Hemin-binding Protein Research Articles

Isolation and Partial Characterization of Hemin-binding Cell Envelope Proteins from Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens

Isolation and Partial Characterization of Hemin-binding Cell Envelope Proteins from <i>Porphyromonas gingivalis</i>, <i>Prevotella intermedia</i>, and <i>Prevotella nigrescens</i>

Identification of a Hemin Utilization Protein of Moraxella catarrhalis (HumA)

Moraxella catarrhalis is a major cause of acute otitis media in young children and has also been implicated as an important cause of exacerbations in adults with underlying pulmonary disease. Due to the considerable level of antibiotic resistance and the high degree of carriage rates in young children, it is likely that the incidence of M. catarrhalis infections will continue to rise. M. catarrhalis is a strict human respiratory pathogen, and this bacterium uses both transferrin and lactoferrin receptors to fulfill the essential iron requirement for survival in vivo. However, these are the only described iron acquisition systems for this organism. In this report we have demonstrated that M. catarrhalis can also utilize hemin as a sole source of iron for growth. In addition, we have identified and characterized an outer membrane protein with homology (26 to 28% similarity) to other known hemin binding and uptake proteins in related gram-negative organisms (i.e., Bordetella and Yersinia spp.). This newly described M. catarrhalis protein, termed HumA, is capable of directly binding to hemin coupled to a solid-phase matrix. M. catarrhalis HumA expressed on the surface of an Escherichia coli hemA-deficient strain (K-12 EB53) is fully capable of complementing the defect and thus restoring the ability of this strain to grow in the presence of hemin. When M. catarrhalis is grown in the presence of hemin, HumA expression is clearly increased as shown by Western blotting with polyclonal antiserum developed against a HumA peptide. In addition, growth analyses revealed that a HumA-deficient mutant of M. catarrhalis (7169::humA) is restricted for growth in the presence of hemin as the sole iron source compared to the wild-type strain. We conclude that HumA is an essential component of a hemin uptake and utilization system previously undescribed for M. catarrhalis, thus providing another mechanism of iron acquisition that may facilitate persistent colonization of the mucosal surface.

A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis

It has been known that Porphyromonas gingivalis has an obligate requirement for hemin or selected heme- or Fe-containing compounds for its growth. In addition, the influence of hemin on the expression of several putative virulence factors produced by this bacterium has also been recently documented; however, the mechanisms involved in hemin uptake are poorly defined. We succeeded in cloning the gene coding for the 35-kDa protein, which was specifically expressed in P. gingivalis and seemed to confer colonizing activities. Recently, we have constructed the P. gingivalis 381 mutant defective in the 35-kDa protein by insertion mutagenesis. The beige mutant exhibited little co-aggregation and the virulence was also decreased. Based on these results and homology search analysis, we focused on assessing the hemin bindings and found the heme regulatory motif (HRM) as a hemin direct binding site. The 35-kDa protein did possess the binding ability of selected protoporphyrins involving the hemin. These results demonstrated that 35-kDa protein is one of the hemin binding proteins in P. gingivalis and suggested that hemin binding ability of 35-kDa protein is important for the expression of virulence in P. gingivalis.

Construction and analysis of hemin binding protein mutants in the oral pathogen Treponema denticola

Treponema denticola, a periodontal pathogen, can use hemin as its sole iron source. The organism synthesizes two low-iron-induced outer-membrane hemin-binding proteins, HbpA and HbpB. To characterize genetically the function of these two novel proteins, standard recombinant DNA procedures and electroporation were used to construct T. denticola strains in which the genomic copies of either hbpA or both hbpA and hbpB were interrupted with an erythromycin resistance cassette. Northern blot and RT-PCR analyses verified that the normal hbpA transcripts were missing in both mutants. The hbpA mutation also had a polar effect on the transcription of hbpB and thus neither mutant strain transcribes the downstream hbpB gene. The parental and hbp mutant strains had similar growth properties in normal media, but the mutants reached a lower cell density than parental cells in iron-restricted media. The results indicate that HbpA and/or HbpB are required for efficient iron utilization but that there is an additional system that can help T. denticola acquire iron. The growth defect of the mutants was totally restored by lactoferrin but only partially restored by adding exogenous hemin or inorganic iron. Thus, hbpA and/or hbpB specifically facilitate hemin and iron utilization under low iron conditions and are presumably important for T. denticola virulence in the host environment.

Cloning and expression of two novel hemin binding protein genes from Treponema denticola.

Treponema denticola does not appear to produce siderophores, so it must acquire iron by other pathways. Indeed, T. denticola has been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding ability. To characterize the HbpA protein, its gene was cloned from genomic DNA libraries of T. denticola. Sequence analysis of the hbpA open reading frame indicated that it encoded a 42.8-kDa protein with a 23-amino-acid signal peptide. HbpA has no significant homology to any proteins in the databases. Southern blot analysis demonstrated that hbpA is present in several T. denticola ATCC strains and clinical isolates, but not in Treponema pectinovorum, Treponema socranskii, or Escherichia coli. HbpA, expressed as a recombinant protein in E. coli and purified by antibody affinity chromatography, has hemin binding activity as determined by lithium dodecyl sulfate-polyacrylamide gel electrophoresis with tetramethylbenzidine staining. Northern blot analysis showed that there were two hbpA-containing transcripts, of approximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced. Interestingly, the 2.6-kb mRNA also encoded a second protein with significant homology to hbpA. This downstream gene, called hbpB, was cloned and sequenced and its product was expressed as a fusion protein in E. coli. The hbpB gene product is 49% identical to HbpA and binds hemin. Thus, T. denticola has two novel hemin binding proteins which may be part of a previously unrecognized iron acquisition pathway.

Hemin-binding surface protein from Bartonella quintana.

Bartonella quintana, the agent of trench fever and a cause of endocarditis and bacillary angiomatosis in humans, has the highest reported in vitro hemin requirement for any bacterium. We determined that eight membrane-associated proteins from B. quintana bind hemin and that a approximately 25-kDa protein (HbpA) was the dominant hemin-binding protein. Like many outer membrane proteins, HbpA partitions to the detergent phase of a Triton X-114 extract of the cell and is heat modifiable, displaying an apparent molecular mass shift from approximately 25 to 30 kDa when solubilized at 100 degrees C. Immunoblots of purified outer and inner membranes and immunoelectron microscopy with whole cells show that HbpA is strictly located in the outer membrane and surface exposed, respectively. The N-terminal sequence of mature HbpA was determined and used to clone the HbpA-encoding gene (hbpA) from a lambda genomic library. The hbpA gene is 816 bp in length, encoding a predicted immature protein of approximately 29.3 kDa and a mature protein of 27.1 kDa. A Fur box homolog with 53% identity to the Escherichia coli Fur consensus is located upstream of hbpA and may be involved in regulating expression. BLAST searches indicate that the closest homologs to HbpA include the Bartonella henselae phage-associated membrane protein, Pap31 (58.4% identity), and the OMP31 porin from Brucella melitensis (31.7% identity). High-stringency Southern blots indicate that all five pathogenic Bartonella spp. possess hbpA homologs. Recombinant HbpA can bind hemin in vitro; however, it does not confer a hemin-binding phenotype upon E. coli. Intact B. quintana treated with purified anti-HbpA Fab fragments show a significant (P < 0.004) dose-dependent decrease in hemin binding relative to controls, suggesting that HbpA plays an active role in hemin acquisition and therefore pathogenesis. HbpA is the first potential virulence determinant characterized from B. quintana.

Isolation and Characterization of a Putative Hemin-binding Protein from Prevotella intermedia

Iron is essential for the growth, reproduction, and survival of all prokaryotic and eukaryotic species1-3). Iron has also been shown to be essential for the initiation and progression of several infectious diseases1). In order to survive therefore, prokaryotes and eukaryotes have devised unique mechanisms to compete for, and sequester the small amounts of iron which are available to them. Eukaryotic cells compete for iron by synthesizing large number of high-affinity iron-binding proteins (i.e., transferrin, lactoferrin), hemoglobin-binding protein (haptoglobin), heme-binding proteins (hemopexin, albumin), and iron-storage proteins (ferritin, hemosiderin) which are capable of competing for, and storing iron1,4-8). Therefore, in order for host-associated bacteria to survive, they must be capable of competing with the host’s large number of highaffinity iron-binding proteins. Prokaryotic cells compete with eukaryotes for this iron by employing two mechanisms: synthesizing their own high-affinity binding and transport proteins, siderophores, and outer membrane associated iron-binding protein. The siderophores are low molecular weight proteins, which bind iron by chelating it from the host, and transferring it to the specific outer membrane receptors which then transport it directly into the cell9). Several Gram-negative bacteria contain, in addition to, or in place of siderophores, iron (hemin) binding proteins in their outer membrane1021). These outer membrane-associated iron-binding proteins are capable of binding iron directly, and transporting it across the outer membrane into the cell. For the most part, these outer membrane expressed iron-binding proteins are expressed during iron stress, and are formed in response to very low levels of iron in the environment. While there are several studies which have dealt with the hemin-binding and hemin-regulated proteins in the oral pathogen, Porphyromonas gingivalis20,22-25), there is no such studies which has studied hemin-binding in Prevotella intermedia. In the study presented here, we have identified, purified, and characterized a putative hemin-binding protein in Prevotella intermedia.

Kinetics of hemin distribution in plasma reveals its role in lipoprotein oxidation

Hemin is a powerful in vitro inducer of low-density lipoprotein (LDL) oxidation, implicated in development of atherosclerosis. To support the proposed role of hemin in atherogenesis, the question of whether hemin has any chance of getting together with LDL in vivo, must be addressed. A stopped-flow technique was employed in order to investigate the fast kinetics of hemin binding to LDL and to other plasma hemin-binding proteins: high-density lipoprotein (HDL), albumin and hemopexin. Based on the measured rate constants of hemin association with and dissociation from each of these proteins, time-dependent hemin distribution in plasma was analyzed. The analysis shows that as much as 80% of total hemin binds initially to LDL and HDL, the plasma components which are most susceptible to oxidation. Only then hemin partially transfers to the antioxidants albumin and hemopexin. The half time of the hemin–LDL complex in plasma, initially comprising 27% of total hemin, was more than 20 s. Not only transient, but also oxidatively active steady-state hemin–lipoprotein complexes in plasma were both predicted from the kinetic analysis and found in experiment. Our data suggest that the hemin–LDL complex may exist in vivo and that its oxidative potential should be considered pro-atherogenic.

Characterization of the Gene for the Hemin-Binding Protein from Porphyromonas Gingivalis

Characterization of the Gene for the Hemin-Binding Protein from <i>Porphyromonas Gingivalis</i>

Hemin regulation of hemoglobin binding by Porphyromonas gingivalis.

Hemoglobin binding to chemostat-grown hemin-excess and hemin-limited cells of Porphyromonas gingivalis W50, and to cells of the avirulent, beige-pigmenting variant W50/BE1, was quantified. Hemin-excess W50 bound more hemoglobin than hemin-limited W50, mirroring the hemin-binding ability of these cells [Microb Ecol Health Dis 7:9-15, 1994]. In contrast to hemin, hemoglobin binding was not enhanced by sodium dithionite. The hemoglobin-binding capacity of hemin-excess W50/BE1 was below that of hemin-limited W50 and only observed under oxidizing conditions. Scatchard analysis revealed similar affinity constants for hemin-excess and hemin-limited W50, and confirmed a lower binding maximum for the latter. Hemin-excess W50/BE1 displayed cooperative binding of hemoglobin. These differences in binding were reflected in the binding of a horse radish peroxidase-conjugated hemoglobin (HHRPO) in a dot-blot assay. However, neither the 32-kDa hemin-binding protein, nor its 19-kDa heat-modified form, from either hemin-limited W50 or hemin-excess W50/BE1, bound this conjugate. These data indicate that hemoglobin binding by P. gingivalis is hemin-regulated and occurs via a mechanism different from hemin binding.

Inhibition of nuclear envelope reconstitution in Xenopus interphase egg extract by hemin.

Addition of hemin to the nuclear reconstitution system of Xenopus interphase egg extract using sperm head chromatin resulted in abnormal pseudonuclei exhibiting flattened membrane patches randomly distributed both on the surface and inside the nuclei. The structures that resembled nuclear pores were observed on these flattened membrane patch structures. Although the nucleosome structure was formed as revealed by the micrococcal nuclease digestion, the B-type lamin uptake into the nuclei was inhibited by hemin. Using heminagarose affinity chromatography, we isolated several hemin-binding proteins from fully reconstituted pseudonuclei. Some of the hemin-binding proteins bound concanavalin A (Con A). Comparison of hemin-binding proteins with those isolated from both fractions of supernatant and pellet separated by high speed centrifugation of the egg extract showed that the hemin-binding proteins of pseudonuclei were supplied from both fractions. The uptake of nuclear hemin-binding proteins did not occur in the incompletely reconstituted nuclei resulting from addition of excess sperm chromatin to the system. These results suggest that the hemin-binding proteins participate in the late steps of nuclear reconstitution during formation of the nuclear envelope.

A monoclonal antibody directed against the 97-kilodalton gonococcal hemin-binding protein inhibits hemin utilization by Neisseria gonorrhoeae.

Neisseria gonorrhoeae expresses two hemin-binding proteins (HmBPs) of 97,000 and 44,000 in molecular weight. A murine monoclonal antibody (MAb) produced against the 97-kDa HmBP from N. gonorrhoeae PID543 specifically inhibited in a concentration-dependent manner the ability of hemin to promote growth. The anti-97-kDa HmBP MAb competitively inhibited binding of the 97-kDa HmBP to a hemin-agarose affinity column. In Western immunoblots, the MAb recognized the 97-kDa homologs from a limited survey of clinical gonococcal isolates. These results support the contention that the 97-kDa HmBP is involved in the gonococcal hemin acquisition pathway.

Characterization of hemin binding activity of Streptococcus pneumoniae.

Streptococcus pneumoniae is a causative agent of bacterial pneumonia, otitis media, meningitis, and bacteremia. It causes considerable morbidity and mortality throughout the world, especially among children, the elderly, and immunocompromised individuals. We have demonstrated previously that the growth of S. pneumoniae is limited under iron-depleted conditions and can be restored by the addition of either hemin or hemoglobin. In the present study, we showed that S. pneumoniae had the ability to bind hemin and that the level of hemin binding activity was not affected by supplementation of the growth medium with iron. Approximately 70 to 80% of the hemin binding activity was mediated by proteinase-resistant components, and the remainder was mediated by proteins. Hemin binding proteins were located in both soluble extract and envelope fractions of pneumococcal cells. By batch affinity chromatography, a major hemin binding polypeptide with an apparent molecular mass of 43 kDa was identified in the cell lysate of S. pneumoniae. Polyclonal antibodies against this polypeptide were raised. By immunoblot analysis, this hemin binding polypeptide was localized in the envelope and did not exhibit any variation in molecular weight among all serotypes tested. The subcellular distribution of hemin binding activity may have functional implications.

Immunologic Epitope, Gene, and Immunity Involved in Pneumococcal Glycoconjugate

Pneumococcal infection persists as a major cause of pneumonia, otitis media, and meningitis in infants. Children less than 2 years of age show the highest incidence of pneumococcal diseases.Production of monoclonal antibody (MAb) to polysaccharide (PS) and binding characteristics to PS epitopes were studied. Removal of the O-acetyl group from 9V PS by alkali hydrolysis resulted in a decreased binding with rabbit 9V antiserum (AS). However, the binding reaction with 9V MAb was less affected by the loss of O-acetyl content. Type 9V IgG MAb provided passive protection and enhanced the opsonophagocytic activity of polymorphonuclear (PMN) leukocytes to kill type 9V pneumococci.The pathogenecity of pneumococci is attributed to various virulence factors distributed on the cell surface, including capsular polysaccharide and protein antigens, for example, pneumolysin, autolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesion (PsaA), and hemin binding protein. Some of these protein antigens may be used as a component to combine with pneumococcal PS vaccine or as a carrier of conjugate vaccine.Clinical trials of pneumococcal conjugate vaccines showed that covalent linkage of capsular PS to protein carriers improved the immunogenicity of the PS. Development of glycoconjugate vaccine for selected pneumococcal types will help solve the problem of poor immunogenecity of PS vaccine in young children used for prevention of pneumococcal infection.

Iron acquisition by oral hemolytic spirochetes: isolation of a hemin-binding protein and identification of iron reductase activity.

Oral anaerobic spirochetes (OAS) have been implicated in the etiology of periodontal disease. To adapt to the environment of the subgingiva, OAS must be able to acquire iron from limited sources. OAS have previously been shown not to produce siderophores but are beta-hemolytic and can bind hemin via a proteinaceous 47-kDa outer membrane sheath (OMS) receptor. Present studies show that [3H]hemin is not transported into the cytoplasm, that hemin and ferric ammonium citrate, as the sole iron sources, can support the growth of OAS and that protoporphyrin IX and Congo red are inhibitory, thereby implying an important in vivo role for hemin as an iron source. Treponema denticola ATCC 35405 produces an iron reductase. The iron reductase can reduce the central ferric iron moiety of hemin. The 47-kDa OMS hemin-binding protein has been purified to apparent homogeneity by methanol-chloroform extraction of cellular lipoproteins and the use of a hemin-agarose bead affinity column. A model of iron acquisition by OAS is presented.

Isolation and characterization of a hemin-binding cell envelope protein from Porphyromonas gingivalis

A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is both hemin and iron regulated was identified and purified in Porphyromonas gingivalis381. A strong hemin-binding function was found by LDS-PAGE and TMBZ staining when cells were grown under hemin (iron)-limited conditions. N-terminal amino acid sequence analysis of CNBr-digested 24 kDa hemin binding protein revealed that this protein belongs to a new, so far undescribed hemin-binding class of proteins.

Lipoprotein e(P4) is essential for hemin uptake by Haemophilus influenzae.

Heme uptake is a common means of iron and porphyrin acquisition by many pathogenic bacteria. The genus Haemophilus includes several important pathogenic bacterial species that characteristically require hemin-, protoporphyrin-, or heme-substituted proteins as essential growth factors under aerobic conditions. However, the mechanism of heme transport is not understood for Haemophilus. We have cloned a DNA fragment from H. influenzae that allows an Escherichia coli hemA mutant to employ exogenous hemin or protoporphyrin IX as sole sources of porphyrin. DNA sequencing of the cloned DNA fragment suggested that a previously characterized gene (hel) encoding an antigenic, outer membrane lipoprotein e(P4) was responsible for the complementation activity. Construction of hel insertion mutations in strain H. influenzae Rd demonstrated that hel is essential for growth under aerobic conditions but not under anaerobic conditions. The aerobic growth defect of hel mutants could be reversed by providing exogenous hemin in the presence of outer membrane. The analysis of hybrids between e(P4) and beta-lactamase demonstrated that a domain of e(P4) near its NH2' terminus was required for its function in hemin use. Within this domain is a short amino acid sequence that displays similarity to H. influenzae hemin binding protein HbpA, hemin-binding motifs present in eukaryotic transcription activator heme-activated protein, and the heme containing proteins hemoglobin (alpha-chain) and cytochrome C3, suggesting that this region may be involved in hemin binding and/or transport.

Utilization of hemin and hemoglobin as iron sources by Vibrio parahaemolyticus and identification of an iron-repressible hemin-binding protein

Several clinical isolates of Vibrio parahaemolyticus were examined for their ability to utilize either hemin or hemoglobin as a sole source of iron. Both compounds appeared to be equally good iron sources. Maximum growth was obtained at 5 μM hemin or 1.25 μM hemoglobin under the conditions tested. Using a hemin-agarose batch affinity method, the hemin-binding protein was isolated from crude total membranes of a hemin-utilizing strain, WP1, grown under iron-deficient but not under iron-sufficient conditions. This protein was identical to the 83 kDa outer membrane protein which was expressed in response to iron limitation. The protein was susceptible to proteinase K cleavage in whole cells, indicating its exposure at the cell surface. Hemin and hemoglobin, but not protoporphyrin IX, inhibited binding of the protein to hemin-agarose.

Utilization of hemin and hemoglobin as iron sources by Vibrio parahaemolyticus and identification of an iron-repressible hemin-binding protein.

Several clinical isolates of Vibrio parahaemolyticus were examined for their ability to utilize either hemin or hemoglobin as a sole source of iron. Both compounds appeared to be equally good iron sources. Maximum growth was obtained at 5 microM hemin or 1.25 microM hemoglobin under the conditions tested. Using a hemin-agarose batch affinity method, the hemin-binding protein was isolated from crude total membranes of a hemin-utilizing strain, WP1, grown under iron-deficient but not under iron-sufficient conditions. This protein was identical to the 83 kDa outer membrane protein which was expressed in response to iron limitation. The protein was susceptible to proteinase K cleavage in whole cells, indicating its exposure at the cell surface. Hemin and hemoglobin, but not protoporphyrin IX, inhibited binding of the protein to hemin-agarose.

Cloning and expression of hemolysin genes from Treponema denticola strains ATCC 35404 (TD-4) and human clinical isolate GM-1 in Escherichia coli

The oral spirochete, Treponema denticola is a putative etiologic agent in adult periodontitis, and acute necrotizing ulcerative gingivitis. In vitro , the oral treponeme produces several factors including proteases, hemolysins, hemin-binding proteins, which could potentially be involved in the virulence of this spirochete. Our laboratory has been investigating the pathobiology of T. denticola, and has demonstrated the production of several hemolysins by T. denticola. In this report two hemolysin genes from T. denticola strains ATCC 35404 (TD-4) and GM-1 were isolated by screening genomic DNA libraries of T. denticola on sheep blood agar plates. Physical maps of the insert fragments were not identical. Southern blot analyses suggested some degree of homology in the nucleotide sequence. Maxicell analyses of [35S]-methionine-labeled polypeptides from the recombinant plasmids have suggested the synthesis of an approximately 62.5 kDa polypeptide. Biochemical characterization of the T. denticola hemolysin genes indicated the activity to be inhibited by Mg2+, Ca2+ and Zn2+ but not by EDTA. Dithiothreitol and glutathione moderately enhanced the hemolytic activity of the recombinant plasmids. Iron partially inhibited the hemolytic activities. Addition of 2-2′ bipyridyl moderately enhanced the activities, possibly by iron limitation. These results suggest the isolation of an identical hemolysin gene from T. denticola strains TD-4 and GM-1.