in a blinded fashion, Animal Care and Veterinary Services personnel scored the condition of the kidneys as follows; 0, no visible abscesses; 1, 1 small abscess; 2, several abscesses; and 3, severely abscessed kidneys. Kidneys were then homogenized in sterile phosphate-buffered saline containing 0.1% Triton X-100 using a PowerGen 700 homogenizer. The homogenates were serially diluted and plated on TSB agar to enumerate recovered bacteria. Data were expressed as the log CFU recovered per mouse.

Computer analyses.

DNA sequence analysis and PCR oligonucleotide primer design were performed using the Vector NTI Suite 7 software package (Informax, Inc.). Microsoft Excel and SigmaPlot (SPSS Inc.) were used for data analysis and graphing applications.

Statistics.

In vivo data were analyzed by the Student unpaired t test. A P value of <0.05 was considered to be statistically significant.

RESULTS

S. aureus ΔfhuCBG mutants are unable to utilize iron(III)-hydroxamates.

The S. aureus iron-regulated fhuCBG operon was previously described by workers in our laboratory (41) and by Xiong et al. (52) and was shown to be necessary for transport of iron(III)-hydroxamates, including aerobactin, coprogen, ferrichrome, Desferal, and rhodotorulic acid, in S. aureus since mutations in either fhuB or fhuG eliminated transport (8, 41). The operon is present in all S. aureus genomes that have been sequenced (43). As part of our laboratory's ongoing studies to elucidate the mechanism of iron(III)-siderophore transport in S. aureus using the fhu system as a model, we constructed H1068, an S. aureus strain with the RN4220 genetic background that contained an fhuCBG operon deletion (see Materials and Methods). The mutation was mobilized by phage transduction into the RN6390 and Newman genetic backgrounds, creating strains H1071 and H1074, respectively. In agreement with previous results indicating that loss of either fhuG or fhuB resulted in a complete inability to utilize any iron(III)-hydroxamate complexes for iron-restricted growth, plate bioassays showed that H1071 and H1074 were unable to utilize Desferal, ferrichrome, coprogen, and aerobactin (data not shown). The mutants were able to utilize all of the hydroxamate siderophores mentioned above for iron acquisition in siderophore plate bioassays when the fhuCBG operon was provided in trans on plasmid pFhuCBG (data not shown).

Mutation of fhuC, but not mutation of other fhu genes, in S. aureus results in an iron-restricted growth defect.

Previously unpublished results from our laboratory indicated that S. aureus strains with fhu mutations, including RN6390 fhuG::Tn917 (H287) and RN6390 fhuD1::Km fhuD2::Tet (H431), did not have an obvious growth defect in iron-deficient media, suggesting that hydroxamate siderophores are not produced by S. aureus in response to iron starvation; in agreement, S. aureus culture supernatants were negative in the Czaky test (36) for hydroxamates (data not shown). Surprisingly, we observed that the growth of H1071 (RN6390 ΔfhuCBG::ermC) was significantly retarded compared to the growth of wild-type strain RN6390, H287 (RN6390 fhuG), and H431 (RN6390 fhuD1 fhuD2) in iron-deficient TMS (Fig. (Fig.1A)1A) and was retarded even more in TMS containing 50 μM 2,2′-dipyridyl, a nonmetabolizable iron chelator (Fig. (Fig.1B).1B). Addition of 50 μM ferric chloride to TMS restored the growth of H1071 to wild-type levels, demonstrating that the impaired growth was due solely to the level of iron available to the bacteria (Fig. (Fig.1A1A and and1B).1B). The iron-restricted growth defect exhibited by H1071 could be complemented by introduction of a plasmid carrying the operon (pFhuCBG). However, more surprising was the observation that fhuC alone in trans, present on plasmid pFhuC, complemented the iron-restricted growth deficiency of strain H1071, indicating that the growth defect of H1071 was a result of the inability to express fhuC. 55Fe-Desferal uptake assays were performed, and they showed that H1071 was incapable of transporting 55Fe-Desferal (Fig. (Fig.2,2, inset), corroborating the bioassay results (see above). Of note, however, was the observation that although pFhuC could complement the growth deficiency of H1071 (as shown in Fig. Fig.1),1), it could not restore the ability of H1071 to transport 55Fe-Desferal (Fig. (Fig.2),2), corroborating previous results that showed that FhuB and FhuG were also required for iron(III)-hydroxamate uptake (8, 41) and also indicating that an additional iron(III)-siderophore transport system, one that is required for iron-restricted growth in S. aureus, was interrupted in H1071.

FIG. 1.
Growth of RN6390 (wild type) (•), H1071 (RN6390 ΔfhuCBG::ermC) (○), H1071 (RN6390 ΔfhuCBG::ermC) with 50 μM FeCl3 ([filled triangle]), H306 (RN6390 sirA::Km) ([down-pointing small open triangle]), H431 (RN6390 fhuD1::Km fhuD2::Tet) ([filled square]), ...
FIG. 2.
55Fe-Desferal-mediated iron transport by S. aureus RN6390 and H1071 derivatives grown in TMS containing 50 μM 2,2′dipyridyl. •, RN6390; ○, H1071/pFhuC; [filled triangle], H1071/pFhuCBG; [down-pointing small open triangle], RN6390 treated with 20 mM KCN ...

FhuC ATPase activity is required for growth on iron(III)-hydroxamates and for iron-restricted growth.

S. aureus FhuC exhibits significant similarity to ATPases and possesses all the hallmarks (signature sequences) of an ATP-binding cassette protein (41). Indeed, FhuC possesses conserved Walker A (36GPNGCGKST44) and Walker B (160IIFLDE165) motifs and the ABC family signature motif, 140LSGGQRQR147; mutations in these sequences often severely reduce or eliminate ATPase activity and transport (for a recent review of bacterial ABC transporters, see reference 16). The Walker A motif, sometimes referred to as the P loop, forms a loop that binds to ATP. The conserved lysine in the Walker A motif in both mammalian and bacterial ABC transporters has been found to be critical for ATP binding and hydrolysis (17, 27, 32, 35). In addition, studies on the E. coli FhuC protein showed that mutation of this conserved lysine eliminated transport (4). Therefore, we mutated the conserved Walker A lysine in S. aureus FhuC (residue 42) by constructing a K42N substitution, equivalent to the E. coli MalK K42N substitution that was studied in detail previously (17). FhuC K42N, expressed from plasmid pFhuC(K42N)BG along with wild-type copies of FhuB and FhuG, did not complement the inability of H1071 to grow on iron(III)-hydroxamates as a sole source of iron (data not shown). Moreover, FhuC K42N, expressed from pFhuC-K42N, did not complement the iron-restricted growth defect of H1071. In both cases, the results indicated that FhuC couples ATP binding and the energy of ATP hydrolysis to the transport of iron chelates in S. aureus.

FhuC is required for iron(III)-staphylobactin transport in S. aureus.

Analysis of the S. aureus genome resulted in identification of several genetic loci whose predicted products exhibit significant similarity to iron(III)-siderophore ABC-type transporters and iron(III)-siderophore binding proteins. At least three of these loci, sirABC, isdEF, and SA1977 to SA1979 (N315 genome designations), lack a gene whose predicted product exhibits similarity to ATPase components of ABC transporters. Thus, it is possible that FhuC interacts with one or more of these other S. aureus ABC transporters to transport an iron complex that is required for growth under iron limitation conditions. In our previous studies we characterized the phenotype of sirA and sirB mutants (15). Mutation of either gene results in a strain with a growth-impaired phenotype under iron limitation conditions, and expression of both genes was shown to be required for transport of iron(III)-staphylobactin. Thus, we tested H1071 for the ability to utilize staphylobactin in 55Fe-staphylobactin transport assays. The results showed that H1071 could not utilize staphylobactin (Fig. (Fig.3,3, inset). Moreover, complementation of this mutant with pFhuCBG and, most notably, pFhuC resulted in significant staphylobactin transport (Fig. (Fig.3),3), indicating that FhuB and FhuG are not involved in staphylobactin transport and that FhuC (as the ATPase), together with SirABC (SirA, binding protein; SirB and SirC, permease) (15, 23), is involved in staphylobactin transport. We did not detect a reduction in the amount of siderophore in H1071 culture supernatants compared to the amount in RN6390 culture supernatants, indicating that loss of the ability to transport staphylobactin in H1071 did not adversely affect the ability to produce siderophore.

FIG. 3.
55Fe-staphylobactin-mediated iron transport by S. aureus RN6390 and H1071 derivatives grown in TMS containing 50 μM 2,2′-dipyridyl. •, RN6390; ○, H1071/pFhuC; [filled triangle], H1071/pFhuCBG; [down-pointing small open triangle], RN6390 treated with 20 ...

The iron-restricted growth defect of S. aureus fhuCBG is more severe than that of S. aureus sirA or sirB mutants.

We previously showed that an S. aureus sirA mutant was not capable of transporting staphylobactin and that this mutant had an iron-deficient growth phenotype since it could not grow in TMS containing 250 μM 2,2′-dipyridyl (15). However, the lack of fhuC in H1071 (although in H1071 fhuCBG is deleted, recall that plasmid pFhuC complements the growth defect in this strain) results in a more attenuated growth defect in response to iron starvation than a sirA or sirB mutation results in. Figure Figure11 shows the compromised growth of H1071 in TMS containing 50 μM 2,2′-dipyridyl, whereas the growth of H306 (RN6390 sirA::Km) was not affected compared with the growth of parent strain RN6390; H1071 failed to grow in TMS containing 100 μM 2,2′-dipyridyl, whereas H306 grew as well as wild-type strain RN6390 (data not shown). Our use of iron-restricted media, such as TMS, which contains enough contaminating iron to allow growth of iron uptake mutants, and the ability to add increasing concentrations of 2,2′-dipyridyl, a nonmetabolizable iron chelator, provided the opportunity to distinguish the contributions of various different iron transporters. Taken together, our results suggest not only that FhuC interacts with SirABC to transport staphylobactin, but also that FhuC interacts with an additional transporter (or transporters) that allows the transport of as-yet-undetermined iron chelates.

The ΔfhuCBG::ermC mutant is only moderately compromised in a mouse kidney abscess model.

We have previously shown that siderophore biosynthesis is important for S. aureus virulence in a mouse kidney abscess model (14), and since we showed in this study that H1071 (RN6390 ΔfhuCBG::ermC) was compromised for staphylobactin uptake, we were interested in determining if the mutation in this strain had an effect on the virulence of S. aureus. S. aureus strain Newman colonizes mice better than RN6390 colonizes mice (14), so the ΔfhuCBG::ermC mutation was mobilized into the S. aureus Newman genetic background to create strain H1074. We confirmed that in the Newman genetic background, the ΔfhuCBG::ermC mutation had the same negative effect on iron-restricted growth that it had in the RN6390 genetic background (data not shown); the iron-restricted growth defect of H1074 could be complemented by introduction of plasmid pFhuC (data not shown).

Groups of Swiss-Webster mice were challenged with 107 CFU of S. aureus Newman (seven mice) or H1074 (Newman ΔfhuCBG::ermC) (six mice) via the tail vein, and the mice were monitored daily in a blinded fashion for alertness, activity, and coat condition. Mice that were challenged with H1074 were significantly less moribund than mice that were challenged with S. aureus Newman (Table (Table2)2) (the clinical scores were 2.7 and 0.5 [P < 0.0001], respectively; a completely healthy mouse would have had a clinical score of 0) (see Materials and Methods). On day 7 the mice were sacrificed. In contrast to the difference in morbidity between the mice in the group challenged with strain Newman and the mice in the group challenged with strain H1074, the data for other clinical parameters, such as kidney abscess score, percentage of weight loss, and bacterial CFU in the kidneys, revealed no statistically significant differences between the mutant and the parent strain (Table (Table22).

TABLE 2.
Clinical characteristics of mice infected with S. aureus Newman and H1074

DISCUSSION

Members of the ABC-ATPase protein family exhibit striking sequence similarity over a stretch of more than 200 amino acids, a region which includes the Walker A and Walker B motifs. Indeed, the ATP binding subunits of ABC transporters are “conserved components” and in different transporters exhibit significantly higher levels of similarity to one another than the transmembrane domains or the binding protein components of transporters exhibit with one another (6). The ATPase activity of the nucleotide binding domains is critical for the transport function and has been studied in several mammalian and bacterial members of this protein family (17, 31, 32, 35, 48). Evidence that S. aureus FhuC functions like other members of this family (i.e., by binding and hydrolysis of ATP to drive substrate translocation across a membrane) comes from our data which show that mutation of the conserved Walker A lysine residue in FhuC eliminates growth on iron(III)-hydroxamates as a sole source of iron and also results in an iron-restricted growth defect that we found was the result of an inability to transport iron in a complex with a staphylococcal siderophore.

Hydroxamate-mediated iron acquisition in S. aureus requires participation of FhuCBG and FhuD1 and/or FhuD2 (41-43), whereas staphylobactin-mediated iron acquisition in S. aureus requires SirABC (15). Based on the results of this study, it is apparent that the ATPase FhuC is required for iron acquisition from both hydroxamate siderophores and the structurally uncharacterized staphylobactin siderophore. To our knowledge, this is the first example of an ABC-ATPase that can function in one organism with more than one set of transmembrane domains to drive transport of different substrates. Although we have not yet shown that there is a direct interaction between FhuC and FhuBG or SirBC, finding that FhuC interacts with both sets of transmembrane domains would not be completely surprising since the total levels of similarity between the four transmembrane domains range from 50 to 57%.

It is clear that abrogation of staphylobactin-mediated iron acquisition in the ΔfhuCBG mutant, and not abrogation of hydroxamate-mediated iron acquisition in this mutant, accounts for the inability of the mutant to grow in iron-restricted conditions. The fact that the iron-restricted growth defect displayed by ΔfhuCBG mutants is more severe than the iron-restricted growth defect of either sirA or sirB mutants during growth in low-iron conditions (Fig. (Fig.1)1) suggests that fhuC may be involved in the transport of an additional iron chelate via another ABC transporter. In this regard, it is noteworthy that the S. aureus genome encodes other putative iron transporters (e.g., IsdEF and SA1977 to SA1979) without a genetically linked ATPase-encoding gene. In future studies we will elucidate the repertoire of substrates whose transport is affected in H1071 and H1074.

In our previous studies, we identified an operon, the sbn operon, whose products exhibit significant similarity with siderophore biosynthetic enzymes expressed by other bacteria (14). Insertional inactivation of sbnE resulted in a mutant strain which failed to produce the staphylobactin siderophore but which still produced siderophore activity (14), indicating that at least two siderophores are produced by the RN6390 and Newman strains. In future studies we will examine the possibility that FhuC is required for transport of this additional siderophore and that loss of transport of staphylobactin and the additional siderophore in fhuCBG mutants accounts for the difference that we observed in growth impairment between fhuCBG mutants and sirA mutants.

The ability to obtain iron is one of the key factors that determines whether there is successful establishment of an infection of a host organism by a bacterial pathogen. Indeed, inactivation of iron acquisition systems in S. aureus and many other bacteria can have a detrimental effect on virulence (3, 9, 14, 18, 24, 30, 38, 45-47, 51), while the loss of some iron acquisition functions in some bacteria does not seem to affect virulence, probably due to redundancy of acquisition systems (29, 38). For S. aureus, previous studies have demonstrated the importance of siderophore production for full virulence of the organism in a mouse model of infection (14), and Skaar et al. have recently shown that heme acquisition by this bacterium is also important for virulence in mice (44). Together, these results indicate that the ability of S. aureus to employ multiple iron acquisition strategies is a key to its success as a pathogen. Results of this study demonstrate that the S. aureus Newman ΔfhuCBG::ermC strain caused significantly less morbidity in mice than S. aureus Newman caused; however, the decrease in virulence potential occurred without significant differences in kidney abscess formation or bacterial load in the kidneys. Unfortunately, we did not examine the bacterial load in other organs, such as the spleen or liver. Our results also indicate that the lack of the potential to express FhuCBG (in vivo expression of fhu genes has not been demonstrated thus far but has been inferred based on the iron-regulated expression profile in vitro) does not affect the ability of S. aureus to persist in vivo (at least in the kidneys). The finding that expression of fhuCBG, both in iron-restricted media and in the kidney abscess model, is required for growth and the full virulence potential of S. aureus, respectively, correlates with the conservation of this operon in all S. aureus genomes sequenced to date (43), despite our previous findings which suggested that fhuCBG may only be important for utilization of hydroxamate siderophore produced by other bacteria or fungi.

The in vivo results presented in this paper contrast with previous results which demonstrated that there was a reduction in bacterial load in the kidneys by 5 days postinfection when a Newman sbnE::Km mutant (unable to produce staphylobactin siderophore) was used (14). Here, we showed that the fhuCBG deletion mutant, at least in vitro, was still able to produce siderophore at wild-type levels and that in vivo this ability may have an impact on the ability of the bacteria to persist in tissues, perhaps through cytopathic effects on the surrounding tissues; cytotoxic effects have been demonstrated for some iron chelates (5, 11). It is also interesting that mariner transposon insertion into fhuCBG did not alter the heme preference of S. aureus (44); unfortunately, however, the position of the mariner insertion was not described, and fhuC may still be expressed. Additional in vivo studies with the fhuCBG mutant and other iron transporter mutants, in addition to siderophore-deficient mutants, are required to examine whether there is an in vivo and infection-site-specific preference for any iron transporter or siderophore in S. aureus.

Given the increasing reports in the literature describing how mutations in iron transport functions impair the virulence of gram-positive bacteria, combined with the prevalence of antibiotic resistance, targeting iron uptake systems offers an attractive route to explore in the search for novel antimicrobials.

Acknowledgments

This research was supported by an operating grant to D.E.H. from the Canadian Institutes of Health Research (CIHR). D.E.H. was supported by a CIHR New Investigator Award, and C.D.S. and S.E.D. were supported by Natural Sciences and Engineering Research Council postgraduate scholarships. We are grateful for infrastructure support from the Canada Foundation for Innovation and the Ontario Innovation Trust.

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