- Research article
- Open Access
ABC transporter FtsABCD of Streptococcus pyogenes mediates uptake of ferric ferrichrome
© Hanks et al; licensee BioMed Central Ltd. 2005
- Received: 10 September 2005
- Accepted: 14 October 2005
- Published: 14 October 2005
The Streptococcus pyogenes or Group A Streptococcus (GAS) genome encodes three ABC transporters, namely, FtsABCD, MtsABC, and HtsABC, which share homology with iron transporters. MtsABC and HtsABC are believed to take up ferric (Fe3+) and manganese ions and heme, respectively, while the specificity of FtsABCD is unknown.
Recombinant FtsB, the lipoprotein component of FtsABCD, was found to bind Fe3+ ferrichrome in a 1:1 stoichiometry. To investigate whether FtsABCD transports Fe3+ ferrichrome, GAS isogenic strains defective in lipoprotein gene ftsB and permease gene ftsC were generated, and the effects of the mutations on uptake of Fe3+ ferrichrome were examined using radioactive 55Fe3+ ferrichrome. FtsB was produced in the wild-type strain but not in the ftsB mutant, confirming the ftsB inactivation. While wild-type GAS took up 3.6 × 104 Fe3+ ferrichrome molecules per bacterium per min at room temperature, the ftsB and ftsC mutants did not have a detectable rate of Fe3+ ferrichrome uptake. The inactivation of ftsB or ftsC also decreased 55Fe3+ ferrichrome uptake by >90% under growth conditions in the case of limited uptake time. Complementation of the ftsB mutant with a plasmid carrying the ftsB gene restored FtsB production and 55Fe3+ ferrichrome association at higher levels compared with the parent strain. The inactivation of mtsA and htsA and Fe-restricted conditions enhanced the production of FtsB and Fe3+ ferrichrome uptake.
The FtsB protein bound Fe3+ ferrichrome, and inactivation of ftsB or ftsC, but not htsA or mtsA, diminished Fe3+ ferrichrome uptake, indicating that FtsABCD, but not HtsABC and MtsABC, is the transporter that takes up Fe3+ ferrichrome in GAS. Fe acquisition systems are virulence factors in many bacterial pathogens and are attractive vaccine candidates. The elucidation of the FtsABCD specificity advances the understanding of Fe acquisition processes in GAS and may help evaluating the GAS Fe acquisition systems as vaccine candidates.
- Necrotizing Fasciitis
- Isogenic Mutant
- Radioactive 55Fe3
- Ferrichrome Uptake
Ferric iron (Fe3+), the stable iron form in an oxidative environment, has extremely low solubility in water under physiological conditions, and mammalian hosts thus do not have sufficient free Fe3+ to support bacterial growth . The major sources of iron in vivo for bacteria are host heme-proteins and other iron complexes [1, 2]. Many bacterial pathogens secrete low-molecular-weight iron chelators called siderophores to assimilate iron from host environments . Ferrisiderophores formed are then transported across the cytoplasmic membrane by specific ATP-binding cassette (ABC) type transporters. ABC transporters consist of a solute-binding protein, a membrane protein (permease) encoded by one or two genes, and an ATPase . The solute-binding proteins are located in the periplasmic space in Gram-negative bacteria and are lipoproteins in Gram-positive organisms. Siderophores can be divided into several types based on chemical structures . Ferrichrome  belongs to the hydroxamate type.
Streptococcus pyogenes or Group A Streptococcus (GAS) is an important Gram-positive human pathogen causing both invasive and non-invasive infections . Non-invasive infections, including pharyngitis, and post-infection sequelae, such as acute rheumatic fever, rheumatic heart disease, and glomerulonephritis, result in substantial morbidity and economic loss globally. Invasive GAS infections, such as necrotizing fasciitis and streptococcal toxic shock syndrome, are associated with high mortality rates. GAS can take up heme from hemoglobin and haptoglobin-hemoglobin complexes . Exogenously-supplied heme and host heme proteins (hemoglobin, myoglobin, and catalase), but not iron-loaded transferrin and lactoferrin, support in vitro growth of GAS under iron-restricted conditions .
Iron acquisition processes in GAS are poorly understood, although progress has recently been made by us [9–11] and other groups [12–14]. GAS genomes [15–17] encode three ABC transporters, namely, HtsABC [9–11] or SiaABC , MtsABC [13, 14], and one encoded by spy0383 to spy0386  (designated FtsABCD), which are homologues of ABC transporters involved in iron acquisition. HtsABC and the cell-surface protein Shp [9–12] are believed to make up the machinery for heme acquisition. The lipoprotein component MtsA of MtsABC binds Fe3+, Zn2+, and Cu2+ , and MtsABC is important for acquisition of Mn2+ and Fe3+ . The transcription of fts ABCD is up-regulated under iron-restricted conditions . However, recombinant FtsB was heme-free and did not contain Fe, Mn, or Zn . The specificity of FtsABCD is thus not known. It is not known whether GAS can use ferrisiderophores such as ferric ferrichrome (Fe3+ ferrichrome) as an iron source.
We found that recombinant FtsB bound Fe3+ ferrichrome, suggesting that FtsABCD is involved in the acquisition of Fe3+ ferrichrome. To test this hypothesis, GAS isogenic strains defective in lipoprotein gene ftsB and permease gene ftsC were generated, and the ftsB and ftsC inactivation dramatically decreased the uptake of Fe3+ ferrichrome, indicating that FtsABCD is the transporter that takes up Fe3+ ferrichrome in GAS.
Binding of Fe3+ ferrichrome by FtsB
If FtsB binds Fe3+ ferrichrome, Fe3+ should co-migrate with the protein on the G-25 column. To test this idea, Fe3+ ferrichrome containing 1.1% 55Fe3+ of total Fe3+ was used to repeat the gel filtration experiment, and 55Fe3+ radioactivity was monitored. As expected, one of two 55Fe3+ peaks co-migrated with the FtsB peak, and the other peak corresponded to free Fe3+ ferrichrome (Fig. 1D). On the basis of 55Fe3+ percentage of total iron and specific activity and protein content, the FtsB peak fraction contained 37 μM FtsB and 36 μM Fe, consistent with the 1:1 binding stoichiometry determined above. These results confirmed that FtsB binds Fe3+ ferrichrome in a 1:1 molar ratio.
Effects of ftsB, ftsC, mtsA, and htsA inactivation on Fe3+ ferrichrome uptake
Effect of Fe3+ ferrichrome on GAS growth
Since FtsABCD is involved in the uptake of Fe3+ ferrichrome, Fe3+ ferrichrome should be an iron source of GAS. This idea was tested by comparing GAS growth curves in THY treated with Chelex 100 to remove metal ions and supplemented with MgCl2 (DTHYMg) in the absence and presence of Fe3+ ferrichrome. The growth curve in the presence of 10 μM Fe3+ ferrichrome shifted to the left by about 40 min compared with that in the absence of Fe3+ ferrichrome under otherwise identical conditions. This small but repeatable stimulatory effect suggests that GAS can use Fe3+ ferrichrome as an iron source. Fe3+ ferrichrome had similar stimulatory effect on the growth of the mutants as that on the growth of the wild type strain, suggesting that either residual uptake of Fe3+ ferrichrome in the ftsB and ftsC mutants were enough to induce the stimulatory effect or an additional process was involved in the uptake of the iron extracted from Fe3+ ferrichrome under the growth conditions.
Effects of ftsB and ftsC inactivations on the uptake of Fe3+ ferrichrome under growth conditions
Effects of mtsA and htsA inactivation and Fe-restricted conditions on Fe3+ ferrichrome uptake and FtsB production
Evidences from this study indicate that FtsABCD is the transporter that takes up Fe3+ ferrichrome in GAS. The evidences include the binding of Fe3+ ferrichrome to FtsB, the effect of the insertional disruption of ftsB or ftsC on uptake of 55Fe3+ ferrichrome, and the non-involvement of MtsABC and HtsABC in Fe3+ ferrichrome uptake. Complementation data of the ftsB mutant with the ftsB gene expressed in trans indicated that the effect of ftsB inactivation was due to the lack of FtsB. MtsABC [13, 14] and HtsABC [9–12] target free Fe3+ and heme, respectively. The specificity of the FtsABCD transporter elucidated in this study resolved the last piece of the puzzle regarding the roles of ABC transporters in Fe acquisition in GAS.
Uptake time was critical to the effects of ftsB and ftsC inactivation on 55Fe3+ activity associated with bacteria under growth conditions. The inactivation had a dramatic effect (>90% decrease in 55Fe3+ activity compared with wild-type strain) when uptake was performed for only 30 min. This decrease was reduced to about 50% when uptake was performed for 3 h. Although the reasons for this reduction are not known, the reduction is unlikely due to the existence of another transporter for Fe3+ ferrichrome. The other uptake results are not consistent with the existence of another Fe3+ ferrichrome transporter. Another possible reason is that 55Fe3+ ferrichrome exchanged its 55Fe3+ with another ferric complex in THY, resulting in a non-ferrichrome 55Fe3+ complex that could be taken up by another transporter. GAS has a putative secreted Fe binding protein (Spy1063). It is not known whether this protein can extract Fe3+ from Fe3+ ferrichrome.
The transcription of ftsABCD is up-regulated under Fe-reduced conditions . Consistent with this observation, Fe-restricted conditions enhanced the production of FtsB and uptake of Fe3+ ferrichrome, further supporting the role of FtsABCD in Fe acquisition. Inactivation of htsA and mtsA also enhanced FtsB production and Fe3+ ferrichrome uptake. Apparently, all the three ABC transporters contributed to Fe3+ acquisition in GAS grown in THY, and inactivation of either mtsA or htsA might result in lower intracellular Fe levels and, in turn, enhanced the expression of ftsABCD. The results also suggest that the expression of ftsABCD, htsABC, and mtsABC is coordinately regulated.
Some Enterococcus faecium clinical strains do not produce siderophores but can acquire iron using exogenous siderophores produced by other bacteria living in the same habitats . GAS is not known to produce siderophores, and GAS genomes [15–17] do not have genes encoding homologues of siderphore-production systems. We could not detect siderophore production in GAS under iron-restricted conditions. Therefore, GAS may not produce siderophors. However, GAS takes up Fe3+ ferrichrome, suggesting that GAS could acquire Fe3+ by using siderophores produced by other bacteria in the pharynx and skin, the noninvasive GAS infection sites.
Fe acquisition systems are virulence factors in many bacterial pathogens [20–24] and are attractive vaccine targets [25–30]. Elucidation of the specificities of the Fe transporters in GAS will facilitate determination of their relative importance in various infections and choose appropriate animal infection models to evaluate their efficacy as vaccine candidates. For examples, HtsABC could be more important in invasive GAS infection since heme should be the Fe source, and FtsABCD could be important in non-invasive infections because exogenous ferric siderophore complexes should be available.
In summary, we found that FtsB bound Fe3+ ferrichrome and that ftsB or ftsC inactivation dramatically decreased Fe3+ ferrichrome uptake. The results indicate that FtsABCD is the transporter that acquires Fe3+ ferrichrome in GAS.
Iron chelating agent 2,2'-dipyridyl was obtained from Aldrich. Sephadex G-25, iron-free ferrichrome A from Ustilago sphaerogena, Chelex 100, and other chemicals were purchased from Sigma (St. Louis, MO). 55Fe3+-labeled ferric chloride was purchased from RI Consultants LLC (Hudson, NH). Purified recombinant FtsB (Spy0385) and MtsA were prepared as described previously [10, 31].
Five female outbred CD-1 Swiss mice (4- to 6-week-old) (Charles River Laboratories, Wilmington, MA) were immunized subcutaneously with 50 μg of recombinant FtsB, MtsA, or PGK suspended in 200 μL of saline emulsified in 44 μL of monophosphoryl lipid A-synthetic trehalose dicorynomycolate adjuvant (Corixa, Hamilton, MT). Mice were boosted at weeks 2 and 4. Immune sera were collected 5 days after the second boost.
Bacterial strains and growth
Serotype M1 GAS strain MGAS5005 has been described previously . MGAS5005 and its isogenic mutants were grown routinely at 37°C in 5% CO2 in Todd-Hewitt broth (Difco Laboratories, Detroit, MI) supplemented with 0.2% yeast extract (THY). Spectinomycin (150 mg/L) was added into THY for mutant strains. Iron-restricted conditions were achieved by adding 0.3 mM 2,2'-dipyridyl into THY and by treating THY with the chelating resin Chelex 100 and supplementing it with 0.4 mM MgCl2 (DTHYMg). Tryptose agar with 5% sheep blood (Becton Dickinson, Cockeysville, MD) and THY agar were used as solid media.
Binding of Fe3+ ferrichrome to FtsB
Gel filtration was used to detect the binding of Fe3+ ferrichrome to FtsB. FtsB (0.3 ml of 0.18 mM) was incubated with 0.9 mM Fe3+ ferrichrome for 20 min at room temperature, loaded onto a Sephadex G-25 column (1.5 × 18 cm), and eluted with 20 mM Tris-HCl buffer, pH 8.0. Eluant was collected as fractions of 0.7 ml. The absorbance of each fraction attributable to protein and Fe3+ ferrichrome was measured. MtsA as a negative control was similarly tested for the binding of Fe3+ ferrichrome. The experiment was repeated with Fe3+ ferrichrome in which 1.1% of total Fe3+ was 55Fe, and A280 and 55Fe3+ radioactivity of each fraction were measured. 55Fe3+ radioactivity was measured using a window of 0–6 keV with a Packard 1500 Tri-Carb Liquid Scintillation Analyzer.
GAS isogenic mutants
Primers used in gene inactivation and confirmation
confirmation of ftsB inactivation
confirmation of ftsC inactivation
confirmation of mtsA inactivation
aad-specific, paired with primer 7, 9, or 11 for inactivation confirmation
pFWaad-specific, paired with primer 8, 10, or 12 for inactivation confirmation
Each suicide plasmid was introduced into MGAS5005 by electroporation at 1.8 kV and 400 Ω. One ml of THY was added into the sample immediately after electroporation. The sample was incubated at 37°C for 2 h and plated on THY agar plates supplemented with 150 mg of spectinomycin per liter to select insertional mutants. The plates were incubated in 5% CO2 at 37°C for two days, and the colonies obtained were screened by PCR analysis using the primers listed in Table 1. Gene interruptions were then confirmed by sequencing the PCR products.
Construction of ftsB-complementing plasmid pCMVftsB
Plasmid pJRS525  was modified to replace the spectinomycin-resistant gene with the chloramphenicol-resistance gene. A 2545-bp fragment without the spectinomycin-resistant gene was amplified from pJRS525 with primers 5'-TCGTGGATCC AAGCTTCACCATGG-3' and 5'-AAGAATTCCTTGCATAGACTTTCGTCAG-3'. The fragment containing the chloramphenicol-resistant gene was amplified from pFW14  with primers 5'-GGAATTCCGGATGCATATGCATG-3' and 5'-GTCCGGATCCTCGAGCTCTAGATC-3'. The PCR products were digested with BamH I and EcoR I and ligated together to yield plasmid pCMV. A DNA fragment containing ftsB and its ribosome-binding site was amplified from MGAS5005 using primers 5'-CGGATCCAATAACTTTATTCTAGGAGAATTAG-3' and 5'-AGGGATCCTTAGTTTTCACTTGATAAGATTG-3'. The PCR product was digested with BamH I and ligated into pCMV at the BamH I site, yielding pCMVftsB containing ftsB. The cloned gene was sequenced to rule out spurious mutations and confirm the desired orientation. The resulting pCMVftsB was introduced into the ftsB mutant by electroporation, and the complement strain (designated ftsB/pCMVftsB) was selected by spectinomycin and chloramphenicol and confirmed for the existence of pCMVftsB by colony PCR using primers 5'-CAATTTCACACAGGAAACAGC-3' (pCMV-specific) and 5'-AGGGATCCTTAGTTTTCACTTGATAAGATTG-3' (ftsB-specific)
Uptake of Fe3+ ferrichrome by GAS
Fe3+ ferrichrome uptake by GAS was monitored using radioactive 55Fe3+ ferrichrome under non-growth and growth conditions. Under the non-growth conditions, wild-type MGAS5005 and its ftsB, ftsC, or mtsA mutant strains were harvested from the mid-exponential growth phase (OD600 of 0.4) by centrifugation. The bacterial pellets were washed with 10 ml of THY and resuspended in 1 ml of THY. To prepare 55Fe3+ ferrichrome working solution, 4.7 nmole 55FeCl3 was incubated with 14.1 nmole Fe-free ferrichrome A in 50 μL of Tris-HCl for 10 min, and the complex was diluted with 22 ml of THY. The 1-ml bacterial suspensions were mixed with 3 ml of the 55Fe3+ ferrichrome solution (final Fe3+ ferrichrome concentration 0.16 μM) to initiate the uptake process and rotated in a 10-ml tube from end to end at room temperature. A triplet of 0.2 ml samples were taken from each mixture at the indicated times, and bacteria were immediately pelleted and washed twice with 0.4 ml of THY. For uptake under growth conditions, 0.16 μM 55Fe3+ ferrichrome was added into the cultures of wild-type, ftsB, and ftsC strains at early exponential or mid-exponential growth phase. A triplet of 1-ml culture samples were taken at the indicated times after the 55Fe3+ ferrichrome addition and treated as described above. The pellets obtained were resuspended in 0.2 ml of THY and mixed with 3 ml of scintillation liquid. 55Fe3+ radioactivity associated with the bacteria was measured as described above.
FtsB and MtsA production
Production of FtsB or MtsA in wild-type and mutant strains was monitored by Western blotting analysis. To prepare samples for the analysis, bacteria were harvested from cultures at the indicated volumes in exponential phase. The bacterial pellets were washed twice with 1.3 ml of PBS, resuspended in 100 μl of PBS, and treated with 200 units of mutanolysin at 37°C for 2 h. The samples were briefly sonicated and mixed with equal volume of 2x SDS-PAGE loading buffer. Proteins in 10 μl of the samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The proteins were detected by Western blotting using specific mouse antiserum as previously described .
Other procedures and measurements
Chromosomal DNA was isolated with the Puregene DNA Isolation kit (Gentra Systems, Minneapolis, MN). Sequence data were obtained with an ABI 310 automated DNA sequencer (Applied Biosystems, Inc., Foster City, CA). Absorbance and optical spectra were obtained with a SPECTRAmax 384 Plus spectrophotometer (Molecular Devices, Sunnyvale, CA). Protein concentrations were determined with the modified Lowry protein assay kit purchased from Pierce (Rockford, IL) with bovine serum albumin as a standard. Intensities of the bands in Western blotting analysis were determined using an AlphaImager 2000 Documentation & Analysis System (Alpha Innotech Corp.).
This work was supported in part by grants P20 RR-16455 and P20 RR-020185 from the National Center for Research Resources, K22AI057347 from National Institutes of Health, and the Montana State University Agricultural Experimental Station. We thank Dr. Kevin McIver for providing plasmid pJRS525.
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