1 Department of Biology, College of Arts and Sciences, Georgia State University, 50 Decatur St, Atlanta, GA 30303, USA
2 Department of Microbiology and Immunology, Wayne State School of Medicine, 540 East Canfield Ave, Detroit, MI 48201, USA
Correspondence
Zehava Eichenbaum
zeichen{at}gsu.edu
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ABSTRACT |
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INTRODUCTION |
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The acquisition of iron from host proteins has been studied in a handful of Gram-positive microbes. Some species employ surface receptors for host proteins like transferrin or lactoferrin (Hartford et al., 1993; Modun et al., 1998
) or siderophores to obtain ferric iron (Coulanges et al., 1998
; Courcol et al., 1997
; De Voss et al., 1999
; Russell et al., 1984
; Sebulsky & Heinrichs, 2001
). The use of haem and host haemoproteins has been demonstrated in Corynebacterium diphtheriae (Schmitt, 1997
, 1999
), Staphylococcus aureus (Mazmanian et al., 2003
), and several streptococci (Bates et al., 2003
; Brown et al., 2001a
; Eichenbaum et al., 1996
; Francis et al., 1985
; Podbielski et al., 1999
). Haem uptake in Gram-positive organisms seems to be mediated by dedicated surface receptors for haem or haemoproteins, while the production of haemophores (secreted haem-binding proteins found in several Gram-negative bacteria) has not been reported (Wandersman & Delepelaire, 2004
).
The principal machinery involved in the uptake of free or complex iron in Gram-positive bacteria is ABC transporters, which consist of a substrate-binding lipoprotein, one or two membrane permease subunits, and a hydrophilic ATPase (Brown & Holden, 2002; Gilson et al., 1988
; Higgins, 1992
; Wandersman & Stojiljkovic, 2000
). Haem and siderophore transporters share significant homology and belong to a defined cluster of ABC transporters. In addition, Gram-positive pathogens carry ABC metal transporters, which are part of a separate cluster of transporters and have affinity for multiple metals (Brown & Holden, 2002
; Claverys, 2001
). In some of these multi-metal transporters, the metal binding receptors function as bacterial adhesins as well (Dintilhac et al., 1997
; Elsner et al., 2002
; Oligino & Fives-Taylor, 1993
; Spellerberg et al., 1999
).
Streptococcus pyogenes is a haemolytic pathogen capable of producing a diverse array of skin and mucous membrane infections as well as aggressive deep tissue diseases and streptococcal toxic shock syndrome. Untreated streptococcal infections can lead to the serious complications of rheumatic fever and acute glomerulonephritis (Bisno et al., 2003; Cunningham, 2000
). Under laboratory conditions Strep. pyogenes can use haem and a variety of haemoproteins such as haemoglobin-haptoglobin, haemoglobin, myoglobin, haem-albumin and catalase as a source of iron, but it cannot use transferrin or lactoferrin (Eichenbaum et al., 1996
; Francis et al., 1985
; Podbielski et al., 1999
). Strep. pyogenes possesses a multi-metal transporter encoded by mts (Janulczyk et al., 1999
) and two transporters from the iron-complex family: sia (streptococcal iron acquisition) (Bates et al., 2003
) or hts (Lei et al., 2003
) and a transporter which we name here siu (streptococcal iron uptake). The siaABC genes were suggested to function as a haem transporter, and SiaA (or HtsA), the binding protein homologue, was shown to bind haemoglobin and haem (Bates et al., 2003
; Lei et al., 2003
). On the other hand, the mts transporter is involved in uptake of manganese and ferric iron (Janulczyk et al., 2003
), where MtsA binds iron, zinc and manganese in vitro (Janulczyk et al., 1999
). The ligand and the function of the siu transporter have not yet been defined. In this study, we investigated the role of the siu transporter in iron acquisition and disease production.
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METHODS |
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ToddHewitt broth (no yeast extract, TH) was also treated for 3 or 20 h with 5 % (w/v) Chelex-100 (Bio-Rad). The pH of the resin-treated medium was adjusted to 7·65, autoclaved, and 0·5 mM CaCl2 and 0·9 mM MgSO4 were added before inoculation. Inductivity-coupled plasma mass spectrometry (ICP-MS) analysis (at the Laboratory for Environmental Analysis, University of Georgia at Athens) demonstrated that TH contains about 17·5±6·5 µM iron, 0·53±0·2 µM manganese and 15·5±0·2 µM zinc, depending on the batch and manufacturer. Chelex-100 treatment for 3 h resulted in 2·7 µM iron, less than 0·18 µM manganese and about 0·3 µM zinc in the medium. Treatment with Chelex-100 for 20 h did not significantly change its iron content in comparison to 3 h of treatment.
Strep. pyogenes was also grown in a chemically defined medium (CDM) (Podbielski et al., 1999; van de Rijn & Kessler, 1980
). CDM was also treated with 3 % (w/v) Chelex-100 for 6 h and filter-sterilized (CxCDM). ICP-MS analysis showed that CxCDM contains 1·6 µM iron and 0·43 µM zinc; the manganese concentration is below the detection level. For cell growth, CxCDM was supplemented with 33 µM MgCl2 and 68 µM CaCl2. All experiments done with Strep. pyogenes cells growing in CDM or CxCDM were inoculated using mid-exponential-phase cells, which were prepared as follows: cells cultured in ZTH medium were harvested at the exponential phase (OD600 0·6), washed twice with PBS, and stored in small frozen aliquots in 16 % (v/v) glycerol. All glassware used for streptococcal growth was soaked for 30 min in a chromic/sulfuric acid solution (Fisher Scientific) and rinsed with double-distilled water (ddH2O). When necessary, the antibiotics spectinomycin and erythromycin were used for E. coli at 100 µg ml1 and 500 µg ml1, respectively. For Strep. pyogenes, spectinomycin and erythromycin were used at 100 µg ml1 and 1 µg ml1, respectively. Optical density was measured with a Beckman DU640 spectrophotometer (600 nm) or with a Scienceware 800-3 Klett colorimeter (640700 transmission filter).
Construction of strains ZE4913, ZE4914 and ZE4915.
The primers used in this study are listed in Table 1. Mutants with insertional inactivation of the siuG and siaB genes were constructed in Strep. pyogenes NZ131 (M49 type) using primers designed according to the Strep. pyogenes SF370 genome database (Ferretti et al., 2001
) (NCBI and TIGR complete genome databases). All of the constructed chromosomal mutations were verified by PCR analysis. The siuG mutant (ZE4915) was constructed by amplifying a 2·9 kb fragment from NZ131 chromosomal DNA using primers fhuX-S and fhuX-A. The PCR fragment, which included the 3'-end of siuB, the entire siuG gene, and a region downstream of siuG, was digested with AatII and SalI and ligated into pBR322, generating the plasmid pSaAa. The ermAM gene (erythromycin resistance) from pFW15 (Podbielski et al., 1996
) was amplified using primers erm-S and erm-A and cloned into the EcoRI site of the siuG gene in pSaAa, generating pSaAaerm. A fragment containing the siuG : : ermAM allele and flanking region was released by AatII/SalI digestion and electroporated into Strep. pyogenes NZ131. Allelic replacement clones were selected on THY agar plates containing erythromycin. A siuG siaB double mutant (ZE4914) was constructed by introducing a disrupted siaB copy in the background of ZE4915. For this purpose, a 2 kb fragment including the 3'-end of siaA, the entire siaB gene and the 5'-end of siaC was amplified from NZ131 chromosomal DNA using primers stoj5 and stoj6. The siaB PCR product was cloned into the XmnI site of pACYC184, producing plasmid pStoj3. The aad9 gene (spectinomycin resistance) was amplified from pUCSpec (Husmann et al., 1997
) using spc-S and spc-A primers and cloned into the BclI site of siaB, generating plasmid p5spc1. The fragment containing the siaB : : aad9 allele and flanking chromosomal regions was released by XmnI/StuI digestion and electroporated into the ZE4915 strain. Allelic replacement mutants were selected on THY agar containing erythromycin and spectinomycin. The construction of the siaB mutant (ZE4913) was the same as the construction of ZE4914, except that the siaB : : aad9 allele was introduced into the wild-type NZ131 strain.
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Ferric iron uptake assays.
Iron uptake assays were essentially performed as previously described, with small modifications (Brown et al., 2001a; Janulczyk et al., 2003
). CxCDM was inoculated with Strep. pyogenes cells (1/250) and the cultures were grown to mid-exponential phase (35 Klett units) at 37 °C. 55FeCl3 (0·2 µCi µl1, 7·4 kBq µl1; 0·02 µM) was added to 1 ml cultures and incubated at 37 °C. Culture samples (200 µl) were drawn every 30 min and washed twice with 500 µl CxCDM containing 10 mM NTA. The radioactivity associated with the cell pellet and the supernatant was measured as counts per minute (c.p.m.) for 5 min against a 3H standard using a Beckman LS6500 scintillation counter. The culture's OD600 was measured at the same time using a Beckman DU640 spectrophotometer. 55Fe incorporation for each time point was standardized for the cell quantity by dividing the c.p.m. by the culture OD600. Competition assays with iron and manganese were performed as above except that increasing concentrations of non-radioactive FeCl3 or MnSO4 were provided in addition to 55FeCl3. Culture samples were drawn after about 60 min, washed twice, and their radioactivity was measured. For the inhibition of ferric iron uptake, increasing concentrations of haem or protoporphyrin IX were added with the 55FeCl3. Samples were taken after 60 min (OD600 about 1), washed, and the radioactivity measured. 55Fe incorporation was defined as the fraction of c.p.m. of the pellet divided by the sum of the c.p.m. in pellet and the supernatant. Haem was prepared as a 10 mg ml1 stock solution of haemin chloride (Sigma) in 0·1 M NaOH pH 10. Protoporphyrin IX was prepared as a 10 mg ml1 stock solution dissolved in 1 : 1 dimethyl formamide/methanol. As a control, a 10 mg ml1 haem solution was treated with 5 % (w/v) Chelex-100 for 1 h prior to adding it to the cells to remove any unbound iron. When necessary, dilutions were prepared in ddH2O.
Zebrafish care and virulence assays.
Care and feeding of zebrafish (Danio rerio) followed published methods (Neely et al., 2002; Westerfield, 1995
). Streptococci were cultured overnight in THY plus 20 % (w/v) peptone (THYP) at 37 °C, diluted 1 : 100 the next day in THYP, and incubated at 37 °C. The cells were harvested at OD600 0·3, washed once with THYP, and diluted to the appropriate concentration in fresh THYP. Injection of zebrafish followed a previously described method (Neely et al., 2002
). Briefly, streptococcal cells (10 µl of 105 ml1) were aseptically injected into groups of four to six anaesthetized male breeder zebrafish (Scientific Hatcheries). Following intraperitoneal (i.p.) or intramuscular (i.m.) injection, the fish were allowed to recover in 225 ml sterilized ddH2O supplemented with aquarium salts (Instant Ocean; Aquarium Systems) at a concentration of 60 mg l1 in a 29 °C incubator. A control animal group was injected with sterile medium. Infected fish were monitored for 48 h and death recorded in intervals of 12 h. For Strep. pyogenes, the 50 % lethal dose (LD50) for infection of zebrafish was determined by the method of Neely et al. (2002)
, where zebrafish were challenged over a range of 101106 c.f.u. of each streptococcal strain.
Zebrafish tissue analysis.
Selected whole zebrafish were fixed following euthanasia at 40 h after infection and 5 µm thick longitudinal sections of the dorsal muscle were prepared for staining as described previously (Neely et al., 2002). Fixed samples were stained with haematoxylin and eosin and examined with an Olympus BX60 microscope equipped with a digital camera and a motorized stage.
Statistics and data analysis.
Statistical significance was determined by using the two-sample Student t-test. The standard error of the mean (SEM) was calculated by dividing the standard deviation by the square root of n.
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RESULTS AND DISCUSSION |
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The siu transporter is involved in iron acquisition
The iron needs of Strep. pyogenes NZ131 were investigated in various media treated with different resins. NZ131 grew in TH medium treated with the chelating resin Chelex-100 or in CxCDM (see Methods), demonstrating that it can proliferate in media containing only 1·6 µM iron and trace amounts of manganese and zinc. Similar observations were made in THY medium that was treated with Chelex-100 (Janulczyk et al., 2003; Ricci et al., 2002
). RNA analysis showed that growth in CxCDM allows significant expression of both the siu and the sia transporters (data not shown), which are both negatively regulated by iron (Bates et al., 2003
; Lei et al., 2003
; Smoot et al., 2001
). The addition of 20 µM iron (but not of 20 µM manganese) repressed siu expression, confirming that while the iron concentration found in this medium is sufficient to support growth, it is low enough to produce an iron-stress signal. Growth of NZ131 was significantly impaired in a buffered ToddHewitt broth containing 12 mM NTA (ZTH-NTA) that was supplemented with a mix of the bivalent metals calcium, magnesium, manganese and zinc as described by Bates et al. (2003)
(black bar, NTA in Fig. 1
). Similarly, 20 mM NTA was previously used to restrict the growth of a second M49 strain, CS101, in THY (Podbielski et al., 1999
).
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Inactivation of the siu transporter results in decreased Fe3+ utilization
The presence of NTA in the ZTH-NTA medium complicates the study of the cell's use of ferric iron. Therefore, we investigated Fe3+ utilization by the wild-type and the siuG mutant using a 55Fe3+ uptake assay in a low-iron medium that does not contain a chelator. 55FeCl3 was added to cells growing in CxCDM at the early exponential phase and incorporation by the cells was monitored every 30 min. Iron accumulation by both the wild-type and the siuG mutant cells increased over time (Fig. 2), with maximum incorporation by wild-type cells observed after about 30 min incubation. The addition of 2 µM non-radioactive iron (56Fe3+) or manganese (Mn2+) inhibited 55Fe3+ incorporation into NZ131 cells by 30 % and 75 %, respectively. Inhibition reached 62 % and 80 % with 6 µM iron or manganese (data not shown). Inhibition of Fe3+ uptake by manganese suggests that at least some of the Fe3+ uptake in NZ131 is mediated by a multi-metal transporter such as the mts transporter (RT-PCR confirmed the presence of mts transcript in the NZ131 strain; data not shown). Similarly, manganese could compete with Fe3+ uptake in the AP1 (M1 type) strain, in which inactivation of mts reduced accumulation of both 54Mn2+ and 55Fe3+ (Janulczyk et al., 2003
).
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Haem inhibits Fe3+ accumulation by Strep. pyogenes
We studied the effect of haem present in the growth medium on Fe3+ accumulation by Strep. pyogenes NZ131. Haem at a concentration as low as 0·75 µM inhibits the accumulation of 55Fe3+ by 55 % as compared to the uptake in the absence of haem, and the addition of 6 µM haem results in about 76 % inhibition (Fig. 3a, black bars). Treatment of haem with Chelex-100 to remove free iron possibly present in the solution did not change the percentage inhibition of 55Fe3+ uptake by haem (Fig. 3a
, white bar). This is consistent with the proposal that it is the haem, and not free iron, that inhibits 55Fe3+ uptake. On the other hand, the addition of protoporphyrin IX, the core structure of haem, did not significantly interfere with 55Fe3+ incorporation, even at a concentration as high as 6 µM (Fig. 3a
, grey bars). Since iron is important for the ability of haem to hinder ferric transport, we suggest that this inhibition is not the outcome of non-specific interference of the haem moiety, but that the use of haem as an iron source leads to repression of the Fe3+ uptake pathways. Haem may also compete with ferric iron for some of the transporters that contribute to Fe3+ uptake.
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When a higher concentration of haem was used, 55Fe3+ accumulation by the siu mutant was inhibited to a level similar to that observed in the wild-type cells. This suggests that an additional transporter(s) is involved in haem utilization and or is affected by haem. To test this hypothesis, a mutant in siaB (the sia membrane permease component, strain ZE4913) and a siuG siaB double mutant (strain ZE4914) were constructed by the insertional inactivation method using an aad9 cassette. The presence of the siaB : : aad9 allele in both mutant strains was confirmed by PCR; as expected the sia fragment amplified from the siaB and the siuG siaB strains was 1·25 kb larger than that produced from the wild-type strain. To test if the sia transporter contributes to the haem effect seen in the siuG mutant, we repeated the assay with the siaB and the siuG siaB strains. Similar to the siuG mutant, 2 µM haem did not efficiently inhibit 55Fe3+ uptake in the siaB mutant (Fig. 3b, hatched bars, P<0·025, n=6), while 4 µM haem led to a decrease in Fe3+ uptake similar to that seen in the wild-type strain. Inactivation of both siaB and siuG had a cumulative effect at 4 and 6 µM haem. In the presence of 6 µM haem, only 52 % inhibition was observed in the double mutant (Fig. 3b
, grey bar), while about 7680 % inhibition was observed in the wild-type, siuG or siaB strains. While this reduction is not striking, it is statistically significant (P<0·005, n=4) when compared to the wild-type. Based on these observations we suggest that haem utilization is partially impaired if the siu transporter is disrupted and that inactivation of the sia transporter reduces haem usage by the cell even further. Additionally, the ability of haem to reduce 55Fe3+ uptake by 52 % in the double mutant may result from the residual activity of the siu system and the presence of other haem utilization pathways. Redundancy in haem utilization pathways has been demonstrated in several Gram-positive bacteria, such as isdDEF and htsABC in Staph. aureus (Mazmanian et al., 2003
; Skaar et al., 2004
), piaABCD and piuBCDA in Strep. pneumoniae (Brown et al., 2001a
), hmuTUV and an uncharacterized transporter in C. diphtheriae (Drazek et al., 2000
; Schmitt & Drazek, 2001
).
siuG is required for virulence of Strep. pyogenes in zebrafish
Competitive index studies showed that piaA in S. pneumoniae was important in both a pulmonary and a systemic murine model for disease (Brown et al., 2001a), and mice immunized with recombinant PiuA and PiaA were protected against systemic pneumococcal challenge (Brown et al., 2001b
). Likewise, the Strep. pyogenes mtsA and the Staph. aureus hts mutants were attenuated in animal infection models (Janulczyk et al., 2003
; Skaar et al., 2004
). Using a zebrafish animal model we investigated the role of siuG in disease progression by Strep. pyogenes. The zebrafish immune system has many similarities to the mammalian system (Postlethwait et al., 1998
; Trede et al., 2001
) and numerous studies have characterized its cardiovascular components (MacRae & Fishman, 2002
). Recent studies established that the zebrafish is a suitable model to investigate streptococcal infections. I.p. and i.m. injection of Strep. pyogenes HSC5 (M5 type) produced lethal infections in the fish, along with hypopigmented lesions and tissue necrosis (Miller & Neely, 2004
; Neely et al., 2002
). We used this model to investigate the role of iron acquisition in disease production and progression by Strep. pyogenes NZ131.
When zebrafish were injected i.m. with a range of 101106 c.f.u. of the wild-type NZ131, the dose response was similar to that reported for HSC5 (LD50 104 cells ml1) (Neely et al., 2002). I.m. injection of NZ131 also produced a hypopigmented lesion with extensive muscular necrosis. A control animal group, mock injected with sterile medium, showed no signs of distress. Forty hours after injection, infected zebrafish were fixed and longitudinal sections were prepared. Staining of the tissue revealed streptococcal cells arranged in clusters at the site of infection, as well as the appearance of some host immune cells (data not shown). When the fish were injected i.p. with a range of 101106 c.f.u. of the wild-type NZ131, the LD50 was higher (>105 cells ml1) than when they were injected i.m. This observation is different from the observations made with the HCS5 strain, where the LD50 in the i.p. route was lower than that in the i.m. infection (Neely et al., 2002
).
We investigated the role of the siu transporter in virulence by comparing the mutant strain to the parent strain when injected separately by both the i.m. and i.p. routes of infection. Groups of four to six zebrafish were challenged with 105 cells ml1 of Strep. pyogenes wild-type and the siuG mutant and monitored for 2 days. I.m. injection with NZ131 resulted in only 14 % survival of the fish by 48 h (Fig. 4). In the siuG mutant the ability to cause death of the fish was significantly reduced (88 % survival, P<0·0115, n=3). I.p. injection with 105 cells ml1 of Strep. pyogenes wild-type resulted in about 50 % increase in animal survival as compared to the i.m. injection. Still, the siuG was less virulent as compared to the wild-type (data not shown). These results suggest that acquisition of iron is important for Strep. pyogenes pathogenesis in the zebrafish model and that siuG function has an important role in vivo in the establishment of infection. Iron metabolism and erythroid development in zebrafish is analogous to that of higher vertebrates; zebrafish produce haem and haemoglobin, carry out haemoglobin switching during development (Brownlie et al., 2003
), use transferrin receptors (Wingert et al., 2004
) and divalent metal transporter 1 (DMT1, Donovan et al., 2002
) to transport iron into and within the cell's compartments, and employ ferroprotein 1 (Fpr1) as an intestinal and macrophage iron exporter (Donovan et al., 2000
). Therefore, it is likely that the iron acquisition mechanisms used by Strep. pyogenes during infection of zebrafish are relevant for iron acquisition during human infection.
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ACKNOWLEDGEMENTS |
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Received 28 March 2005;
revised 3 August 2005;
accepted 5 August 2005.
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