Phase Variation of Hemoglobin Utilization in Neisseria gonorrhoeae

Most Neisseria gonorrhoeae isolates are unable to use human hemoglobin as the sole source of iron for growth (Hgb⁻), but a minor population is able to do so (Hgb⁺). This minor population grows luxuriously on hemoglobin, expresses two outer membrane proteins of 42 kDa (HpuA) and 89 kDa (HpuB), and binds hemoglobin under iron-stressed conditions. In addition to the previously reported HpuB, we identified and characterized HpuA, which is encoded by the gene hpuA, located immediately upstream of hpuB. Expression of both proteins was found to be controlled at the translational level by frameshift mutations in a run of guanine residues within the hpuA sequence encoding the mature HpuA protein. The “on-phase” hemoglobin-utilizing variants contained 10 G’s, while the “off-phase” variants contained 9 G’s. Insertional hpuB mutants of FA19 Hgb⁺ and FA1090 Hgb⁺ no longer expressed HpuB but still produced HpuA. A polar insertional mutation of the upstream hpuA gene in FA1090 Hgb⁺ eliminated production of both HpuA and HpuB, whereas a nonpolar insertional mutant expressed HpuB only. Insertional mutagenesis of either hpuA or hpuB or both substantially decreased the hemoglobin binding ability of the FA1090 Hgb⁺ variant and prevented growth on hemoglobin plates. Therefore, both HpuA and HpuB were required for the utilization of hemoglobin for growth.     

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.     

Iron-regulated outer membrane protein of Bacteroides fragilis involved in heme uptake.

Under iron starvation, Bacteroides fragilis expresses various iron-regulated outer membrane proteins. In this study, a deferrated minimal medium was used in growth experiments, and the role of one of these iron-regulated outer membrane proteins (a 44-kDa protein) in an iron uptake mechanism which acquires iron from heme compounds was elucidated. When a specific 44-kDa protein antiserum was used in a medium with heme as the only iron source, growth inhibition was observed. These results demonstrate that the 44-kDa outer membrane protein plays an important role in the uptake of heme in B. fragilis.     

Expression of the CopB outer membrane protein by Moraxella catarrhalis is regulated by iron and affects iron acquisition from transferrin and lactoferrin.

The amino acid sequence of the cell-surface-exposed, 81-kDa CopB outer membrane protein of Moraxella catarrhalis was found to be similar to those of TonB-dependent outer membrane proteins of other gram-negative bacteria. Expression of CopB was affected by the availability of iron in the growth medium, and the extent of overexpression of CopB in response to iron limitation varied widely among the M. catarrhalis strains tested. Wild-type M. catarrhalis strains were found to be able to utilize ferric citrate, transferrin, lactoferrin, and heme as sources of iron for growth in vitro. However, an isogenic copB mutant was severely impaired in its ability to utilize transferrin and lactoferrin as sole sources of iron for growth, whereas this same mutant grew similarly to the wild-type parent strain when supplied with ferric citrate as the iron source. The copB mutant was not significantly different from its wild-type parent strain in its ability to bind transferrin and lactoferrin. In addition, the wild-type parent strain and the copB mutant exhibited equivalent rates of uptake of 55Fe from ferric citrate. However, the copB mutant was markedly less able than the wild-type strain to take up 55Fe from transferrin and lactoferrin. These results indicate that lack of expression of the CopB protein exerts a direct or indirect effect on the ability of M. catarrhalis to utilize iron bound to certain carrier proteins.     

Characterization of the hgbA locus encoding a hemoglobin receptor from Haemophilus ducreyi.

Haemophilus ducreyi can bind hemoglobin and use it as a source of heme, for which it has an obligate requirement. We previously identified and purified HgbA, a hemoglobin-binding outer membrane protein from H. ducreyi. In this report, we describe the molecular cloning, expression, DNA sequence, and mutagenesis of the structural gene for HgbA, hgbA. H. ducreyi and recombinant Escherichia coli expressing hgbA bound [125I]hemoglobin, establishing HgbA as a receptor. Insertions or deletions in the cloned hgbA gene abolished expression of HgbA and hemoglobin binding in E. coli. Mutagenesis of H. ducreyi by allelic exchange of insertions into hgbA abolished its ability to bind [125I]hemoglobin or utilize hemoglobin as a source of heme. The deduced protein sequence was similar to those of the TonB-dependent family of outer membrane receptors. The most similar member was HutA (heme receptor) from Vibrio cholerae. Tbp1 and Lbp1 (transferrin and lactoferrin receptors, respectively, from pathogenic Neisseria spp.) also showed very significant homology. Thus, by characterizing the hgbA locus, this work elucidates a potentially important role of HgbA in obtaining heme and/or iron from the host.     

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.     

A pleiotropic iron-uptake mutant of Neisseria meningitidis lacks a 70-kilodalton iron-regulated protein.

We isolated an iron-uptake mutant of Neisseria meningitidis M986-NCV-1 that was severely limited in the ability to use several sources of iron in the form of Fe3+. This mutant, FAM11, grew poorly or not at all with human transferrin (TF) or lactoferrin (LF) as the sole iron source in a defined medium but grew as well as wild-type meningococci with hemin or hemoglobin. Uptake of 55Fe bound to TF, LF, dicitrate complexes, aerobactin, or nitrilotriacetate was reduced to 0 to 4% of the wild-type level. FAM11 did not produce an iron-repressible outer membrane protein (FeRP) of 70 kilodaltons (kDa) found in membranes of iron-stressed M986-NCV-1. Western blot (immunoblot) analysis using rabbit antiserum against this protein revealed that at least 17 of 18 meningococcal and 10 of 14 gonococcal strains produced an FeRP of ca. 70 kDa. The 70-kDa FeRP was shown to be surface exposed by radioimmunoprecipitation with human immune sera. These data suggest that the 70-kDa FeRP is somehow involved in Fe uptake from TF and LF. However, we were unable to transform the iron-uptake phenotype from FAM11 into wild-type meningococci to confirm this. Revertants of FAM11 that grew with TF and LF did not regain the ability to make the 70-kDa FeRP but also did not completely regain the Fe-uptake phenotype of M986-NCV-1.     

Identification of a transferrin-binding protein from Borrelia burgdorferi.

Bacterial pathogens have evolved various strategies to acquire iron from the iron-restricted environment found in mammalian hosts. Borrelia burgdorferi should be no different with regard to its requirement for ferric iron, and previous studies have suggested that transferrin (Tf) may be a source of iron in vivo. By probing blots with Tf conjugated to horseradish peroxidase, we have identified an outer membrane protein (28 kDa) from B. burgdorferi B31 that bound holo-Tf but not apo-Tf. The 28-kDa protein bound human, rat, or mouse Tf and was produced only by low-passage (less than passage 5), virulent isolates of strain B31. In addition, the Tf-binding protein (Tbp) from strain B31 retained the ability to bind Tf after treatment with 2% sodium dodecyl sulfate-1% beta-mercaptoethanol and heating to 100 degrees C for 5 min. These properties are remarkably similar to those of the Tbp of Staphylococcus aureus and Tbp2 from Neisseria meningitidis. B. burgdorferi Sh-2-82 produced an outer membrane protein different in size, i.e., 26 kDa, but with properties similar to those of to the protein from strain B31, suggesting variation in B. burgdorferi Tbps. The exact role of the 28-kDa protein in iron acquisition by B. burgdorferi remains to be determined.     

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.     

Binding and accumulation of hemin in Neisseria gonorrhoeae.

The ability to utilize hemin and hemin-containing compounds for nutritional iron (Fe) uptake has been documented for several pathogenic bacteria. Neisseria gonorrhoeae can utilize free hemin as a source of Fe for growth; however, little is known concerning the mechanisms involved in hemin transport. In this study we have characterized the binding and accumulation of hemin by N. gonorrhoeae and defined the specificity of the gonococcal hemin receptor. N. gonorrhoeae F62 was grown in a chemically defined medium containing the iron chelator Desferal, and hemin transport was initiated by the addition of [59Fe]hemin (4.0 or 8.0 microM; specific activity, 7.0 Ci/mol). 59Fe uptake from radiolabeled hemin by N. gonorrhoeae was energy dependent, and 59Fe was shown to accumulate in the cell at a constant rate during logarithmic growth. However, we observed a decrease in the uptake of 59Fe from radiolabeled hemin when inorganic iron was present in the growth medium. Binding of 59Fe from radiolabeled hemin was inhibited by the addition of either cold hemin, hematoporphyrin, or hemoglobin, but not by ferric citrate. Although [14C]hemin was found to support the growth of N. gonorrhoeae, we did not detect the uptake of 14C from radiolabeled hemin. Extraction of the gonococcal periplasmic ferric binding protein (Fbp) from cultures grown with [59Fe]hemin indicated that a majority of the 59Fe was associated with the Fbp. Taken together, the results presented here indicate that hemin binds to a gonococcal outer membrane receptor through the protoporphyrin portion of the molecule and that following binding, iron is removed and transported into the cell, where it is associated with the gonococcal periplasmic ferric binding protein, Fbp.     

Monoclonal antibodies against the 70-kilodalton iron-regulated protein of Neisseria meningitidis are bactericidal and strain specific.

When grown under iron limitation, Neisseria meningitidis expresses a number of outer membrane proteins (OMPs), one of which is a 70-kilodalton (kDa) major OMP. After immunization of mice with outer membrane preparations of iron-depleted cells of strain H44/76 (B:15:P1.7,16), hybridoma cell lines producing monoclonal antibodies against the 70-kDa OMP were obtained. Some of these monoclonal antibodies demonstrated strong bactericidal activity against the homologous strain H44/76 in the presence of human complement, suggesting potential application of the 70-kDa OMP as a vaccine component. However, none of the 10 selected monoclonal antibodies was able to recognize the corresponding protein from five heterologous strains of various serosubtyping characteristics. A polyclonal anti-70-kDa OMP serum also did not react with the other strains. This result shows that immunodominant surface-exposed epitopes of the meningococcal 70-kDa iron-limitation-inducible OMP are strain specific.     

Molecular characterization of FrpB, the 70-kilodalton iron-regulated outer membrane protein of Neisseria meningitidis.

The structural gene encoding the 70-kDa outer membrane protein FrpB of Neisseria meningitidis was cloned and sequenced. A mutant lacking FrpB was constructed. No difference in iron utilization between the mutant and the parental strain was observed. A minor effect of the mutation on serum resistance was observed. A topology model for FrpB in the outer membrane is proposed.     

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.     

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.     

Pyochelin-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein.

An iron-regulated outer membrane protein of 75,000 daltons was strongly expressed following iron limitation of strains of Pseudomonas aeruginosa which fail to produce pyoverdine. A mutant nonderepressible for this protein (K372) was deficient in pyochelin-mediated iron transport at 150 nM FeCl3, consistent with a role for the 75-kDa protein in ferripyochelin transport. Moreover, ferripyochelin specifically protected the 75-kDa protein against trypsin digestion, supporting an interaction between ferripyochelin and the 75-kDa protein. Previous reports implicated a 14,000-dalton outer membrane protein as the receptor for ferripyochelin (P.A. Sokol and D.E. Woods, Infect. Immun. 40:665-669, 1983) and demonstrated that a mutant (FBP-28) expressing a defective 14-kDa outer membrane protein did not exhibit pyochelin-mediated iron transport (P.A. Sokol, J. Bacteriol. 169:3365-3368, 1987). Nonetheless, we were able to demonstrate (i) that FBP-28 was inducible for the 75-kDa protein under iron-limiting conditions and (ii) that concomitant with the induction of this protein in FBP-28, pyochelin-mediated iron uptake at 150 nM FeCl3 was observed. Interestingly, strain K372 did transport ferripyochelin at higher (750 nM) FeCl3 concentrations, suggesting that a second pyochelin-mediated iron transport system, perhaps involving the 14-kDa outer membrane protein identified previously, operates in P. aeruginosa.     

Molecular characterization of the 98-kilodalton iron-regulated outer membrane protein of Neisseria meningitidis.

When grown under iron limitation, Neisseria meningitidis expresses several additional outer membrane proteins (OMPs), which were studied to assess their vaccine potential. Two monoclonal antibodies were obtained against a 98-kDa OMP of strain 2996 (B:2b:P1.2). Cross-reactivity studies revealed that the two antibodies reacted with 44 and 42 of 74 meningococcal strains, respectively. The antibodies did not block the binding of transferrin or lactoferrin to intact cells. The structural gene for the protein, tentatively designated iroA, was isolated and sequenced. Computer analysis revealed homology to the ferric siderophore receptors in the outer membrane of Escherichia coli and to gonococcal transferrin-binding protein 1 (TbpA). The high degree of cross-reactivity and the results of Southern blot analyses, which showed that the iroA gene is also present in strains that did not react with the monoclonal antibodies, suggest that the 98-kDa OMP is well conserved among meningococci and that it is a suitable vaccine candidate. However, the antibodies were not bactericidal in an in vitro assay with human complement.     

The major anaerobically induced outer membrane protein of Neisseria gonorrhoeae, Pan 1, is a lipoprotein.

Pan 1 is an acidic outer membrane protein of Neisseria gonorrhoeae that is expressed only when gonococci are grown anaerobically. On silver-stained sodium dodecyl sulfate-polyacrylamide gels, Pan 1 migrates as an intense but diffuse 54-kDa protein. The deduced amino acid sequence of Pan 1 from the aniA (anaerobically induced protein) open reading frame reveals a lipoprotein consensus sequence, Ala-Leu-Ala-Ala-Cys, and a processed molecular mass of 39 kDa. Furthermore, there is strong homology at the N terminus and C terminus of Pan 1 to the termini of the gonococcal outer membrane lipoproteins Lip and Laz. [3H]palmitic acid labeling of gonococci grown under oxygen-limited conditions demonstrated specific incorporation of label into Pan 1, suggesting further that Pan 1 is a lipoprotein.     

Identification and characterization of the human lactoferrin-binding protein from Neisseria meningitidis.

Lactoferrin-binding activity in Neisseria meningitidis was detected by a solid-phase binding assay with horseradish peroxidase-conjugated human lactoferrin (HRP-lactoferrin). Expression of lactoferrin-binding activity was regulated by the level of iron in the medium, so that growth in the presence of the iron chelator EDDA (ethylenediamine di-ortho-hydroxyphenylacetic acid) resulted in a greater than 350-fold increase in binding activity, which was reversed by addition of excess iron. A maximal level of expression could be obtained at reasonable culture densities by using either intermediate levels of EDDA or high levels of EDDA and moderate levels of complexed iron sources such as hemoglobin and transferrin. Competition binding assays demonstrated that the binding of lactoferrin was specific for human lactoferrin in that neither bovine lactoferrin, human transferrin, nor human hemoglobin was able to block binding of HRP-lactoferrin. The binding specificity for human lactoferrin correlated with growth studies in which human but not bovine lactoferrin could support the growth of iron-starved cells. Binding of lactoferrin was not dependent on its level of iron saturation, since iron-saturated lactoferrin and apolactoferrin were equally effective at blocking binding of HRP-lactoferrin in competitive binding assays. The lactoferrin-binding protein was identified as a 105,000-molecular-weight iron-regulated outer membrane protein in three different meningococcal strains by a batch affinity method with biotinylated human lactoferrin and streptavidin-agarose.     

Immunogenicity and antigenic heterogeneity of a human transferrin-binding protein in Neisseria meningitidis.

Growing Neisseria meningitidis on an iron restriction medium induces the synthesis of new outer membrane proteins, some of them true iron-regulated outer membrane proteins (IROMPs) and others synthesized because of the stress produced by the iron restriction. Some of these proteins are antigenic and can be considered for the development of vaccines; this is especially desirable in the case of N. meningitidis serogroup B, for which polysaccharide vaccines are not efficient. The antigenicity of N. meningitidis 37- and 70-kDa IROMPs has been studied previously; in this work, we studied the immunogenicity and antigenic heterogeneity of another IROMP, the human transferrin-binding protein 2 (TBP2), which seems to be indispensable for meningococcal growth inside the host. Mice were inoculated with purified outer membrane vesicles (blebs) from 5 selected N. meningitidis strains, and the five serum samples obtained were analyzed for anti-TBP2 antibodies by using the homologous strain and for cross-reactivity with the TBP2 of the 4 other selected strains and another 35 heterologous N. meningitidis strains. The TBP2s of the 5 strains tested were all immunogenic in mice to various degrees depending on the strain, and all five TBP2s shared one or more epitopes with heterologous strains (as shown by the cross-reactivities of the five serum samples), although the number of cross-reacting strains was very variable, ranging from 2 for strain V002 to 35 for strain P391. This suggests that the TBP2 epitopes of different strains differ in nature or in their accessibility to the immune system. Under the iron restriction conditions used, all strains synthesized a non-TBP2 antigenic 56-kDa protein thought to be a stress protein.