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MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Mar. 2004, p. 154171 Vol. 68, No. 11092-2172/04/$08.000 DOI: 10.1128/MMBR.68.1.154171.2004Copyright 2004, American Society for Microbiology. All Rights Reserved.

Iron Transport Systems in Neisseria meningitidisDonna Perkins-Balding,* Melanie Ratliff-Griffin, and Igor Stojiljkovic

Rollins Research Center, Department of Microbiology and Immunology,

Emory University School of Medicine, Atlanta, Georgia 30322

INTRODUCTION ................ ................. ................. .................. ................. ................. .................. ................. ..............154IRON AVAILABILITY IN THE HUMAN HOST...................................................................................................155IRON ACQUISITION MECHANISMS...................................................................................................................156

Transferrin and Lactoferrin Receptors ...............................................................................................................156Genetic arrangement and regulation by Fur ................. ................. .................. ................. ................. ............157Structure predictions..........................................................................................................................................158Structure and functional relationships............................................................................................................158Energy from the TonB-ExBD system ................ ................. ................. .................. ................. .................. ........159Transport through the periplasm.....................................................................................................................159Ferric iron in the cytoplasm..............................................................................................................................160

Hemoglobin Receptor HMBR ...............................................................................................................................160Genetic arrangement and regulation by Fur ................. ................. .................. ................. ................. ............161

Structure predictions..........................................................................................................................................161Structure and functional relationships............................................................................................................161Energy from the TonB-ExbBD system ................. ................. ................. .................. ................. .................. .....161Transport through the periplasm.....................................................................................................................162Heme in the cytoplasm.......................................................................................................................................162

Haptoglobin-Hemoglobin Receptor HPUAB ................ .................. ................. ................. .................. .................163Genetic arrangement and regulation by Fur ................. ................. .................. ................. ................. ............163Structure predictions..........................................................................................................................................163Structure and functional relationships............................................................................................................164Energy from the TonB-ExbBD system ................. ................. ................. .................. ................. .................. .....165Transport through the periplasm.....................................................................................................................165

Other Potential Iron Transporters.......................................................................................................................165 Vaccine Potential and Trials.................................................................................................................................165

CONCLUDING REMARKS......................................................................................................................................166 ACKNOWLEDGMENTS ...........................................................................................................................................167

REFERENCES ............... .................. ................. ................. .................. ................. .................. ................. ................. ..167

INTRODUCTION

Success, in terms of bacterial survival inside the host, de-pends on an organisms ability to scavenge essential nutrients.Acquisition of iron and iron complexes has long been recog-nized as a major determinant in the pathogenesis of Neisseriameningitidis. This ability to acquire iron under iron-restrictedconditions has undoubtedly contributed to its success as one ofthe leading causes of bacterial meningitis in children andhealthy adults in the world.

N. meningitidis is the organism responsible for two serious

human diseases, pyogenic meningitis and meningococcal sep-ticemia. These diseases are secondary infections of the menin-ges, skin, and other body parts, resulting from systemic spreadof meningococci (10). The progression of illness begins withexposure, followed by carriage or colonization, with an occa-sional advance to the disease state. The carriage rate in normalhealthy adults is roughly 10%, and several factors, including

the virulence of the strain, host susceptibility, and environmen-tal influences play a role in the development of invasive disease(10). Despite a low risk of contracting meningococcal disease,it remains a serious health problem worldwide. The prevalenceof meningococcal disease varies with geographical location, with endemic disease occurring in developed countries andepidemic disease predominating in developing countries, suchas the region of sub-Saharan Africa (100, 147). Three factorscontribute to its ominous reputation; these include a high mor-tality rate, a rapid onset of severe disease symptoms, and thepotential for epidemic spread.

Research has well established the dependence of N. menin-gitidis growth on iron availability. There are numerous exam-ples of pathogenic bacteria, including N. meningitidis, whosevirulence is enhanced in experimental infections by the injec-tion of iron compounds into the animal host (see reference 65for a review). The importance of iron uptake for virulence in N.meningitidis was first proposed by Payne and Finkelstein (110).Shortly thereafter, the detrimental effects of iron deprivationon the growth of N. meningitidis were described by Archibaldand DeVoe (14). These effects included both reduced replica-tion and reduced respiration rates. Iron uptake was also shownto be a crucial factor in meningococcal infection by Holbein etal., who injected iron compounds (iron dextran or human fer-

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Emory School of Medicine, Rollins ResearchCenter, 1510 Clifton Rd., Rm. 3152, Atlanta, GA 30322. Phone: (404)727-5968. Fax: (404) 727-3659. E-mail: [email protected].

D.P.-B. and M.R.-G. dedicate this paper to the memory of IgorStojiljkovic (1959-2003).

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ritransferrin) into mice and challenged them with a subsequentintraperitoneal inoculation of live N. meningitidis (6971). Thisresulted in lethal infection, whereas mice injected with only N.meningitidis suffered a transient bacteremia and recoveredquickly. Iron-starved meningococci grown in vitro were alsofound to have increased virulence in vivo (34). A hypoferremicresponse, which reduces the amount of free transferrin by asmuch as 70%, helps control meningococcal infection in mice(and humans) (168). However, this response is ineffective if theiron concentration is artificially raised as in Holbeins experi-ments (70, 168).

IRON AVAILABILITY IN THE HUMAN HOST

The majority of iron in the body is stored intracellularly inferritin (up to 4,500 Fe3 atoms per ferritin molecule) andhemoglobin (57). Extracellular iron found in body fluids isattached to high-affinity iron-binding proteins, such as trans-ferrin in serum and lymph and lactoferrin in milk and secre-tions. In order for N. meningitidis to cause disease, it has to

survive in various environments within the host, including theoropharynx, bloodstream, and cerebrospinal fluid, which con-tain various forms and concentrations of free and complexediron. Of these environments, the cerebrospinal fluid has thehighest content of free iron, at 2.2 mol/liter (57). The majorsource of iron available to the meningococci, therefore, is ironcomplexed to host iron-binding proteins. These host iron-bind-ing proteins have high binding constants for iron, rangingaround 1036, and they are usually only 30 to 40% saturated.This correlates to virtually zero free iron for bacterial growth(67). N. meningitidis has managed to overcome this iron limi-tation through the evolution of iron acquisition systems whichenable it to use transferrin, lactoferrin, hemoglobin, and hap-

toglobin-hemoglobin as iron sources (55, 95, 96, 145).The majority of transferrin is synthesized in the liver and

circulates in human serum, functioning to sequester iron. Thisprevents both the damaging effects of free iron and bacterialgrowth (57). Transferrin can bind ferric iron at locations ofabsorption and storage and transport it to locations of utiliza-tion. Mammalian cells receive iron from transferrin through areceptor-mediated process involving the internalization oftransferrin and the release of iron in endosomal compart-ments.

Human transferrin is a monomeric 80-kDa bilobed glyco-protein having a ferric ion-binding site and bicarbonate anion-binding site in each lobe (57, 145, 167). All members of the

transferrin family have the bilobed structure, with each lobesubdivided into two / domains that are separated by a deepcleft that contains the iron-binding site. The proposed mech-anism of iron binding has come from crystal studies of rabbitserum transferrin (16). Four amino acids (D63, Y95, Y188, andH249) and two oxygen atoms from the bicarbonate anion func-tion to coordinate the ferric ion in the binding pocket (16, 57).The binding of the anion is believed to neutralize the chargesaround the site that might repel the ferric ion and to providethe remainder of the six ligands needed for binding (see ref-erence 57 for a review). The mechanism of iron release fromtransferrin has not been described, but it is believed to involvea conformational change mediated by a hinge-like region thatis located behind the iron-binding site and between the two

domains. Domain opening may be promoted by a specific re-ceptor protein, reduction of Fe3 to Fe2, or lowered pH (16).

Transferrin is not an abundant source of iron on mucosalsurfaces, although, transferrin receptors have been found in allclinical isolates ofN. meningitidis. A transferrin receptor-defi-cient gonococcal strain is avirulent in human model studies,

while the parental strain is virulent (47). This demonstrates theimportance of the transferrin receptor in Neisseria gonorrhoeaeinfection and indicates that the genitourinary mucosal surfacehas enough transferrin to support bacterial growth. Becausethe most abundant source of transferrin is human serum, thissubstrate is likely to be an important source of iron during theinvasive phase of meningococcal infection. However, studies

with the gonococcus suggest that transferrin may be an impor-tant source of iron on mucosal surfaces as well.

Lactoferrin, a member of the transferrin family of proteins,is found mostly in phagocytic cells and in secretions such asmilk, mucus, and tears. Neutrophils actively release lactoferrinat sites of infection (139). The structural and functional fea-tures of lactoferrin are very similar to those of transferrin,

including its tight yet reversible binding to ferric iron, a con-served three-dimensional structure, and almost identical iron-binding sites (7, 17, 150). The sequence identity between lac-toferrins and transferrins is roughly 60% (17). There are,however, unique differences between these related proteins,including a higher affinity of lactoferrin for iron (especiallyevident at low pH) and bactericidal activity associated withlactoferrin (17). Lactoferrin also has some additional proper-ties that are derived from its surface characteristics, includingthe ability to bind anionic molecules such as DNA, heparin,and glycosaminoglycans. It can also bind to certain cells andmodulate their activity, act proteolytically on certain sub-strates, and enter cells via nonconventional routes (16). All

meningococcal strains tested have been shown to utilize lacto-ferrin, and supplemental lactoferrin can enhance the virulenceof meningococcal infection in mice (95, 140). Utilization oflactoferrin is not essential for causing gonococcal colonizationor invasive disease (47). However, recent studies of humangonococcal infection indicate that in the absence of the trans-ferrin receptor, expression of the lactoferrin receptor is suffi-cient for initiating infection (8). In addition, the expression ofboth transferrin and lactoferrin receptors results in a compet-itive advantage over a strain expressing only the transferrinreceptor in mixed infections (8).

One important aspect of transferrin and lactoferrin utiliza-tion by the meningococcus is that there is a preference for

human transferrin and human lactoferrin. Even though there isconsiderable homology between the transferrins, such as por-cine, bovine, sheep, and ovotransferrin, N. meningitidis candiscriminate against these non-human-derived transferrins (85,141, 144). This correlates well with the role of N. meningitidisas an obligate human pathogen.

Another source of iron for N. meningitidis is hemoglobin. Inaddition, meningococci are capable of using haptoglobin-he-moglobin and free heme, but not heme-hemopexin or heme-albumin, as sources of iron (55). Hemoglobin is the best char-acterized of host iron-binding proteins (see reference 53 for areview). Briefly, hemoglobin is the oxygen-binding and trans-port protein of erythrocytes, functioning to deliver oxygenfrom areas of high concentration to areas of low concentration

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throughout the host. The iron component of hemoglobin is thecentral atom within a heme moiety. The heme groups of he-moglobin are located in heme-binding clefts within each offour subunits and interact hydrophobically with amino acids inthe cleft. The heme iron interacts with six ligands within theiron-binding cleft of hemoglobin: four nitrogen atoms from theporphyrin moiety of heme, the imidazole side chain of a histi-dine residue, and oxygen, when present. Increased concentra-tion of carbon dioxide in the peripheral tissues, which is rapidlyconverted to carbonic acid, lowers the pH and favors the re-lease of oxygen as the sixth ligand of the heme iron.

Hemoglobin is not readily available for use by N. meningiti-dis because of compartmentalization within the erythrocytes.Spontaneous hemolysis results in small amounts of hemoglo-bin being released into normal human serum (80 to 800 nM).However, this free hemoglobin rapidly dissociates into twodimers and is rapidly complexed to circulating haptoglobin andtransported to the liver. Like that of transferrin, the level ofhemoglobin on the mucosal surface of the oropharynx is nor-mally very low, yet meningococci express two different hemo-

globin receptors (156). It is possible that a small amount oflocal hemolysis would make a large difference in the amount offree hemoglobin and haptoglobin-hemoglobin complexesavailable on mucosa for bacterial growth (145). In addition,these receptors participate in heme utilization by meningo-cocci (61, 102). Other heme-requiring pathogens have beenshown to colonize the oropharynx and survive on the availableheme (145). Recently, two additional members of hemoglobinfamily of proteins have been discovered: neuroglobin, which isexpressed predominantly in the brain, and cytoglobin, withstrongest expression in heart, stomach, bladder, and small in-testine (37). Presently, it is not known whether these hemo-globins can be used as a source of iron or heme for bacteria.

IRON ACQUISITION MECHANISMS

Two general mechanisms of iron acquisition in bacteria havebeen described: siderophore-mediated iron acquisition by cog-nate receptors and receptor-mediated iron acquisition fromhost iron-binding proteins (3133, 42). One important differ-ence between these two mechanisms of iron acquisition is thatsiderophores and hemophores are taken into the cell whole,

whereas iron and heme must be stripped away from host car-rier proteins prior to transport. N. meningitidis possess severaliron uptake systems that rely on high-affinity receptors foriron-bound host proteins, including transferrin, lactoferrin,and hemoglobin (Fig. 1). Each of these systems is discussed indetail below. In situations where information was not availablefor N. meningitidis iron transport systems, data from experi-ments using the closely related N. gonorrhoeae may have beenincluded, and this is indicated appropriately below; for anexcellent review ofN. gonorrhoeae iron transport see reference133.

Many enteric bacteria use siderophore-mediated iron up-take, and the characterization of these systems has precededdescriptions of receptor-mediated iron acquisition systems. In N. meningitidis, however, no low-molecular-mass compoundsthat scavenge insoluble ferric iron have been identified (13,171). Original experiments demonstrating the ability of N.

meningitidis to use iron from human transferrin demonstratedthat transferrin-bound iron could not be used if the transferrin

was contained in dialysis tubing; this suggests that even if N.meningitidis produces siderophores, they cannot remove ironfrom transferrin and may not have a significant impact in vivo(15). Studies indicate that meningococci may be able to useheterologous siderophores secreted by other bacteria (136).For some time it has been reported that the gonococci couldutilize ferric enterobactin and aerobactin as iron sources (38,170, 171). Recently in N. gonorrhoeae, an outer membraneprotein named FetA (formerly FrpB) has demonstrated lowbinding affinity and transport of ferric enterobactin (21, 38, 54,113). The binding constant of FetA for enterobactin was muchlower than for other enterobactin receptors, and it was there-fore proposed that this receptor could interact with high affin-ity to a yet-unidentified, phenolate siderophore (38). A homol-ogous protein, with 91% identity to gonococcal FetA, has beenidentified in N. meningitidis and presumably functions similarly(113, 165).

Transferrin and Lactoferrin Receptors

Two proteins, transferrin-binding protein A (TbpA) andtransferrin-binding protein B (TbpB), function as the trans-ferrin receptor in N. meningitidis (44, 49, 63, 142). TbpA andTbpB are induced along with several other proteins in theouter membranes ofN. meningitidis under iron-restricted con-ditions (22, 86, 144). Initially, an affinity isolation procedureusing biotinylated transferrin was employed to demonstratethe presence of two transferrin-binding proteins in N. menin-gitidis (144). The proteins which bound transferrin were TbpA(formerly Tbp1), which was 98 kDa, and TbpB (formerly

Tbp2), which was 68 kDa (66, 104, 143). Among differentmeningococcal isolates, the molecular masses of TbpA and -B vary, with TbpA ranging from 93 to 98 kDa and the moreheterogeneic TbpB varying from 68 to 85 kDa (58, 66, 143).TbpA can be found on all strains, both invasive and carrier,and depending on the strain, there are roughly 700 to 4,700copies per cell (117).

Although it has not been characterized as well as the trans-ferrin receptor, the lactoferrin receptor is believed to be animportant virulence factor of the meningococci. The entry siteof N. meningitidis into the body is the nasopharynx, wherelactoferrin predominates as the main source of iron. Addition-ally, lactoferrin has been shown to cross the blood-brain bar-rier during acute inflammation and may also serve as a sourceof iron during the invasive phase of infection (73, 115). Sup-porting this role, the meningococcal lactoferrin receptor hasbeen found on all strains tested to date (95, 115). In contrast,the gonococcal lactoferrin receptor has been found on onlyhalf of clinical isolates (8, 95). This difference may be relatedto the differences in the niches they occupy, with lactoferrinutilization being an essential function of the meningococci.

The lactoferrin receptor of N. meningitidis, like the trans-ferrin receptor, consists of two protein components, LbpA andLbpB. Initial experiments using affinity isolation by lactoferrinidentified a 98-kDa lactoferrin-binding protein named LbpA,formerly known as IroA (112, 123, 143). Insertional inactiva-tion of lbpA in N. meningitidis eliminated lactoferrin binding,

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lactoferrin utilization, and expression of the 98-kDa outermembrane protein (23, 123). Under lower-stringency isolationconditions, a second 84-kDa lactoferrin-binding protein wasidentified and named LbpB (139).

Genetic arrangement and regulation by Fur.The

tbplocus isa bicistronic operon located on the chromosome of N. menin-

gitidis. It consists of open reading frames tbpB and tbpA, whichare separated by an 87-bp intergenic region (86). Unlike thecase for many other genes of N. meningitidis, there is no phase

variation of the transferrin receptor due to slipped-strand mi-spairing. Therefore, the transferrin receptor could potentiallybe expressed at all times under in vivo conditions of ironlimitation. A putative promoter containing regulatory regionsof significant homology to Fur-binding sites precedes the tbplocus (86). The Fur protein is a global repressor protein whichnegatively regulates iron-controlled genes at the transcrip-tional level (see reference 68 for a review). Although the Fur-binding site at the tbp locus has not been characterized, iron

limitation has been shown to be the only signal required forexpression of the Tbp receptor (144). Derepression of thetransferrin receptor in N. gonorrhoeae results from mutating itsfur gene (160). The N. meningitidis Fur homologue has also

been identified, but studies involving Fur regulation of themeningococcal tbp operon have not been reported (76, 159).Similarly, the genes for the lactoferrin receptor in N. men-

ingitidis are on a transcriptional operon, with lbpB locatedupstream oflbpA (24, 90, 115, 139). The existence of an operon

was predicted, since there are no 10 and 35 sequencesupstream of lbpA and the stop and start codons of the genesoverlap (115). Primer extension, reverse transcriptase PCR,and insertional inactivation of lbpB with a cassette containingtranscriptional terminators, which disrupted the expression ofboth LbpA and LbpB, confirmed that the genes constitute anoperon (90, 139). The promoter region directly upstream oflbpB also has a predicted Fur-binding sequence, which over-laps the 10 sequence, thus implying that these genes are

FIG. 1. Schematic representation of iron acquisition systems in N. meningitidis. Under iron-limiting conditions, meningococci express severalproteins involved in the uptake and processing of iron and heme. Several of these are outer membrane receptor proteins, which recognizetransferrin, lactoferrin, hemoglobin, and hemoglobin-haptoglobin. Depicted are the TonB-dependent receptors, TbpAB, LbpAB, HmbR, andHpuAB. All consist of a pore-forming membrane protein and, except for HmbR, a lipoprotein that is involved in substrate binding. Anuncharacterized TonB-independent heme uptake system is not shown. The FbpABC transport proteins are involved in the periplasmic transportof ferric iron, and an undescribed system may exist for the periplasmic transport of heme. In the cytoplasm, ferric iron and heme can be storedin bacterioferritin. Heme can also be converted to biliverdin and CO in the cytoplasm by heme oxygenase. Dimeric forms of TonB and the FbpBCproteins from E. coli have been described but are not depicted as such here. LOS, lipooligosaccharide



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regulated by the Fur repressor and iron availability. Westernblotting and mRNA analysis have confirmed that transcriptionand LbpA and -B expression are iron regulated, consistent withthe upstream Fur-binding site (90, 115).

Structure predictions. Both Tbp proteins and both Lbp pro-teins are expressed as precursors carrying amino-terminal sig-nal peptides and are localized to the outer membrane. Initially,

the translated sequence of TbpB suggested that it was a li-poprotein, since it has a signal II protease cleavage site anddoes not appear to have membrane-spanning regions. LbpB

shares 33% amino acid identity to TbpB (115). Protease sus-ceptibility assays done on the LpbB homologue from Moraxellacatarrhalis suggested that the protein is largely exposed at thecell surface (139). Like TbpB, LbpB contains a signal peptidase

II recognition sequence with an amino-terminal cysteine resi-due, which implies that it undergoes lipidation (139). Biochem-ical studies have confirmed that TbpB has a 20-amino-acidsignal sequence that is cleaved, followed by lipidation of thefirst amino acid (cysteine) of the mature protein (91). Morethan likely, it is anchored to the outer membrane via its N-terminal lipid (145). The crystal structure of TbpB has not yet

been determined, but it is predicted to be a bilobed protein likeits transferrin substrate since it contains two transferrin-bind-ing domains and has internal amino acid repeat regions (94,

126). A high-affinity transferrin-binding domain which remainsstable after exposure to heat and sodium dodecyl sulfate islocated in the amino-terminal domain, and a second trans-

ferrin-binding site is located in the carboxyl-terminal domain(45, 126, 166). A unique feature of LbpB, not shared by TbpB,is the presence of two long stretches of acidic amino acidresidues. These domains are predicted to be important in the

interaction with lactoferrin, which is a positively charged mol-ecule (115).

TbpA and LbpA share 25% amino acid identity and a high

degree of similarity (63). They also exhibit low homology toother TonB-dependent transport proteins. The predictedstructures of TbpA and LbpA are based on their sequencehomology with the ferric siderophore receptors (see reference

42 for a review). Homology to this family of transporters sug-gests that the ancestral meningococcal transferrin and lacto-ferrin receptors may have been a single-unit transporter like

the siderophore receptors; in fact, homologous single-unittransferrin receptors have recently been described for Pasteu-

rella multocida and Histophilus ovis (56, 106). The two-dimen-sional models of TbpA and LbpA depict 22 transmembrane-spanning regions that form an extended -sheet which is

predicted to fold into an amphiphillic -barrel pore structuresimilar to the structures of all of the characterized bacterialouter membrane proteins (36, 80). The models predict 11extracellular loops of various sizes. The sizes of both TbpA andLbpA are roughly 20 kDa greater than those of the sid-

erophore receptors, and they are predicted to have larger sur-face loops (145). Some of these predicted loops have beenexamined experimentally (29, 122). For LbpA, the cell surfaceexposure of some of these predicted loops has been analyzedby using antisera against synthetic LbpA peptides (122). Pre-

dicted loops shown to be surface accessible included loop 4(L4), L5, L7, L10, and L12 (122). Additionally, the amino-terminal ends of these proteins may extend into the pores of

the predicted barrel structures from the periplasmic side, sim-ilar to the plug domains of the ferric siderophore receptors.

TbpA and TbpB function together in the outer membrane of

N. meningitidis as a transferrin receptor complex. Mutants de-ficient in both proteins are unable to bind or utilize iron fromtransferrin (74). Furthermore, mutants deficient in eitherTbpA or TbpB are also unable to utilize iron from transferrin,

but they have transferrin-binding capabilities (74). In N. gon-

orrhoeae, however, mutants deficient in TbpB alone could uti-lize iron from transferrin at roughly 20% of wild-type levels,

suggesting that TbpA requires the cooperative activity of TbpBto be fully functional (9). Several lines of evidence are consis-tent with TbpA and TbpB colocalization in the outer mem-brane, including protease susceptibility assay of TbpB (46, 59,

121). Purification and in vitro experiments further supportthese results. When transferrin affinity chromatography is usedto purify the Tbp proteins, TbpA and TbpB can be isolated

together under high-ionic-strength conditions (48). TbpA and-B have been examined by photon correlation spectroscopy,

which demonstrated that in vitro, they will form a complex insolution (27).

Likewise, evidence suggests that LbpA and LbpB form acomplex in the outer membrane similar to the TbpAB com-plex. Two-dimensional gel electrophoresis demonstrated the

comigration of LbpA and LbpB, whereas under denaturingconditions, the LbpAB complex is not formed (122). Further,

when isolated from an isogenic LbpA mutant, LbpB migrates

to a different position under the same native electrophoreticconditions (122).

Structure and functional relationships. The affinity of bind-ing of the wild-type gonococcal receptor to transferrin is com-

parable to estimates for the mammalian transferrin receptor,with a Kd of 5 to 20 nM (48). The bacterial transferrin receptor,however, is both genetically and antigenically distinct from the

mammalian transferrin receptor (44). No studies which pre-cisely measure the affinity or kinetics of binding for lactoferrin

with the lactoferrin receptor have been reported. However,competitive blocking experiments with unlabeled lactoferrin

ligand and the intact lactoferrin receptor complex indicate thatthe inhibition of binding occurs at concentrations similar tothose used for transferrin in transferrin receptor interaction

studies. The binding affinities are therefore in the same rangeas those of the transferrin receptor, between 10 and 50 nM (A.Schryvers, personal communication).

The specific interactions between TbpA and human trans-ferrin and LbpA and lactoferrin remain to be described. The

process must involve the high-affinity binding of transferrin tothe receptor complex, the stripping of iron from transferrin,and the transport of iron through the outer membrane into theperiplasm. Neither transferrin or lactoferrin is internalized inthis process (149).

Data support an interaction between TbpA and the C lobeof transferrin, which is also the domain most highly saturated

with iron (5, 177). This interaction is not dependent on thepresence of carbohydrate chains on transferrin, indicating thatit is a direct protein-protein interaction (108). Recombinant

human-bovine transferrin hybrid proteins were designed todetermine regions of contact with the transferrin receptor(127). Amino acid residues (aa) 346 to 588, within the carbox-



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yl-terminal end of transferrin, were found to be responsible for

interacting with the meningococcal receptor; in addition, a

region very near the carboxyl terminus has an influence on theavidity of the interaction (127).

Regions of the lactoferrin receptor that interact with lacto-

ferrin have not yet been determined. Attempts to identify

regions of lactoferrin that interact with the receptor proteinhave been made. The N lobe and C lobe fragments of lacto-

ferrin were separated by tryptic digestion, and their binding to

the lactoferrin receptor was assessed (174, 176). Both lobes of

lactoferrin were bound by the receptor; recent studies have

identified two binding regions within the C lobe of humanlactoferrin (174). This is in contrast to the case for transferrin,

of which only the C lobe interacts with the transferrin receptor.

Specific sites of interaction remain to be determined.Likewise, studies directed at identifying the transferrin-bind-

ing domain of TbpA and other important functional domains

have begun. Since the crystal structure of TbpA has not been

determined, structure-function studies for TbpA have relied

on the predicted two-dimensional model. The binding domainfor transferrin is most likely located in the extracellular loops

of TbpA. Experiments involving the deletion of predicted ex-

tracellular loops suggest that L4 and L5 are involved in trans-

ferrin binding (29). Consistent with these experiments, over-

expressed loop regions, L4 and L5 combined and L5 alone,

have been shown to specifically bind transferrin in solid-phasein vitro assays (93). An effort has also been made to demon-

strate that L5 and other predicted loops, including L2, L3, L7,

and L10, are surface exposed, by using the insertion of detect-

able epitopes (M. K. Yost and C. N. Cornelissen, presented at

the Thirteenth International Pathogenic Neisseria Conference,

Olso, Norway, 2002). Additionally, loop deletion analysis has

indicated that L8 may be involved in the utilization of ironfrom transferrin, since loss of this predicted extracellular loop

results in a transferrin utilization-deficient but binding-profi-cient phenotype (29).

The exact roles of TbpB and LbpB in transferrin and lacto-

ferrin utilization by N. meningitidis remain unclear. TbpA

alone is capable of removing iron from transferrin (61); how-

ever, TbpB appears to act as a facilitator in this process (9).

Similarly, binding analysis of LbpA and LbpB indicates that

both proteins can bind lactoferrin independently, but only an

LbpA mutant is incapable of using iron from lactoferrin (115).

In nutrition assays, TbpB meningococci are unable to grow

on media supplemented with 1 M transferrin (74). TbpB-

deficient gonococci can grow only poorly under the same con-ditions (9). TbpB also has the ability to distinguish between

apo- and holotransferrin and binds the holotransferrin with a

100-fold-higher affinity, whereas TbpA does not (26, 48, 121,125, 128, 176). The specificity and high affinity of TbpB for thecorrect iron-loaded substrate is undoubtedly key to its function

in the transferrin receptor complex.

As mentioned previously, TbpB is a bilobed protein and

both halves are capable of binding transferrin (28, 126, 166).

The interactions of each lobe of TbpB with transferrin appear

to be identical, since experiments demonstrate that the same

peptide derivatives of transferrin are recognized by both bind-

ing domains (129).

Energy from the TonB-ExBD system. Analogous to theTonB-dependent siderophore acquisition systems found in en-teric bacteria, N. meningitidis requires a ton system for utiliza-

tion of transferrin, lactoferrin, hemoglobin, and haptoglobin-hemoglobin (157). The TonB complex, which consists of TonB,ExbB, and ExbD, is located in the inner membrane and func-tions to transduce the energy of the proton motive force into

conformational changes in TonB-dependent outer membranetransporters (see reference 120 for a review). The N. meningi-tidis TonB, ExbB, and ExbD proteins have roughly 30% amino

acid identity with their Escherichia coli homologues (157). Re-constitution of the neisserial hemoglobin utilization system in

E. coli required not only the hemoglobin receptor protein butthe neisserial ton genes as well (157). Recently, the intracellu-

lar replication ofN. meningitidis within epithelial cells has alsobeen shown to be a TonB-dependent process, implicatingTonB in acquisition of intracellular host iron (82). In the infant

rat model of N. meningitidis septicemic infection, tonB, exbB,and exbD were found to be essential (158).

More is known about the interactions between TonB and the

gonococcal transferrin receptor than about the meningococcaltransferrin receptor. TonB has been copurified with the gono-coccal TbpA protein in the presence and absence of TbpB(77). No chemical cross-linkers were used to artificially main-tain this interaction. Furthermore, immunoprecipitation stud-ies allowed the isolation of TbpA-TonB complexes (77). Trans-ferrin was not necessary to detect the interaction, implying that

TonB binding occurs in the absence of ligand binding (77).However, the TbpA TonB box, which is a short region ofhomology near the amino termini of TonB-dependent recep-tors, was shown to be essential (77). Mutating the TbpA TonB

box left the receptor de-energized and insensitive to TonB,similar to the case for TonB box mutations in other TonB-dependent receptors. This TonB box mutant was not affected

in transferrin binding, but it did not transport iron and did notefficiently release transferrin from the cell surface. Further,mutations in the TonB box of TbpA were shown to have an

effect on the protease susceptibility profile of TbpB (46). Thisfurther suggests an interaction between TbpA and TbpB andindicates that this interaction is somehow influenced by theinteraction between TbpA and TonB and most likely by the

energy state of TbpA (46).Transport through the periplasm. Several different routes

for the translocation of iron across the cytoplasmic membraneare possible in bacteria (see reference 81 for a review). Theimport of ferric iron acquired from transferrin and lactoferrin

into the cytoplasm of N. meningitidis requires the Fbp (ferric-binding protein) system (79). Only heme iron can be utilized by

fbp-deficient meningococci, and not the ferric iron derivedfrom transferrin, lactoferrin, or iron chelates (79). This systemis distinct from the periplasmic iron transport systems involved

in the transport of iron-siderophore complexes. The Fbp sys-tem has low but significant homology to the respective com-ponents of ATP-binding cassette (ABC) transporters that areinvolved in the utilization of sulfate, spermidine, and pu-trescine (81). Homologous systems include the hitABC operon

of Haemophilus influenzae and the sfuABC operon of Serratia

marcescens (12, 137). ABC transporters couple ATP hydrolysiswith substrate translocation across biological membranes. The



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Fbp transporter consists of FbpA, a substrate-binding protein;FbpB, a putative inner membrane component of the permease;and FbpC, a putative ATPase (78). An iron-regulated operon,

with Fur-binding sites, encodes the genes for the Fbp system(51, 78).

FbpA is one of the major iron-regulated proteins and hasbeen identified in all pathogenic Neisseria species (20, 99, 101).It has an apparent molecular mass of 37 kDa and is very basic(pI of 9.35) (97, 98). The role of Fbp in the periplasmictransport of iron was first indicated by its localization to theperiplasm and its transient association with 55Fe acquired from55Fe-labeled human transferrin (19, 40). In vitro studies usingpurified TbpA, TbpB, and FbpA have shown that FbpA isabsolutely unable to acquire transferrin-bound iron unless themetal is freed by the interaction of transferrin with the TbpABcomplex (61). No studies describing exactly how FbpA acquiresferric iron from the transferrin receptor or how it relinquishesthe iron for transport across the inner membrane have beenreported.

The crystal structure of FbpA from H. influenzae has been

determined, and interestingly, the protein resembles a singlelobe of transferrin (35). FbpA and transferrin share some ad-ditional characteristics. They both have the ability to reversiblybind ferric iron, and they share similar binding affinities (40).

Also, only a single ferric ion binds per lobe (two for transferrinand one for FbpA), and the ferric iron-binding sites are similar(97, 103). The crystal structure revealed that identical aminoacid residues in transferrin and FbpA, i.e., two tyrosines, ahistidine, and a glutamic acid, coordinate the iron atom butthat these amino acids arise from different regions of FbpAthan of transferrin (42). Additionally, the FbpA protein com-pletes the octahedral coordination of Fe3 with oxygens froma phosphate and a water molecule, whereas transferrin utilizes

the two oxygens from a carbonate molecule (42).The FbpB protein is a 56-kDa inner-membrane-spanning

protein which has some internal homology and a putative in-teraction domain proposed to bind the 38-kDa FbpC compo-nent (81). FbpB may interact with FbpA as well as conferspecificity of binding for ferric iron, based on similarity to theinner membrane component of the bacterial permease formaltose, MalF (72, 102). FbpC is expected to provide theenergy for transport through the FbpB cytoplasmic permease.FbpC is a putative ATPase and has a walker box motifcharacteristic of the ATPase family of proteins. No detailedmolecular studies have been done with the FbpB and FbpCcomponents of the periplasmic transporter system.

Ferric iron in the cytoplasm.Iron serves many essentialfunctions in biological systems. The large majority of bacteria

use iron-dependent enzymes in metabolic processes such asDNA replication, electron transfer, and the metabolism ofoxygen, peroxide, and superoxide (65). However, in the pres-ence of oxygen, iron can form free radicals which are toxic tothe cell. Therefore, sequestration, mobilization, and storage ofiron in a nontoxic, bioavailable form are very important to acell. Little is known about what happens to iron in the cyto-plasm. Two types of iron storage proteins have been identifiedin bacteria, i.e., bacterioferritin (Bfr), which stores both hemeiron and nonheme iron, and ferritin, which stores only iron andnot heme (11). No iron storage proteins have been describedfor N. meningitidis; however, Bfr has been identified recently in

the closely related N. gonorrhoeae (41). No homologues offerritin were found in N. gonorrhoeae. Most Bfrs are high-molecular-weight homopolymers, consisting of many subunits;however, the gonococcal Bfr has two similar but nonidenticalsubunits (41). The genes for the gonococcal Bfr were identi-fied; they are more than likely transcribed as an operon (41).Regulation by the Fur repressor is indicated by the presence ofan upstream Fur box sequence; however, recent studies dem-onstrate that transcription of bfrA and bfrB increases in thepresence of iron, as should be expected for iron storage pro-teins (64).

The Hemoglobin Receptor HMBR

Every environment within the host, where N. meningitidismust survive in order to cause disease, has only trace amountsof heme iron and heme-containing proteins (60, 84, 156). Yet,two independent heme transport systems which recognizeheme-containing proteins, hemoglobin (HmbR) and/or hapto-globin-hemoglobin (HpuAB), have been identified in N. men-

ingitidis (87, 153). Over half of the clinical meningococcal iso-lates from serogroups A, B, C, and Y examined expressed bothhemoglobin receptors, which suggests an important role forthese receptors in causing human disease (130). The first he-moglobin receptor identified in N. meningitidis was HmbR(153). The insertional inactivation of hmbR prevented the useof hemoglobin, but not transferrin, lactoferrin, or iron-citrate,as an iron source (153). HmbR is responsible for bindinghemoglobin with a high affinity, stripping heme from hemoglo-bin, and transporting the heme into the periplasm. Only heme,and not hemoglobin, is internalized in this process (153). Inaddition, HmbR preferentially recognizes but is not limited tohuman hemoglobin, unlike the transferrin and lactoferrin re-

ceptors, which are human species specific (155). Furthermore,a mutation in the hemoglobin receptor gene, hmbR, had pro-found effects on the survival of meningococci in an infant ratmodel of infection, indicating that hemoglobin utilization isimportant for N. meningitidis virulence (153).

Even a small amount of hemolysis would significantly alterthe amount of hemoglobin and haptoglobin-hemoglobin avail-able for bacterial growth. There are many possibilities for howmeningococci could have access to free hemoglobin: (i) lowlevels of hemoglobin can be found in normal serum due tospontaneous hemolysis; (ii) a manifestation of meningococcaldisease known as disseminated intravascular coagulation, in-

volving the activation of blood clotting mechanisms throughout

the body, results in some erythrocyte lysis; and (iii) meningo-cocci, like other bacterial pathogens, may secrete hemolysinsand cytolysins. Two proteins belonging to the RTX family ofproteins have been identified in N. meningitidis (161163).FrpA and FrpC contain typical RTX domains found in cyto-toxins from other gram-negative pathogens. The secretion ofFrpA by the heterologous RTX transport system of E. coli hasdemonstrated its relationship to this family of proteins (161,163). FrpA, however, is rarely found in clinical isolates. Incontrast, FrpC has been found in all invasive and most carriermeningococcal strains tested to date (107). Furthermore, hightiters of both immunoglobulin G (IgG) and IgA that recog-nized FrpC were found in the convalescent-phase sera of pa-tients with meningococcal disease (107). If these proteins are



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involved in cytotoxicity or hemolysis, the possession of hemo-globin utilization mechanisms by meningococci would be ex-tremely advantageous.

Genetic arrangement and regulation by Fur. The gene en-coding the hemoglobin receptor, hmbR, is located 178 bpdownstream of a gene involved in the catabolism of heme,hemO (179). Several potential transcriptional starts and a Furbox sequence can be found in the intergenic region; however,several lines of evidence suggests a transcriptional linkage be-tween the genes. HmbR expression is down-regulated in hemOmutants and can be induced in hemO conditional mutants(179). The hemO gene also has a Fur box regulatory sequencein its promoter region. Fur regulation of hmbR was demon-strated by using a Fur titration assay in E. coli. The upstreamFur box sequence and intergenic promoter region of hmbR,

when present in multicopy, were able to titrate Fur away froma Fur-regulated reporter gene (153). In addition, hemoglobinbinding by N. meningitidis was shown to be iron regulated butnot heme regulated (155). The Fur repressor from the closelyrelated N. gonorrhoeae has recently been isolated and shown by

electrophoretic gel shift assays to bind to the promoter regionsof hmbR and hemO (148).

In contrast with the transferrin receptor of N. meningitidis,the expression of HmbR undergoes phase variation due toslip-strand mispairing of poly(G) tracts within the hmbR gene(60, 86). The obvious advantage associated with phase-varyingsurface proteins is evasion of the host immune response. In-terestingly, among clinical isolates of serogroups A, B, and C,the phase variation frequency of hemoglobin receptors hasbeen found to vary dramatically, ranging from approximately106 to 102 CFU1. Frequencies of phase variation wereshown to be the genetic traits of particular strains, where twounlinked hemoglobin receptors phase vary with similar fre-

quencies within a given isolate (131). The elevated mutationrates found in a study of epidemic serogroup A isolates wereattributed to defects in mismatch repair pathways (131). Thesestudies found a relatively high frequency of hypermutablestrains among epidemic isolates, suggesting that the ability tomutate at high frequency may be advantageous to the menin-gococci (131).

Structure predictions. HmbR is an 89-kDa protein that lo-calizes to the outer membrane of N. meningitidis. A signalpeptidase I recognition sequence is located in the amino-ter-minal region, and amino acid sequencing of the mature proteindemonstrates cleavage of a 23-amino-acid peptide (111).HmbR shares some weak amino acid identity, between 21 and

22%, with the neisserial transferrin and lactoferrin receptors(153). It also shares homology with heme transporters fromother species, including HemR from Yersinia enterocolitica(152).

Since the structure of HmbR has not yet been obtained, atwo-dimensional model has been developed based on its inclu-sion in the TonB-dependent family of iron transporters (Fig.2). The two-dimensional model of HmbR consists of 22 puta-tive transmembrane -sheets and 11 surface-exposed loops(111). Two putative disulfide bonds are located in L6 and L8.The putative L7 contains an invariable histidine residue andamino acid motifs FRAP (YRVP in HmbR) and NPNL, ahighly conserved domain present in all heme-hemoglobin re-ceptors (156). The amino-terminal end of the protein is pre-

dicted to extend into the pore of the barrel structure, like theplug domains of the ferric siderophore receptors. Additionally,three-dimensional modeling of HmbR on the structures offerric siderophore receptors FepA, FhuA, and FecA has alsobeen performed (111). The predicted cork domain of HmbR

was superimposed on the cork domain of FecA, and the aminoacids which joined the cork region to the beginning of the firsttransmembrane region corresponded to residues P150 to R152of HmbR, as indicated in the two-dimensional model. The loopregions of HmbR, L7 and L8, and the intervening transmem-brane region corresponded well to the homologous region onFepA. It was also noted that the L7-L8 region of HmbR in thethree-dimensional model was marked by a high electrostaticpotential, with several glutamic acid residues (E494, E548, andE549) in close proximity to each other.

Structure and functional relationships. The binding kineticsfor the interaction between hemoglobin and HmbR have beenmeasured with purified proteins in vitro by using surface plas-mon resonance. HmbR has a high binding affinity for hemo-globin, with a Kd of around 13 nM. The association rate, or rate

of HmbR-Hb complex formation, is 4.4 105 M1 s1, andthe stability, or dissociation rate, of the complex is 6.8 103

s1 (D. Perkins-Balding, unpublished data).The region of hemoglobin that directly interacts with HmbR

has not yet been identified. However, functionally importantdomains of HmbR, including a region putatively involved inhemoglobin binding, have been discerned by using a series ofHmbR deletions, insertions, and site-directed mutations (111).Mutations exhibiting similar phenotypes in N. meningitidis fellinto two groups. The first group of mutations affected hemo-globin binding and localized to the middle region of the pro-tein; two putative extracellular loops, L2 (aa 190 to 232) andL3 (aa 254 to 284), marked this putative Hb-binding region.

The second group of mutations resulted in a failure to utilizehemoglobin but did not affect proficiency in hemoglobin bind-ing. These mutations localized to a region in the middle tocarboxyl-terminal end of HmbR; the putative extracellularloops involved included L6 (aa 420 to 462), L7 (aa 486 to 516),and L10 (aa 661 to 700).

Deletion analysis also revealed that the highly conservedprotein motif found in all heme-hemoglobin receptors, withinputative extracellular L7 of HmbR, is essential for hemoglobinutilization but is not required for hemoglobin binding. Thisfinding suggests a mechanistic involvement of this motif inheme removal from hemoglobin. In addition, an amino-termi-nal deletion in the putative cork-like domain of HmbR affected

hemoglobin usage but not hemoglobin binding. This resultsupports a role of the cork domain in utilization steps that aresubsequent to hemoglobin binding.

Energy from the TonB-ExbBD system. Hemoglobin utiliza-tion mediated by HmbR is a TonB-dependent process. Theamino-terminal end of the mature HmbR protein contains aputative six-amino-acid TonB box, a characteristic shared by allTonB-dependent transporters (153). Further, studies haveshown that HmbR requires expression of the N. meningitidisTon system to function as a hemoglobin receptor in E. coli(157). In addition, a specific interaction with the neisserialTonB protein has been demonstrated in vitro, both by im-munoprecipitation analysis and by surface plasmon reso-nance. The binding kinetics of the HmbR-TonB interaction



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were examined by surface plasmon resonance, and the bind-ing affinity for neisserial TonB is high, with a Kd of 76 nM.The rate of HmbR-TonB complex formation is 1.3 105

M1 s1, and the rate of dissociation of the complex is 9.7 103 s1 (Perkins-Balding, unpublished data).

Transport through the periplasm.Little is known about thetransport of heme from the periplasm through the inner mem-

brane ofN. meningitidis. The Fbp system is not involved, how-ever, since the use of iron from heme and hemoglobin isunimpaired in an fbpABC mutant strain (79). Once HmbRtransports heme into the periplasm, it is most likely deliveredto the cytoplasm by HemTUV-like proteins. The HemTUVsystem ofY. enterocolitica is a model system for heme-specific

ABC transporters in gram-negative bacteria. The transporterconsists of a heme-specific periplasmic binding protein, anintegral inner membrane permease, and a cytoplasmic AT-Pase. Even though the complete genome of N. meningitidis hasbeen sequenced, proteins homologous with periplasmic hemetransporters have not been identified.

Heme in the cytoplasm. Until recently, very little has beenknown about how microorganisms utilize iron that is seques-tered in heme. Some bacteria have been shown to possessenzymes homologous to human heme oxygenase, HO-1. Thisenzyme is capable of degrading heme to -biliverdin, with the

liberation of free iron and CO (Fig. 3). HO-1 is an inducibleisoform of human heme oxygenase which has been found torespond to a diverse group of stimuli. These include heat shockand the presence of heavy metals as well as the substrate andcofactor, heme (114). The response to these stimuli suggeststhat HO-1 may play a significant role in protection againstoxidative stress. Within the last several years, heme oxygenaseshave been characterized for three human pathogens: N. men-ingitidis (HemO), Pseudomonas aeruginosa (PigA), and Coryne-bacterium diphtheriae (HmuO) (124, 138, 179). Thus, it is alsoplausible that microbial pathogens capable of utilizing hemeand hemoglobin as a source of iron may have heme oxygenase-like enzymes for the degradation of heme molecules, not onlyfor the acquisition of iron but perhaps also for heme detoxi fi-

FIG. 2. Topological model of HmbR. The size of each -sheet was arbitrary taken to be 10 aa residues. L1 through L11 indicate putativesurface-exposed loops of HmbR. Numerals indicate amino acid residues of HmbR.



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cation. N. meningitidis hemO mutants were found to be hyper-susceptible to heme, supporting this idea (179).

HemO is essential to the utilization of heme and hemoglo-bin as the sole sources of iron in N. meningitidis, as a hemO-deficient mutant is unable to utilize either (180). Mechanisti-cally, HemO is identical to C. diphtheriae HmuO; bothenzymes convert heme to -biliverdin, with the liberation ofiron and carbon monoxide (179). Interestingly, PigA from P.aeruginosa, which is 37% identical to HemO, may represent anovel bacterial heme oxygenase. PigA was found to produce astructurally different biliverdin than all previously character-ized heme oxygenases (124). Instead of -biliverdin, PigA

yields 70% -biliverdin and 30% -biliverdin.The crystal structure of HemO from N. meningitidis has been

determined (Fig. 4) (146). The distal pocket of the heme-HemO complex is significantly smaller, having a solvent-acces-sible volume of 7.5A, compared to 53.5A for the hemeHO-1complex. The distal helix has a kink of approximately 50o

directly over the heme provided by the glycines of the highlyconserved sequence 116Gly-Ser-Asn-Leu-Gly-Ala121 in HemO,

corresponding to 139Gly-Asp-Leu-Ser-Gly-Gly144 in HO-1.This kink in the helix is proposed to provide flexibility to thehelix, which is required for binding of the substrate (heme) andrelease of the product (biliverdin). The helix closely ap-proaches the heme, with direct backbone contacts from Gly-139 and Gly-143 in HO-1. The heme propionates and theirinteraction with the protein are critical in binding and correctlyorienting the -meso carbon of the heme for hydroxylation(146). Residues possibly involved in interactions with the hemein HO-1 are Lys-18, Lys-22, Lys-179, and Arg-183, as well asTyr-134. In the heme-HemO complex, only Lys-16 and Tyr-112, corresponding to Lys-18 and Tyr-134, are absolutely con-served in the structure. Lys-22 is replaced by Thr-20, and

Trp-53 largely fills the space occupied by Arg-183 and Lys-179.Mutagenesis studies on Arg-183 of HO-1 have suggested thatthis residue is involved in orienting the heme within the activesite through direct hydrogen bonding to the propionate group.The critical proximal helix histidine residue of HemO (His-23)is absolutely conserved among all heme oxygenases (82).

It remains to be seen whether other bacteria possess hemeoxygenases that are not homologous with HmuO, HemO, andPigA or, alternatively, use iron from heme by a different mech-anism. Iron acquisition systems are very redundant in bacteria,and this would not be an unexpected solution to the problem ofiron limitation.

Haptoglobin-Hemoglobin Receptor HPUAB

The second heme-specific transporter in N. meningitidis isHpuAB. HpuAB is a two-component transporter, like thetransferrin and lactoferrin receptor proteins of the meningo-cocci. Although it seems that the preferred substrate of theHmbR receptor is human hemoglobin, the HpuAB receptorhas a broader spectrum of ligands. HpuAB binds hemoglobin,haptoglobin, and hemoglobin-haptoglobin complexes (87).Studies with non-human-derived hemoglobins have demon-strated no discrimination between different hemoglobins byHpuAB (134). Interestingly, HpuAB also shows enhancedbinding of oxidized methemoglobin over reduced hemoglobin(134). Methemoglobin may be an abundant source of iron for

meningococci when erythrocytes lyse. Production of methemo-globin is due to dissociation of the tetrameric hemoglobin intohemoglobin 11 dimers, the change of ferrous iron to ferriciron in heme, and loss of the liganded oxygen atom (134).

Some meningococci express either HpuAB or HmbR, whileothers express both (155). Unlike in the meningococci, HpuABis more than likely the only hemoglobin receptor expressed inthe gonococci due to a premature stop codon in hmbR (155).The reason that meningococci have redundant hemoglobinreceptor proteins is unclear, but it suggests that acquisition ofheme, perhaps from various source proteins, is important inpathogenesis.

Genetic arrangement and regulation by Fur. The hemoglo-bin-haptoglobin receptor HpuAB is encoded on a bicistronicoperon. An insertional mutant of hpuA was shown to exert apolar effect on downstream hpuB, eliminating HpuB expres-sion, and the bicistronic mRNA for hpuAB was isolated (89).Primer extension analysis was used to identify the transcrip-tional start site of the hpuAB operon, located 33 nucleotidesupstream of the translational start for hpuA (89). The hpuB

gene does not contain its own promoter. The promoter regionof hpuAB contains a Fur box sequence, and the mRNA tran-script was shown to be iron repressible (89). Thus, like forother iron-transport systems of N. meningitidis, regulation ofthe hemoglobin-haptoglobin receptor appears to be under thecontrol of the Fur protein.

Like that of the HmbR receptor and many other virulence-related proteins in N. meningitidis, expression of HpuAB variesdue to translational frameshifting resulting from slip-strandmispairing of a poly(G) tract within the coding sequence ofhpuA (88). The rate of phase variation of hpuAB is similar tothe rate for hmbR within any given strain, provided that thenumber of repeat sequences in the poly(G) tract is equal (131,

132). Interestingly, the frameshifting mutation in hpuA corre-lates not only with a lack of HpuA expression but with a lackof HpuB as well (88).

Structure predictions. The hemoglobin-haptoglobin recep-tor consists of lipoprotein HpuA and the transmembrane pro-tein HpuB. Labeling of meningococci with [3H]palmitic aciddemonstrated that HpuA is acylated (89). This lipoprotein isbelieved to be anchored to the outer membrane via its lipidmoiety and to be largely surface exposed (89). HpuA is 37 kDa,

approximately one-half of the size of the lipoprotein compo-

nents of the transferrin and lactoferrin receptors, TbpB and

LbpB. It shares almost no sequence similarity with these pro-

teins (88). In fact, no homologues have been identified for

HpuA.Conversely, HpuB is an 85-kDa outer membrane protein

that shares 25% identity and 40% similarity with the TbpA and

LbpA components of the ferric iron transporters of meningo-

cocci (134). Based on its high degree of similarity to the TonB-

dependent receptor family, HpuB is predicted to be a -barrel

structure, forming a pore in the meningococcal outer mem-

brane, which is filled with an amino-terminal plug domain(134). No structural model has been reported for HpuB.

Data suggest that HpuA and HpuB interact, probably form-ing a receptor complex in the outer membrane. HpuB can bepurified by hemoglobin affinity chromatography (39). HpuA,on the other hand, can be isolated only by using hemoglobin



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agarose in the presence of HpuB, indicating a possible inter-

action (39). Also, protease susceptibility assays confirmed thesurface accessibility of both proteins and indicated a physical

interaction between HpuA and HpuB (134). A 21-kDa hydro-

philic domain of HpuA, containing numerous protease cleav-

age sites, was protected from cleavage in the presence, but not

absence, of HpuB and remained membrane associated after its

proteolytic release from the amino-terminal anchor (134).

Structure and functional relationships. Experimental data

suggest that ligand recognition and binding by the two-compo-

nent HpuAB receptor function differently from those of the

two-component transferrin and lactoferrin receptors of N.

FIG. 3. Chemical steps in heme degradation as defined by studies of eukaryotic heme oxygenases and C. diphtheriae HmuO.



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meningitidis. Unlike the accessory function that both TbpB andLbpB serve in transferrin and lactoferrin utilization, HpuA is

absolutely essential for meningococcal growth on hemoglobinor hemoglobin-haptoglobin as sole sources of iron (134). Anal-

ysis of hemoglobin binding by HpuAB indicates that only onehigh-affinity binding site exists (134). Conversely, both TbpAand TbpB have been shown to independently bind transferrin

with high affinity (48). The affinity of binding of hemoglobin tothe single bipartite HpuAB receptor was estimated at 150 nM(134). The single binding site of HpuAB is consistent with thephenotypic observation that mutants deficient in either HpuAor HpuB demonstrate almost no binding to hemoglobin (39,134). Optimal binding and heme utilization require both pro-teins. Other receptor functions, however, such as removal ofthe iron or heme from host carrier proteins, transport through

the outer membrane, and interactions with the TonB-ExbBDsystem, may be similar for HpuAB and the transferrin andlactoferrin receptors.

Energy from the TonB-ExbBD system. Transport of hemeiron by HpuAB requires the meningococcal TonB system (88).

A TonB box sequence has also been identified in the amino-terminal region of HpuB (89). Recent studies which investi-gated the roles of TonB and the proton motive force in thebinding of hemoglobin to HpuAB and in the conformation ofHpuAB in the outer membrane have been done (134). Theabsence of TonB or a proton motive force had an influence onthe protease susceptibility of HpuB, suggesting that TonBcauses a conformational change in HpuB. Additionally, thepresence of TonB and the proton motive force was not abso-

lutely required for hemoglobin binding, but binding was de-creased by 10-fold in a de-energized cell. The binding affinityhad not decreased; therefore, a decrease in the number ofbinding-competent receptors was indicated. In this system, it

was proposed that TonB serves to enhance a productive inter-action between HpuA and HpuB, which would yield moretwo-component, single-binding-site receptors, in a binding-competent state at the cell surface (134).

Transport through the periplasm. Heme transportedthrough the HpuAB receptor into the periplasm is expected touse the same system for delivery of heme into the cytoplasm asheme transported through HmbR (see above).

Other Potential Iron Transporters

Seven potential genes with products having homology toTonB-dependent transporters have been identified from theNeisseria genome sequencing projects (164). Two out of threegenes which have been examined are present in the N. menin-

gitidis DNA sequence; these are tdfF and tdfH (164). OnlyTdfH appears to be expressed as determined by Western blotanalysis (164). Based on homology to the FpvA pyoverdintransporter of P. aeruginosa and the FhuE coprogen trans-porter of E. coli, TdfF is predicted to be a siderophore trans-porter, and TdfH exhibits low homology to the HasR hemo-phore receptor of S. marcescens (164). In addition, two outermembrane proteins, which bind heme, have been identified in N. meningitidis; these remain uncharacterized (83).

Vaccine Potential and Trials

There are various reasons to consider the transferrin recep-

tor of N. meningitidis a good vaccine candidate. Two of themost obvious are localization to the outer membrane, where itis surface accessible, and the lack of phase variation. In addi-tion, the transferrin receptor has been identified on every clin-ical meningococcal isolate studied to date (47). There is alsogood evidence that the transferrin receptor is expressed in vivoduring infection. N. meningitidis taken directly from cerebro-spinal fluid of a patient with meningitis demonstrated bindingto gold-labeled human transferrin and was expressing TbpB(3). Meningococcal infection in mice can be enhanced by add-ing iron-saturated human, but not bovine, transferrin, indicat-ing that the receptor-mediated uptake of iron is working in

vivo and transferrin is effective in supplying the required iron

in this environment (69, 140). In addition, antisera from pa-tients recovering from meningitis contain cross-reactive anti-bodies to TbpA and -B (4, 62). Further, asymptomatic carriersof N. meningitidis have high antibody titers to the Tbps,

whereas healthy adults have almost none (62).More attention has been given to TbpB as a vaccine candi-

date than to TbpA. Antibody recognition to TbpA is confor-mation dependent, and antibodies to the protein are not bac-tericidal (18, 50). However, recently TbpA was shown toprotect against meningococcal infection in a mouse model

when administered as the sole immunogen (169). The advan-tage of using TbpA over TbpB is that it is less variable, and itmay be protective against a wider range of meningococcalstrains (169).

FIG. 4. Crystal structure of N. meningitidis heme oxygenase,HemO, with bound heme.



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TbpB has been shown to be protective in animal modelsafter both active and passive immunization (1, 50, 92). Anti-bodies to TbpB, unlike those to TbpA, elicit strong comple-ment-mediated bactericidal activity, and humans mount bac-tericidal antibody responses to TbpB upon infection (2, 4, 92,118, 119). Again the disadvantage of using TbpB as an im-munogen is its heterogeneity, but there is evidence for con-served epitopes among different TbpBs (173). At least twoTbpB representatives will be required in a vaccine for broad-spectrum coverage (135, 145). Recently, a mucosal deliverysystem was examined for efficacy with TbpB (43). The highlyefficient secretion mechanism for filamentous hemagglutininfrom Bordetella pertussis was used to secrete a recombinantTbpB; the recombinant TbpB was created as a fusion to theamino-terminal end of filamentous hemagglutinin, which con-tains essential secretion signal sequences (43). The recombi-nant B. pertussis was then used to deliver TbpB intranasally inmice. Antigen-specific antibodies, IgG and IgA, which hadcomplement-mediated bactericidal activity against N. meningi-tidis were detected (43).

Whether the meningococcal lactoferrin receptor proteinscan elicit a protective immune response in the host needs to beevaluated. The lactoferrin receptor has been considered apromising vaccine candidate for many of the same reasons asthe transferrin receptor; it is surface accessible and is ex-pressed on all meningococcal strains examined to date. How-ever, unlike the transferrin receptor, the lbpAB operon mayexperience phase variation (151). The LbpA portion of thelactoferrin receptor appears to be antigenically stable, basedon the cross-reactivity to monoclonal antibodies with 70 dif-ferent meningococcal strains (112). The human antibody re-sponse to LbpA has been analyzed by using two LbpA proteinsfrom different N. meningitidis strains and 70 samples of conva-

lescent-phase sera from patients with meningococcal disease(75). The findings indicated that fewer than 50% of sera con-tained IgG that recognized LbpA, and the response was notuniform between the two different LbpA proteins. Hence,LbpA, like its homologue TbpA, does not elicit a highly cross-reactive antibody response.

The human immune response to meningococcal LbpB hasnot been reported, but data from LbpB of M. catarrhalis indi-cate that patient sera elicit a strong immune response to theLbpB of this pathogen (25, 139). Further, the TbpB homo-logue of LbpB is reported to elicit a strong bactericidal anti-body response in humans. Unfortunately, TbpB has beenfound to have a high degree of sequence variability (47 to 82%

identity) between strains. When the sequences of LbpB in fiveserogroup B strains were compared, there was 70 to 80%amino acid identity, which is a considerable degree of variabil-ity and is disadvantageous for vaccine development (116).

The vaccine potentials of HmbR and HpuAB have not beenexplored, in part because there are no obvious advantages inusing the hemoglobin receptors over other iron transport pro-teins, specifically the transferrin receptor. The benefits of usingHmbR are much the same as with the transferrin receptor,including its surface accessibility and its presence on all inva-sive strains tested to date. Analysis of a limited number ofconvalescent-phase serum samples demonstrated reactivity toHmbR, suggesting its expression in vivo (I. Stojiljkovic, unpub-lished data). Although the expression of HmbR in vivo has not

been unambiguously confirmed, the expression of other hemo-globin receptors in vivo, such as those of certain Haemophilusspecies, has been reported. The in vivo expression of the outermembrane hemoglobin receptors in H. influenzae has beendemonstrated in the human model for acute otitis media, andthe hemoglobin receptor for Haemophilus ducreyi has beenshown to be essential for infection in a human model system (6,172). A strong disadvantage associated with both HmbR andHpuAB as vaccine candidates is the phase variation of theirexpression and sometimes a very high frequency of switching tooff. The best fit for these receptors with respect to vaccinedevelopment would be as a second immunogen, perhaps en-hancing the efficacy of a vaccine that confers less than idealprotection.

Studies of hemoglobin receptors in N. meningitidis may notultimately lead to a protective vaccine against the pathogenicmeningococci; however, knowledge of these systems has led tothe development of bactericidal compounds which use hemetransporters for delivery into the cell (30, 154). Heme trans-porters are not unique to the meningococci. Many other patho-

genic bacteria have heme iron acquisition systems, and othersalmost certainly remain to be described (84). The developmentof broad-spectrum antibiotics that use a common portal ofentry, such as heme transport systems, warrants attention in atime when antibiotic resistance is ever increasing. Nonironmetalloporphyrins, such as gallium protoporphyrin IX, havedemonstrated strong and broad antibacterial activity and arecurrently under investigation (154).

CONCLUDING REMARKS

Four identified iron acquisition systems, the ability to usemultiple sources of iron for survival, the redundancy and phase

variation of heme iron transport systems, and the regulation ofknown virulence factors by iron attest to the importance of ironfor N. meningitidis. It has been further demonstrated that thepathogenesis of meningococci is negatively affected by the ab-sence of the transferrin and hemoglobin receptor proteins in

vivo. The essential requirement for iron and its role in patho-genesis, however, are not unique for N. meningitidis; manyother bacterial pathogens have demonstrated an essential re-quirement for iron. There are several aspects of meningococcaliron acquisition that are different from most other bacterialsystems, as well as some common themes shared with otherbacteria.

Some of the uncommon features of meningococcal iron

transport systems include two-component receptors, iron re-moval from host proteins, a lack of siderophores, the phasevariation of heme transport systems, and the existence of hemeoxygenase. (i) N. meningitidis and other closely related bacteria(such as other Neisseria species and Pasteurella species) are theonly species to express two-component outer membrane re-ceptors. Most other bacteria have single-unit transporters inaddition to secreted molecules, such as siderophores or hemo-phores (42). This is not a uniform feature, however, since themeningococcal hemoglobin receptor, HmbR, consists of a sin-gle protein unit. (ii) Also unusual, but not unique, is the lack ofsiderophore production by meningococci accompanied by thepotential use of siderophores secreted by other microorgan-isms (38). Although the search for a meningococcal sid-



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erophore has spanned many years, the possibility remains thatone exists but has not yet been identified. (iii) The iron trans-porters of meningococci are different from other transportersbecause they must remove iron or heme from high-affinityassociations with host carrier proteins, in addition to theirtransport functions. As mentioned previously, the interactionsbetween host carrier proteins and iron are extremely strong,and it remains unclear how meningococcal receptors are ableto destabilize or disrupt these interactions. (iv) Like manyother genes of the neisseriae, the hemoglobin and haptoglobin-hemoglobin receptors are subject to phase variation; this is nottypical of other bacterial iron transport systems. (v) Finally, thepresence of a heme oxygenase in meningococci, which servesto catabolize and detoxify heme, is currently an uncommonfeature of bacteria. Many bacteria are expected to possessunidentified heme oxygenases, and two others, including onefrom a gram-positive bacterium, have been identified already.

Some of the commonalities, and primary features, betweeniron transport in N. meningitidis and other bacterial iron trans-port systems include regulation by iron via the Fur repressor

protein, transport through the periplasm by ABC-like trans-porters, membership in the TonB-dependent family of recep-tor proteins, and TonB-dependent transport functions. (i)Thus far, all of the described iron transporters in N. meningi-tidis appear to be iron regulated through the Fur repressorprotein. The Fur regulon in N. meningitidis may in fact consistof a broad array of genes, since genes involved in major cata-bolic, secretory, and recombination pathways of the closelyrelated N. gonorrhoeae have been identified as part of the Furregulon (148). (ii) Ferric iron transport, and possibly hemeiron transport, requires an ABC transport system, consisting ofa periplasmic binding protein, transmembrane permease, andinner-membrane-associated ATPase (FbpABC) (see above).

Ferric siderophore transporters and related transporters usesimilar ABC transport proteins, of which the FhuBCD systemis the best characterized. (iii) Limited, but significant, homol-ogy to the ferric siderophore receptors places a component ofeach meningococcal receptor, namely, TbpA, LbpA, HmbR,and HpuB, in this family of proteins. Three members of thisfamily, i.e., FepA, FhuA, and FecA, have been crystallized.Structures reveal monomeric transmembrane -barrels thatform pores, which are plugged from the periplasm by a glob-ular amino-terminal domain. No crystal structure data areavailable for the meningococcal iron transporters; therefore,predictions of structure are made based on their inclusion inthis protein family. (iv) All of the iron transporters in N. men-ingitidis

have shown dependence on the energy-transducingTon system. This is characteristic of all members of this proteinfamily. Important questions remain unanswered for all TonB-dependent iron transport systems, not only those found in N.meningitidis. How does the Ton system transduce energy fromthe proton gradient of the cytoplasmic membrane to the outermembrane, and how do outer membrane transporters receiveand respond to this energized signal?

Despite the large volume of information known about irontransport in N. meningitidis, our understanding remains shal-low. So many questions remain unanswered. Some of these areas follows. (i) What are the structures of these different trans-porters? (ii) What is the mechanism of iron stripping andtransport? (iii) How is TonB involved? (iv) How is the cell

protected from the toxicity of free iron and free heme? (v)Why are there so many different iron transport systems inmeningococci? (vi) Why are there seemingly redundant sys-tems? (vii) Why is there phase variation of the hemoglobin andhaptoglobin-hemoglobin receptors? (viii) What is the mecha-nism of TonB-independent heme uptake reported for menin-gococci, and does there exist a second set of TonB-ExbBDproteins, as identified in species such as V. cholerae, P. aerugi-nosa, and S. marcescens, which functions in this process (52,105, 109, 157, 178)? Answers to these questions will enhanceour understanding of iron transport and utilization by N. men-ingitidis and perhaps lead to the development of an effective

vaccine.

ACKNOWLEDGMENTS

We thank William Shafer and Gordon Churchward for their sup-port. We also thank Andy Rasmussen for useful discussions of themanuscript.

This work was supported by Public Health Service grants AI472870and AI045883.

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