Receptor in Haemophilus influenzae Type b

Vol. 62, No. 1

INFECTION AND IMMUNITY, Jan. 1994, p. 48-59 0019-9567/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Identification and Characterization of an Iron-Regulated Hemopexin Receptor in Haemophilus influenzae Type b JOHANN C. Y. WONG,' JULIE HOLLAND,' TINA PARSONS,' ANN SMITH,2 AND PAUL WILLIAMS'* Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom,' and School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 641102 Received 18 August 1993/Returned for modification 28 September 1993/Accepted 7 October 1993

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 hemehemopexin 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 hemehemopexin 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-ammonioJ-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.

Of the many complex interactions which occur between a pathogen and its host, one common essential component of the infectious process involves the multiplication of the invading microorganism within host tissues. Such growth is critical to the establishment of an infection and depends, in part, upon the ability of the pathogen to scavenge certain essential nutrients (1, 43). For almost all pathogens, iron is an indispensable nutrient. Although there is an abundance of iron in the extracellular body fluids, the free ionic iron concentration (10-18 M), because of the presence of the iron-binding glycoproteins transferrin (in serum) and lactoferrin (on mucosal surfaces and in polymorphonuclear leucocytes), is far too low to support the growth of most microorganisms (7, 43, 44). Since most pathogens multiply successfully in body fluids and cause infection in the absence of exogenously supplied iron, they clearly possess efficient iron-scavenging systems. The best understood of these are based on low-molecularmass, high-affinity iron chelators (siderophores) and are employed by gram-negative pathogens such as Escherichia coli, Klebsiellapneumoniae, Shigella spp., Salmonella spp.,

and the pseudomonads (3, 7, 8). Siderophores are able to remove iron from transferrin, regardless of the species of transferrin. The iron-siderophore complex is internalized by the bacterial cell after recognition and binding by specific outer membrane receptor proteins. However, it has recently become apparent that not all bacteria employ siderophores. Several important human and animal pathogens, including Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, and Actinobacillus pleuropneumoniae, acquire transferrin-bound iron via a siderophore-independent, receptor-mediated process (14, 19-22, 26, 44) analogous to the iron transport systems employed by mammalian cells. For the utilization of iron-transferrin, a direct interaction between outer membrane transferrin-binding proteins and the iron-glycoprotein complex is required. The system is further distinguished from siderophore-mediated iron uptake mechanisms by its preference for the species of transferrin of its natural host. For example, humans are the only known natural reservoir for H. influenzae, which exhibits considerable specificity for iron bound to human transferrin (20, 21). Heme and some heme-protein complexes, including hemoglobin, which are abundant in the vertebrate host can also act as an alternative iron source for pathogens such as yersiniae, N. gonorrhoeae, Pleisiomonas shigelloides, and Vibrio cholerae (4, 5, 35, 36). For H. influenzae, heme serves

* Corresponding author. Phone: (0602) 515047. Fax: (0602) 515102.

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to satisfy not only the organism's need for iron but also its essential requirement for the porphyrin macrocycle (2, 24, 37, 45). This is because this microorganism lacks the enzymes of the heme biosynthetic pathway, although most strains possess the terminal ferrochelatase enzyme, which forms heme from protoporphyrin IX (PPIX) (42). However, in extracellular tissue fluids, free heme, like iron, is not readily available to an infecting microorganism. In plasma, tetrameric hemoglobin liberated by hemolysis or tissue damage dissociates into dimers which are then bound by haptoglobin and taken up into liver parenchymal cells via receptor-mediated endocytosis, and both hemoglobin and haptoglobin are catabolized (15). Depletion of haptoglobin results in the circulation of hemoglobin, which becomes oxidized to methemoglobin, from which heme dissociates. This free heme becomes available for binding by hemopexin and albumin, the major serum heme-binding proteins (27). Human albumin binds heme with one high-affinity binding site (Kd, 10 nM) and two lower-affinity binding sites (Kd, ca. 1 ,uM). Hemopexin binds heme even more avidly (Kd, jj

X

^^-

a

a

-

-

i0

97D> _W) > 66D

438

4 _7 4

457

3>345D

429

29D 1

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3

4

5

6

7

8

FIG. 9. Isolation of the H. influenzae type b strain 706705 hemopexin receptor by affinity chromatography on heme-human hemopexin-Sepharose 4B. Lanes 1-6, SDS-PAGE of the proteins isolated from bacteria grown in sBHI (lanes 1, 3, and 5) and in sBHI plus EDDA (lanes 2, 4, and 6). Lanes 1 and 2 show whole-cell envelope proteins, lanes 3 and 4 show the proteins extracted from cell envelopes following treatment with CHAPS, and lanes 5 and 6 show the proteins obtained after affinity chromatography on hemopexin-Sepharose 4B. Lanes 7 and 8, Western blots of the proteins shown in lanes 5 and 6, respectively, probed with biotinylated human heme-hemopexin. The positions of the molecular mass markers (in kilodaltons) are indicated by the open arrowheads to the left of lane 1. The closed arrowheads denote the positions and molecular masses (in kilodaltons) of the iron-regulated proteins isolated by affinity chromatography on heme-hemopexin-Sepharose 4B.

2

3

FIG. 10. Western blot of the cell envelope proteins (lane 1) and affinity-isolated hemopexin-binding proteins (lane 2) of H. influenzae type b strain 706705 grown in sBHI plus EDDA and probed with MAb T11-30. MAb T11-30 was also immobilized on protein A-agarose beads and used to purify the 57-kDa protein from a CHAPSsoluble extract prepared from 706705. Lane 3 shows a Western blot of the protein eluted from the bound MAb and probed with biotinylated heme-human hemopexin. The positions and masses (in kilodaltons) of the proteins are indicated by the arrowheads.

expression of which is not affected by the presence of excess PPIX in the iron-restricted growth medium (46). Control of hemopexin receptor expression by PPIX (and also heme) may therefore be subservient to that of iron, since PPIX starvation in iron-sufficient medium (also a heme-deficient medium) did not induce hemopexin receptor expression. Neither heme, PPIX, hemoglobin, nor transferrin competitively inhibited hemopexin in the binding assays, providing convincing evidence for the specificity of this iron-regulated receptor for heme-hemopexin. The H. influenzae human heme-hemopexin receptor is therefore distinct from the well-established transferrin receptor. However, in contrast to the hemopexin receptor on mammalian cells, the H. influenzae hemopexin receptor binds apo-hemopexin or heme-hemopexin with similar affinities. The ability of apohemopexin to bind to the receptor was confirmed by performing direct binding assays using the biotinylated apoprotein. On binding heme, hemopexin undergoes a major ligand-induced conformational change in which the hemebinding N-terminal domain (domain I) assumes a more compact shape which enhances interaction between domains I and II and renders the protein more resistant to proteolysis (27). This conformational change appears to be essential for recognition by the hemopexin receptor on liver parenchymal cells but not for recognition by the H. influenzae receptor. Presumably, regions of the bound glycoprotein which are unaffected by the heme-induced conformational change are recognized by H. influenzae. An analogous situation exists for the H. influenzae transferrin receptor, which is unable to distinguish between the different conformations presented by iron-loaded and apo transferrin (21). H. influenzae has no nonhuman hosts and shows considerable preference for human, rather than other mammalian, transferrins even though these iron-binding proteins are highly homologous (20, 21). In the case of hemopexin, both rabbit and human heme-hemopexins donate heme to H. influenzae and bind to whole cells. Rat apo- and hemehemopexins were reported not to bind to type b strains grown anaerobically in the absence of heme (10). Further work is currently under way to establish whether rat hemehemopexin donates heme via the iron-regulated hemopexin receptor reported in the present paper. A mutant of H. influenzae type b strain DM42 which is unable to make the

H. INFLUENZAE HEMOPEXIN RECEPTOR

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A

57

57

7

7

38~~~~~~3 29

29

29 2

3

4

B 16

FIG. 11. (A) SDS PAGE of the hemopexin-binding proteins of H. influenzae type b strains 706705 (lane 1), NCTC8468 (lane 2), Eagan (lane 3), and DM42 (lane 4) isolated from CHAPS-soluble extracts prepared from bacteria grown in sBHI plus EDDA by affinity chromatography on human heme-hemopexin-Sepharose 4B. The arrowheads denote the positions and molecular masses (in kilodaltons) of the iron-regulated proteins isolated by affinity chromatography. (B) Western blot of the cell envelope proteins of 706705 (lane 1), NCTC8468 (lane 2), Eagan (lane 3), and DMA2 (lane 4) prepared from bacteria grown in sBHI plus EDDA and probed with MAb Tll-30. The positions of the molecular mass markers (in kilodaltons) are indicated to the right of lane 4 by the open arrowheads. The closed arrowheads denote the positions of the 57and 30-kDa proteins recognized by MAb Tll-30.

100-kDa hemopexin receptor is nevertheless as virulent as the parent in the infant rat model (10), currently the best model of H. influenzae meningitis in humans. This suggests either that the 100-kDa protein is not needed for growth and virulence in the in vivo environment or that H. influenzae has additional mechanisms for acquiring heme during growth in vivo, perhaps involving receptors for hemoglobin, hemo-

globin-haptoglobin, or an alternative hemopexin receptor. H. influenzae 706705 did not express a 100-kDa hemopexin-binding protein after anaerobic or iron-restricted growth. Although DML2 produced the 100-kDa protein after anaerobic growth, the protein was not induced following growth in iron-restricted media. Since (i) both strains bound hemopexin after growth in iron-restricted medium and (ii) in both strains trypsin abolished this hemopexin binding, these data suggested the involvement of an alternative outer membrane protein receptor. By hemopexin-Sepharose 4B affinity chromatography, three proteins of 57, 38, and 29 kDa from cell envelopes of H. influenzae 706705 grown under

iron-restricted conditions were isolated which were absent from envelopes prepared from bacteria grown in iron-plentiful medium. Similar results were obtained for DM42 and for H. influenzae Eagan and 8468, which are known to belong to phylogenetic lineages different from that of 706705 (16, 34). On Western blots, the 57- and 29-kDa proteins renatured to

57

bind heme-hemopexin or apo-hemopexin, providing that they were solubilized in SDS-PAGE sample buffer at temperatures at or below 37°C. Furthermore, omission of 2-mercaptoethanol from the sample buffer did not influence the migration of the three proteins on SDS-PAGE gels, showing that they are not disulfide-bridge-linked subunits of a single receptor protein. These data suggest that the iron-regulated binding of hemopexin by H. influenzae is mediated through an outer membrane transport and receptor system consisting of at least three proteins. The proteins isolated by hemopexin affinity chromatography could not be differentially eluted from the resin and therefore appear to form a complex. This iron-regulated hemopexin receptor is therefore distinct from the 100-kDa hemopexin-binding protein previously described by Hanson et al. (10) and from the H. influenzae transferrin receptor, which consists of two ironregulated, outer membrane transferrin-binding proteins of around 90 and 105 kDa (34). Since we could not identify the iron-regulated hemopexin receptor directly on Western blots probed with hemopexin, we attempted to obtain MAbs directed against the receptor which would enable us to identify directly the proteins involved. We generated one MAb, T11-30, which we demonstrated to recognize shared epitopes on the 57-kDa hemopexin-binding protein and on a 30-kDa protein of strain 706705. This MAb was used to probe the 100-kDa hemopexin-binding protein of DML2. No reaction was observed, indicating that the 57-kDa protein was not derived by proteolytic breakdown of the 100-kDa protein. Furthermore, this antibody reacted with a 57-kDa protein on Western blots of cell envelopes prepared from DML2, Eagan, and 8468, strains from which hemopexin affinity chromatography yielded the 57-, 38-, and 29-kDa proteins. These data provide convincing evidence for the expression of two distinct hemopexin receptors by DIA2 and indicate that this strain shares a common iron-regulated receptor with the other H. influenzae strains examined. Interestingly, the cloned DIA2 genetic locus expressing heme-hemopexin-binding activity was reported to encode several other proteins. Linker insertion mutagenesis of this locus revealed that elimination of the expression of a 60-kDa protein exerted a negative effect on hemopexin binding (10). It is possible that the 57-kDa hemopexin-binding protein described in this work may be related to this 60-kDa protein. Hanson et al. (10) also described a mutant of DIA2 which lacks the 100-kDa hemopexin-binding protein and was unable to grow aerobically on BHI-NAD agar around wells containing heme-hemopexin. However, since the medium employed was iron sufficient and since iron availability controls the expression of the hemopexin receptor described in this paper, we presume that although DL42 possesses the iron-regulated receptor, it would not have been induced on BHI-NAD agar. Hemopexin-mediated heme uptake was first observed in vivo in studies using rats (32) and has subsequently been demonstrated to occur in isolated hepatic parenchymal cells (33) as well as in cultured human and mouse hepatoma cells (31), human polymorphonuclear leukocytes (23), HL60 human promyelocytes (40), K562 human erythroleukemic cells (39), and U937 human leukemic cells (39). This transport system involves a saturable, high-affinity receptor which is endocytosed following the binding of heme-hemopexin. Cross-linking studies have provided evidence that the murine hemopexin receptor is an 85- to 90-kDa protein composed of 65- to 70-kDa and 20-kDa disulfide-linked subunits; the 20-kDa protein appears to be part of the hemopexin-

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INFECT. IMMUN.

WONG ET AL.

binding site (29). The porcine receptor is composed of 70and 16-kDa proteins (18), whereas that from the human placenta is an 80-kDa protein (38). Either heme release from receptor-bound hemopexin may occur internally following endocytosis of the heme-hemopexin receptor complex or heme transfer may occur at or near the cell surface, since in isolated rabbit liver plasma membranes, heme is transferred to an integral membrane heme-binding protein (MHBP) having a molecular mass of around 17.5 kDa (27). Heme release is considered to occur after hemopexin undergoes a conformational change induced by binding of the hemehemopexin complexes to the receptor. While heme release is pH dependent, it nevertheless takes place in isolated liver plasma membranes and does not require an acidic pH (27). The MHBP is not the 20-kDa hemopexin receptor ,B subunit (28). Antibodies to MHBP block both hemopexin binding to the receptor and heme transport, suggesting that MHBP is, however, closely associated with the hemopexin receptor

(29). This information raises several possibilities with regard to the mechanism of heme uptake via the H. influenzae hemopexin receptor described here. Since bacteria do not appear to internalize receptor-bound transferrin (19, 44), it is unlikely that the heme-hemopexin receptor complex is internalized. It is therefore possible that the acquisition and internalization of heme from hemopexin may involve transfer of heme to a heme-binding protein at or near the cell surface. Such heme transfer would be facilitated by an acidic

pH. The 57-, 38-, and 29-kDa proteins isolated by affinity chromatography could conceivably constitute either three different hemopexin receptors or a multicomponent transport system. The 57-, and 29-kDa proteins, but not the 38-kDa protein, possess intrinsic hemopexin-binding activity and can be reversibly renatured. The 38-kDa protein may

therefore either be an accessory protein or be irreversibly denatured by treatment with SDS. Interestingly, Lee (17) has recently reported the isolation of a surface-located, iron-regulated outer membrane heme-binding protein of 39.5 kDa from H. influenzae type b; iron-regulated periplasmic proteins of 40 and 31 kDa have also been observed (12). In addition, H. influenzae grown in heme-depleted media induces proteins of 38 and 43 kDa which are repressed during growth in heme-sufficient media (2, 37). It is therefore tempting to speculate that the 38-kDa protein isolated in the present study is analogous to the 39.5-kDa heme-binding protein and is involved either in the removal of heme from hemopexin bound to the 57- or 29-kDa protein or in the transport of heme across the outer membrane. Alternatively, heme may be transferred from the hemopexin receptor to a 51-kDa heme-binding lipoprotein, which is located on the periplasmic side of either the cytoplasmic or outer mem-

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20.

nature and contribution of each protein to the acquisition

21.

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ACKNOWLEDGMENTS This work was supported in part by grants from the National

Meningitis Trust (to P.W.) and from the National Institutes of Health (DK37463) (to A.S.), which are gratefully acknowledged.

We thank G. Denton for assistance with production of monoclonal antibodies.

23.

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