Iron acquisition by Haemophilus influenzae.

The mechanisms for acquisition of iron by Haemophilus influenzae and their role in pathogenesis are not known. Heme and nonheme sources of iron were evaluated for their effect on growth of type b and nontypable strains of H. influenzae in an iron-restricted, defined medium. All 13 strains acquired iron from heme, hemoglobin, hemoglobin-haptoglobin, and heme-hemopexin. Among nonheme sources of protein-bound iron, growth of H. influenzae was enhanced by partially saturated human transferrin but not by lactoferrin or ferritin. Purified ferrienterochelin and ferridesferrioxamine failed to provide iron to H. influenzae, and the supernatants of H. influenzae E1a grown in iron-restricted medium failed to enhance iron-restricted growth of siderophore-dependent strains of Escherichia coli, Salmonella typhimurium, and Arthrobacter terregens. Marked alterations in the profile of outer membrane proteins of H. influenzae were observed when the level of free iron was varied between 1 microM and 1 mM. Catechols were not detected in the supernatants of strain E1a; however, iron-related hydroxamate production was detected by two biochemical assays. We conclude that the sources of iron for H. influenzae are diverse. The significance of hydroxamate production and iron-related outer membrane proteins to H. influenzae iron acquisition is not yet clear.     

Involvement of HxuC outer membrane protein in utilization of hemoglobin by Haemophilus influenzae

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable Haemophilus influenzae (NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobin-haptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic hgbA hgbB hgbC mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the hxuC gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by H. influenzae.     

Effect of multiple mutations in the hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae type b.

Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA and hgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from the hgpA hgpB double mutant. We have cloned and sequenced the gene encoding this third hemoglobin/hemoglobin-haptoglobin binding protein and designate it hgpC. Insertional mutation of hgpC did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An hgpA hgpB hgpC triple mutant constructed by insertional mutagenesis showed a reduced ability to use the hemoglobin-haptoglobin complex but was unaltered in the ability to use hemoglobin. A second class of mutants was constructed in which the entire structural gene of each of the three proteins was deleted. The hgpA hgpB hgpC complete-deletion triple mutant was unable to utilize the hemoglobin-haptoglobin complex and showed a reduced ability to use hemoglobin. We have identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in Haemophilus influenzae. Any one of the three proteins is sufficient to support growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilization. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.     

Identification and characterization of an iron-regulated hemopexin receptor in Haemophilus influenzae type b.

Heme can serve Haemophilus influenzae as a source of both essential porphyrin and iron. In extracellular mammalian body fluids neither free heme nor free iron is available, since they are tightly bound to hemopexin and transferrin, respectively. Since H. influenzae grows in the presence of iron-transferrin and heme-hemopexin and is known to express a saturable receptor for transferrin, we investigated the process by which this pathogen acquired heme from hemopexin for use as an iron source. The ability of human and rabbit hemopexin to donate heme as a source of iron to H. influenzae type b strains was demonstrated by plate bioassays. With a dot enzyme assay with biotinylated hemopexin as ligand, H. influenzae bound heme-hemopexin and apo-hemopexin following growth in iron-restricted, but not in iron-sufficient, medium. Competitive binding studies with heme-hemopexin and apo-hemopexin demonstrated saturability of binding. Neither heme, protoporphyrin IX, hemoglobin, nor transferrin blocked the binding of hemopexin to whole cells, demonstrating the specificity of binding. Treatment of whole H. influenzae cells with trypsin abolished binding. Taken together, these observations suggest that H. influenzae type b expresses an outer membrane protein(s) which acts as a receptor for hemopexin and which is regulated by the availability of iron in the growth medium. In iron-restricted media, H. influenzae 706705 and DL42 did not express the 100-kDa hemopexin-binding protein previously reported (M.S. Hanson, S.E. Pelzel, J. Latimer, U. Muller-Eberhard, and E.J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). The putative iron-regulated hemopexin receptor was solubilized from cell envelopes of H. influenzae 706705, DL42, and Eagan with the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and isolated by affinity chromatography on heme-hemopexin-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins bound to the affinity resin revealed three proteins of 29, 38, and 57 kDa, of which the 57- and 29-kDa proteins bound hemopexin after Western blotting (immunoblotting). A monoclonal antibody to the 57-kDa hemopexin-binding protein of 706705 recognized a 57-kDa protein on Western blots of the cell envelope proteins of 706705, DL42, and Eagan; no reaction was observed with the 100-kDa hemopexin-binding protein of DL42. These data suggest that some H. influenzae strains possess at least two hemopexin receptors, the expression of which is determined by the prevailing growth environment.     

Reduced severity of middle ear infection caused by nontypeable Haemophilus influenzae lacking the hemoglobin/hemoglobin-haptoglobin binding proteins (Hgp) in a chinchilla model of otitis media

Since Haemophilus influenzae lacks enzymes necessary for synthesis of the porphyrin ring, it has an absolute growth requirement for a porphyrin source. This requirement can be satisfied in vitro by hemoglobin and hemoglobin complexed to haptoglobin. The products of the hgp genes mediate the utilization of heme from hemoglobin-haptoglobin. These genes are also involved in the use of heme from hemoglobin, although additional gene products independently mediate the acquisition of heme from this substrate. Different strains of H. influenzae possess one to four hgp genes. A nontypeable H. influenzae mutant lacking all the hgp genes was constructed and compared to the wild-type strain in a chinchilla (Chinchilla lanigera) model of otitis media. Compared to the wild-type strain, the hgp-deficient mutant exhibited a significantly delayed onset of detectable middle ear infection and significantly reduced duration of infection as assessed by both video otoscopy and tympanometry and as evidenced by viable bacterial counts in middle ear effusions. In addition, the maximum bacterial load in the middle ears of chinchillas infected with the mutant strain was significantly reduced when compared to the parent. These data indicate that the hemoglobin/hemoglobin-haptoglobin binding proteins are required for bacterial proliferation during H. influenzae-induced otitis media in chinchillas.     

Effects of serum carrier proteins on the growth of pathogenic neisseriae with heme-bound iron.

The pathogenic neisseriae can use free heme and hemoglobin as an essential source of iron (Fe) for growth in vitro, but it is unknown whether they can utilize heme bound to human hemopexin or to human serum albumin, or hemoglobin bound to haptoglobin. We found that neither Neisseria meningitidis nor Neisseria gonorrhoeae used bound heme, but bound hemoglobin was used as an Fe source by two meningococcal strains and one gonococcal strain. A second gonococcal strain, previously shown to use free hemoglobin poorly or not at all, also did not grow with hemoglobin-haptoglobin complex as an Fe source. These observations suggest that hemoglobin might act as an Fe source in vivo for many pathogenic neisseriae even when in complexed (bound) form, but heme probably would not support growth in vivo if bound to serum carrier proteins.     

Functional characterization of HgbB,a new hemoglobin binding protein of Pasteurella multocida

The biological function and role in pathogenesis of a Pasteurella multocida A:1 strain hemoglobin binding protein was investigated. The hgbB gene from the P. multocida A:1 strain, VP161, was cloned and characterized. hgbB was 2991 bp in length and encoded a mature length protein of 111 kDa. HgbB was predicted to be an outer membrane protein and shared 68 and 69% similarity to the hemoglobin/hemoglobin-haptoglobin binding protein, HI0712 from Haemophilus influenzae Rd and HgpC, from H. influenzae b, respectively. HgbB exhibited features typical of TonB dependent receptors, including seven conserved regions typical of these proteins, and conserved invariant residues. Escherichia coli expressing recombinant HgbB was found to bind hemoglobin in a solid phase dot blot binding assay. However, when a truncated form of the protein was expressed in E. coli, cells could no longer bind hemoglobin. Insertional inactivation of hgbB did not affect the ability of P. multocida to bind hemoglobin, nor its ability to produce disease in a mouse model. In addition, recombinant HgbB did not confer any protection against homologous or heterologous challenge.     

Iron acquisition and hemolysin production by Campylobacter jejuni.

Campylobacter jejuni strains were tested for their ability to acquire iron from various iron sources present in humans. Hemin, hemoglobin, hemin-hemopexin, and hemoglobin-haptoglobin stimulated the growth of C. jejuni strains in low-iron medium. Transferrin, lactoferrin, and ferritin were unable to provide iron to the strains tested. Derivatives of the naturally transformable C. jejuni strain 81-176 were isolated on the basis of their inability to use hemin as an iron source. These mutants were also unable to use hemoglobin, hemin-hemopexin, or hemoglobin-haptoglobin as iron sources. Some mutants lacked a 71,000-Da iron-regulated outer membrane protein, while others appeared to retain all of their outer membrane proteins. Growth curves and a recombination experiment that exploited natural transformation were used to further characterize the mutants. A hemolytic activity was shown to be produced by several C. jejuni strains, but it did not appear to be iron regulated.     

Complex role of hemoglobin and hemoglobin-haptoglobin binding proteins in Haemophilus influenzae virulence in the infant rat model of invasive infection

Haemophilus influenzae requires an exogenous heme source for aerobic growth in vitro. Hemoglobin or hemoglobin-haptoglobin satisfies this requirement. Heme acquisition from hemoglobin-haptoglobin is mediated by proteins encoded by hgp genes. Both Hgps and additional proteins, including those encoded by the hxu operon, provide independent pathways for hemoglobin utilization. Recently we showed that deletion of the set of three hgp genes from a nontypeable strain (86-028NP) of H. influenzae attenuated virulence in the chinchilla otitis media model of noninvasive disease. The present study was undertaken to investigate the role of the hgp genes in virulence of the wild-type serotype b clinical isolate HI689 in the infant rat model of hematogenous meningitis, an established model of invasive disease requiring aerobic growth. Bacteremia of high titer and long duration (>14 days) and histopathologically confirmed meningitis occurred in >95% of infant rats challenged at 5 days of age with strain HI689. While mutations disrupting either the Hgp- or Hxu-mediated pathway of heme acquisition had no effect on virulence in infant rats, an isogenic mutant deficient for both pathways was unable to sustain bacteremia or produce meningitis. In contrast, mutations disrupting either pathway decreased the limited ability of H. influenzae to initiate and sustain bacteremia in weanling rats. Biochemical and growth studies also indicated that infant rat plasma contains multiple heme sources that change with age. Taken together, these data indicate that both the hgp genes and the hxuC gene are virulence determinants in the rat model of human invasive disease.     

Identification of an outer membrane protein involved in utilization of hemoglobin-haptoglobin complexes by nontypeable Haemophilus influenzae.

A recombinant plasmid containing a 6.5-kb fragment of nontypeable Haemophilus influenzae (NTHI) chromosomal DNA was shown to confer a hemoglobin-haptoglobin-binding phenotype on Escherichia coli. Use of a mini-Tn10kan transposon for random insertion mutagenesis of this recombinant plasmid allowed localization of the NTHI DNA responsible for this hemoglobin-haptoglobin-binding phenotype to a 3.5-kb PstI-XhoI fragment within the 6.5-kb NTHI DNA insert. When this mutagenized NTHI DNA fragment was used to transform the wild-type NTHI strain, the resultant kanamycin-resistant mutant exhibited significantly decreased abilities to bind hemoglobin-haptoglobin and utilize it as a source of heme for aerobic growth in vitro. This mutant also lacked expression of a 115-kDa outer membrane protein that was present in the wild-type parent strain. Transformation of this mutant with wild-type NTHI chromosomal DNA restored the abilities to bind and utilize hemoglobin-haptoglobin and to express the 115-kDa outer membrane protein. Nucleotide sequence analysis of the relevant NTHI DNA revealed the presence of a gene, designated hhuA, that encoded a predicted 117,145-Da protein. The HhuA protein exhibited features typical of a TonB-dependent outer membrane receptor and had significant identity with the hemoglobin receptors of both Haemophilus ducreyi and Neisseria meningitidis.     

Detection of phase variation in expression of proteins involved in hemoglobin and hemoglobin-haptoglobin binding by nontypeable Haemophilus influenzae

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study (I. Maciver, J. L. Latimer, H. H. Liem, U. Muller-Eberhard, Z. Hrkal, and E. J. Hansen. Infect. Immun. 64:3703-3712, 1996) indicated that nontypeable H. influenzae (NTHI) strain TN106 expressed a protein that bound hemoglobin-haptoglobin and was encoded by an open reading frame (ORF) that contained a CCAA nucleotide repeat. Southern blot analysis revealed that several NTHI strains contained between three and five chromosomal DNA fragments that bound an oligonucleotide probe for CCAA repeats. Three ORFs containing CCAA repeats were identified in NTHI strain N182; two of these ORFs were arranged in tandem. The use of translational fusions involving these three ORFs and the beta-lactamase gene from pBR322 revealed that these three ORFs, designated hgbA, hgbB, and hgbC, encoded proteins that could bind hemoglobin, hemoglobin-haptoglobin, or both compounds. Monoclonal antibodies (MAbs) specific for the HgbA, HgbB, and HgbC proteins were produced by immunizing mice with synthetic peptides unique to each protein. Both HgbA and HgbB were readily detected by Western blot analysis in N182 cells grown in the presence of hemoglobin as the sole source of heme, whereas expression of HgbC was found to be much less abundant than that of HgbA and HgbB. The use of these MAbs in a colony blot radioimmunoassay analysis revealed that expression of both HgbA and HgbB was subject to phase variation. PCR and nucleotide sequence analysis were used in conjunction with Western blot analyses to demonstrate that this phase variation involved the CCAA repeats in the hgbA and hgbB ORFs.     

Passively released heme from hemoglobin and myoglobin is a potential source of nutrient iron for Bordetella bronchiseptica

Colonization by Bordetella bronchiseptica results in a variety of inflammatory respiratory infections, including canine kennel cough, porcine atrophic rhinitis, and a whooping cough-like disease in humans. For successful colonization, B. bronchiseptica must acquire iron (Fe) from the infected host. A vast amount of Fe within the host is sequestered within heme, a metalloporphyrin which is coordinately bound in hemoglobin and myoglobin. Utilization of hemoglobin and myoglobin as sources of nutrient Fe by B. bronchiseptica requires expression of BhuR, an outer membrane protein. We hypothesize that hemin is acquired by B. bronchiseptica in a BhuR-dependent manner after spontaneous loss of the metalloporphyrin from hemoglobin and/or myoglobin. Sequestration experiments demonstrated that direct contact with hemoglobin or myoglobin was not required to support growth of B. bronchiseptica in an Fe-limiting environment. Mutant myoglobins, each exhibiting a different affinity for heme, were employed to demonstrate that the rate of growth of B. bronchiseptica was directly correlated with the rate at which heme was lost from the hemoprotein. Finally, Escherichia coli cells expressing recombinant BhuR had the capacity to remove hemin from solution. Collectively, these experiments provided strong experimental support for the model that BhuR is a hemin receptor and B. bronchiseptica likely acquires heme during infection after passive loss of the metalloporphyrin from hemoglobin and/or myoglobin. These results also suggest that spontaneous hemin loss by hemoglobin and myoglobin may be a common mechanism by which many pathogenic bacteria acquire heme and heme-bound Fe.     

Identification and purification of a conserved heme-regulated hemoglobin-binding outer membrane protein from Haemophilus ducreyi.

A hemoglobin-binding protein (HgbA) from Haemophilus ducreyi was identified and purified. The 100-kDa HgbA was detected in all strains of H. ducreyi tested, and a somewhat larger hemoglobin-binding protein was found in one strain of Haemophilus influenzae. HgbA was purified and the amino acid sequence of the N terminus of HgbA revealed no significant homologies with known proteins. Two different antisera to HgbA from H. ducreyi 35000 recognized HgbA proteins from all tested H. ducreyi strains; they did not recognize proteins from the H. influenzae strain. Expression of HgbA was regulated by the level of heme but not by iron present in the medium. Animal species of hemoglobin competed with iodinated human hemoglobin for binding to whole cells of H. ducreyi and supported the growth of H. ducreyi. The lack of immunological cross-reactivity and the differences in hemoglobin specificities between the H. ducreyi and the H. influenzae hemoglobin-binding proteins suggest that they are unrelated.     

Identification and purification of a hemoglobin-binding outer membrane protein from Neisseria gonorrhoeae.

The majority of in vitro-grown Neisseria gonorrhoeae strains were unable to use hemoglobin as the sole source of iron for growth (Hgb-), but a minor population was able to do so (Hgb+). The ability of Hgb+ gonococci to utilize hemoglobin as the iron source was associated with the expression of an iron-repressible 89-kDa hemoglobin-binding protein in the outer membrane. The N-terminal amino acid sequence of this protein revealed amino acids, from positions 2 to 16, identical to those of HpuB, an 85 kDa iron-regulated hemoglobin-haptoglobin utilization outer membrane protein of Neisseria meningitidis. Isogenic mutants constructed by allelic replacement with a meningococcal hpu::mini-Tn3erm construct no longer expressed the 89-kDa protein. Mutants could not utilize hemoglobin to support growth but still grew on heme. Thus, the gonococcal HpuB homolog is a functional hemoglobin receptor and is essential for growth with hemoglobin.     

A functional tonB gene is required for both utilization of heme and virulence expression by Haemophilus influenzae type b.

Haemophilus influenzae is nearly unique among facultatively anaerobic bacteria in its absolute requirement for exogenously supplied heme for aerobic growth. In this study, a mutant analysis strategy was used to facilitate identification of H. influenzae cell envelope components involved in the uptake of heme. Chemical mutagenesis was employed to produce a mutant of a nontypeable H. influenzae strain unable to utilize either protein-bound forms of heme or low levels of free heme. This mutant was transformed with a plasmid shuttle vector-based genomic library constructed from the same wild-type nontypeable H. influenzae strain, and a growth selection technique was used to obtain a recombinant clone that could utilize heme. Analysis of the DNA insert in the recombinant plasmid revealed the presence of several open reading frames, one of which encoded a 28-kDa protein with significant similarity to the TonB protein of Escherichia coli. This H. influenzae gene product was able to complement a tonB mutation in E. coli, allowing the E. coli tonB mutant to form single colonies on minimal medium containing vitamin B12. When this H. influenzae gene was inactivated by insertional mutagenesis techniques and introduced into the chromosome of wild-type strains of H. influenzae type b, the resultant transformants lost their abilities to utilize heme and produce invasive disease in an animal model. Genetic restoration of the ability to express this TonB homolog resulted in the simultaneous acquisition of both heme utilization ability and virulence. These results indicate that the H. influenzae TonB protein is required not only for heme utilization by this pathogen in vitro, but also for virulence of H. influenzae type b in an animal model.     

hgpB, a Gene Encoding a Second Haemophilus influenzae Hemoglobin- and Hemoglobin-Haptoglobin-Binding Protein

Haemophilus influenzae requires heme for growth and can utilize both hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified a hemoglobin- and hemoglobin-haptoglobin-binding protein, HgpA, in H. influenzae HI689. Mutation of hgpA did not affect binding or utilization of either heme source. The hgpA mutant exhibited loss of a 120-kDa protein and increased expression of a 115-kDa protein. These data suggested that at least one other gene product is involved in binding of these heme sources by H. influenzae. A 3.2-kbp PCR product derived from HI689 was cloned. The nucleotide sequence indicated a separate, distinct gene with high homology to hgpA, which would encode a 115-kDa protein. Primers were designed for directional cloning of the structural gene in the correct reading frame. Sonicates of induced Escherichia coli harboring the cloned open reading frame bound both hemoglobin and hemoglobin-haptoglobin. An insertion/deletion mutant of H. influenzae at the newly identified locus, designated hgpB, was constructed. The 115-kDa protein was not detected in the mutant after affinity purification using biotinylated hemoglobin. An hgpA hgpB double-mutant strain exhibited a reduced ability to utilize hemoglobin-haptoglobin, although it was unaltered in the ability to utilize hemoglobin. Affinity isolation of hemoglobin-binding proteins from the double mutant resulted in isolation of an approximately 120-kDa protein. Internal peptide sequencing revealed this protein to be a third distinct protein, highly homologous to HgpA and HgpB. In summary a second hemoglobin- and hemoglobin-haptoglobin-binding protein of H. influenzae has been identified and characterized, and the presence of an additional protein of similar function has been revealed.     

Outer membrane protein DsrA is the major fibronectin-binding determinant of Haemophilus ducreyi

The ability to bind extracellular matrix proteins is a critical virulence determinant for skin pathogens. Haemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, binds extracellular matrix components, including fibronectin (FN). We investigated H. ducreyi FN binding and report several important findings about this interaction. First, FN binding by H. ducreyi was greatly increased in bacteria grown on heme and almost completely inhibited by hemoglobin. Second, wild-type strain 35000HP bound significantly more FN than did a dsrA mutant in two different FN binding assays. Third, the expression of dsrA in the dsrA mutant restored FN binding and conferred the ability to bind FN to a non-FN-binding Haemophilus influenzae strain. Fourth, an anti-DsrA monoclonal antibody partially blocked FN binding by H. ducreyi. The hemoglobin receptor, the collagen-binding protein, the H. ducreyi lectin, the fine-tangle pili, and the outer membrane protein OmpA2 were not involved in H. ducreyi FN binding, since single mutants bound FN as well as the parent strain did. However, the major outer membrane protein may have a minor role in FN binding by H. ducreyi, since a double dsrA momp mutant bound less FN than did the single dsrA mutant. Finally, despite major sequence differences, DsrA proteins from both class I and class II H. ducreyi strains mediated FN and vitronectin binding. We concluded that DsrA is the major factor involved in FN binding by both classes of H. ducreyi strains.     

Identification and characterization of a Streptococcus pyogenes operon involved in binding of hemoproteins and acquisition of iron

The hemolytic Streptococcus pyogenes can use a variety of heme compounds as an iron source. In this study, we investigate hemoprotein utilization by S. pyogenes. We demonstrate that surface proteins contribute to the binding of hemoproteins to S. pyogenes. We identify an ABC transporter from the iron complex family named sia for streptococcal iron acquisition, which consists of a lipoprotein (siaA), membrane permease (siaB), and ATPase (siaC). The sia transporter is part of a highly conserved, iron regulated, 10-gene operon. SiaA, which was localized to the cell membrane, could specifically bind hemoglobin. The operon's first gene encodes a novel bacterial protein that bound hemoglobin, myoglobin, heme-albumin, and hemoglobin-haptoglobin (but not apo-haptoglobin) and therefore was named Shr, for streptococcal hemoprotein receptor. PhoZ fusion and Western blot analysis showed that Shr has a leader peptide and is found in both membrane-bound and soluble forms. An M1 SF370 strain with a polar mutation in shr was more resistant to streptonigrin and hydrogen peroxide, suggesting decreased iron uptake. The addition of hemoglobin to the culture medium increased cell resistance to hydrogen peroxide in SF370 but not in the mutant, implying the sia operon may be involved in hemoglobin-dependent resistance to oxidative stress. The shr mutant demonstrated reduced hemoglobin binding, though cell growth in iron-depleted medium supplemented with hemoglobin, whole blood, or ferric citrate was not affected, suggesting additional systems are involved in hemoglobin utilization. SiaA and Shr are the first hemoprotein receptors identified in S. pyogenes; their possible role in iron capture is discussed.     

Molecular Characterization of the Hemin Uptake Locus (hmu) from Yersinia pestis and Analysis of hmu Mutants for Hemin and Hemoprotein Utilization

Sequence analysis of the hemin uptake locus (hmu) of Yersinia pestis revealed five genes, hmuRSTUV, required for use of hemin and hemoproteins as iron sources. The translated gene products have homologies with proteins of the hemin transport genes of several gram-negative bacteria. Promoters were identified upstream of hmuP'R (p1) and upstream of hmuS (p2); p1, which contains a Fur box, is regulated by iron and Fur, while p2 exhibits weak, but constitutive, activity. HmuR, which has homology with TonB-dependent outer membrane (OM) receptors, is localized to the OM of Y. pestis and is required for utilizing hemin and all hemoproteins under iron-depleted conditions. The proposed ABC transporter, HmuTUV, is necessary for use of hemin, hemin-albumin, and myoglobin, but not hemoglobin, hemoglobin-haptoglobin, or heme-hemopexin, as iron sources. In the absence of HmuTUV, HmuS, a cytoplasmic protein, is involved in use of hemoglobin and heme-hemopexin. In mice, the 50% lethal doses of Y. pestis DeltahmuP'RSTUV mutants injected subcutaneously or retro-orbitally did not differ from that of the Hmu(+) parent strain. Thus, the hmu system is not essential for infection in mice via these routes. Growth studies showed that a DeltahmuP'RSTUV mutant could grow in iron-depleted medium containing high concentrations of hemoglobin, suggesting that an Hmu-independent, lower-affinity hemoglobin uptake system may exist.     

Acquisition of iron from host proteins by the group A streptococcus.

To identify mammalian iron-binding proteins that can serve as iron sources for Streptococcus pyogenes, the group A streptococcus (GAS), we used a plate assay. Ferritin, hemin, hemoglobin, myoglobin, and catalase can support growth of GAS on iron-depleted medium. However, growth was not detected when iron was provided as iron-saturated transferrin or lactoferrin or bound to cytochrome c. Therefore, it appears that GAS can use the intracellular iron sources available in the human body, which is consistent with its ability to cause tissue destruction during infection.