INFECTION AND IMMUNITY,0019-9567/99/$04.0010

Feb. 1999, p. 576–580 Vol. 67, No. 2

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Binding and Utilization of Human Transferrin byPrevotella nigrescens

PASCALE DUCHESNE,1 DANIEL GRENIER,1* AND DENIS MAYRAND2

Groupe de Recherche en Ecologie Buccale, Faculte de Medecine Dentaire,1 andFaculte des Sciences et de Genie,2 Universite Laval, Quebec, Canada

Received 17 July 1998/Returned for modification 17 September 1998/Accepted 9 November 1998

To survive and multiply within their hosts, pathogens must possess efficient iron-scavenging mechanisms. Inthe present study, we investigate the capacity of Prevotella nigrescens and Prevotella intermedia to use varioussources of iron for growth and characterize the transferrin-binding activity of P. nigrescens. Iron-saturated hu-man transferrin and lactoferrin, but not ferric chloride and the iron-free form of transferrin, could be used assources of iron by P. nigrescens and P. intermedia. Neither siderophore activity nor ferric reductase activity could bedetected in P. nigrescens and P. intermedia. However, both species showed transferrin-binding activity as wellas the capacity to proteolytically cleave transferrin. To various extents, all strains of P. nigrescens and P. inter-media tested demonstrated transferrin-binding activity. The activity was heat and protease sensitive. The ca-pacity of P. nigrescens to bind transferrin was decreased when cells were grown in the presence of hemin.Preincubation of bacterial cells with hemin, hemoglobin, lactoferrin, fibrinogen, immunoglobulin G, or laminindid not affect transferrin-binding activity. The transferrin-binding protein could be extracted from the cell sur-face of P. nigrescens by treatment with a zwitterionic detergent. Subjecting the cell surface extract to affinitychromatography on an agarose-transferrin column revealed that it contained a protein having an estimatedmolecular mass of 37 kDa and possessing transferrin-binding activity. The transferrin-binding activity of P. ni-grescens and P. intermedia may permit the bacteria to obtain iron for survival and growth in periodontal pockets.

Periodontal diseases affect the tooth-supporting tissues andare initiated by an overgrowth of specific bacterial speciesfound at the gingival margin. A number of research groupshave reported associations between the presence of specificbacterial species in periodontal pockets and the different formsof periodontal diseases (reviewed in reference 16). Althoughthe recent subdivision of strains of Prevotella intermedia intoP. intermedia and Prevotella nigrescens makes earlier microbi-ological studies difficult to interpret, these two species havebeen suggested to play an etiologic role in gingivitis and de-structive periodontitis (16). Recently, Paquet and Mouton (27)showed that strains typed as P. intermedia or P. nigrescens canbe isolated from a variety of clinical situations, including gin-gival health, gingivitis, and periodontitis. This finding suggeststhat these strains may be opportunistic pathogens.

Iron is a constituent of important metabolic enzymes and isessential for the growth of almost all microorganisms (24).Consequently, a critical component of the virulence of micro-organisms is their ability to obtain iron from their hosts. Littleis known about iron sources in the periodontal environment.Iron-containing proteins such as hemoglobin, lactoferrin, andtransferrin are known constituents of gingival crevicular fluid(GCF) (5, 7) and are likely to serve as sources of iron for thegrowth of periodontopathogens in vivo. In the course of peri-odontitis, transferrin may represent one of the most importantsources of iron for periodontopathogens. To support that idea,Curtis et al. (7) showed that transferrin, along with albuminand immunoglobulin G, was the major protein in GCF frompatients with gingivitis. They also reported that transferrin waspresent in large amounts in GCF from patients with destruc-tive periodontitis (7).

There are several different mechanisms by which pathogenicbacteria can acquire iron from human transferrin, thus allow-ing their multiplication in the host. Extracellular low-molecu-lar-mass iron-chelating molecules, also called siderophores,can sequester the iron bound to transferrin and transport it toa specific receptor present on the bacterial cell surface (13–15,24, 33). Some bacterial species can obtain iron from transferrinvia a siderophore-independent system which involves (i) pro-duction of cell surface receptors highly specific for transferrin(15, 24, 26, 32, 33); (ii) proteolytic cleavage of transferrin,resulting in disruption of the iron-binding sites, with the re-lease of free iron (26); or (iii) reduction of exogenous Fe31 andthe consequent release of Fe21 (15, 33).

Studies of sources of iron for and mechanisms of iron ac-quisition by periodontopathogens are crucial to a better under-standing of the virulence of these bacteria. Although a numberof research groups have investigated these aspects for Porphy-romonas gingivalis (2, 30), to our knowledge nothing has beendone concerning other black-pigmented anaerobic bacteria.The aims of this study were to investigate the capacity ofP. nigrescens and P. intermedia to use various sources of ironand to study the transferrin-binding activity of P. nigrescens.

MATERIALS AND METHODS

Bacterial strains and growth conditions. P. nigrescens ATCC 33563, 5W2,Cg1265, R102, T2, and YD22-4; P. intermedia ATCC 25611, A5.4/6, BH20/30,BMH, G8-9K-3, and NY363; Actinomyces viscosus 54.2; Streptococcus mutansATCC 10449; Actinobacillus actinomycetemcomitans ATCC 29522; Capnocyto-phaga ochracea 1956c; Fusobacterium nucleatum 102.3; Peptostreptococcus micros89A; and Treponema denticola ATCC 35405 were used in this study. Mostexperiments were carried out with P. nigrescens ATCC 33563 and P. intermediaATCC 25611. Bacteria were routinely grown in mycoplasma broth base (BBLMicrobiology Systems, Cockeysville, Md.) supplemented with hemin (10 mg/ml),vitamin K (1 mg/ml), and glucose (20 mg/ml) (MBB-glucose).

Growth studies were carried out with the above medium not supplementedwith hemin but treated with the chelating resin (3 g/100 ml) Chelex 100 (SigmaChemical Co., St. Louis, Mo.) for 2 h at room temperature with constantagitation. This iron-poor medium was supplemented with either hemin, ferricchloride, human lactoferrin (iron-saturated form; 1,500 mg of iron/g), human

* Corresponding author. Mailing address: Groupe de Recherche enEcologie Buccale, Universite Laval, Cite Universitaire, Quebec, Can-ada G1K 7P4. Phone: (418) 656-7341. Fax: (418) 656-2861. E-mail:Daniel.Grenier@greb.ulaval.ca.

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apotransferrin (iron-free form; #30 mg of iron/g), or human holotransferrin(iron-saturated form; 1,200 to 1,600 mg of iron/g), all at 10 mM and obtainedfrom Sigma. Human serum was also tested for its capacity to support bacterialgrowth. All cultures were incubated at 37°C in an anaerobic chamber (N2-H2-CO2, 80:10:10). To evaluate the capacity of the bacteria to use the different ironsources, cultures were serially transferred into fresh media (1% inoculum) for upto 10 successive subcultures. An optical density of 0.5 at 660 nm was requiredbefore bacteria were transferred. A compound that could sustain bacterialgrowth for at least 10 subcultures was considered an efficient source of iron.Doubling times were estimated from the semilogarithmic plot of growth curvedata.

Electrophoretic analysis of transferrin. Proteolytic cleavage of transferrinduring the growth of P. nigrescens ATCC 33563 and P. intermedia ATCC 25611was evaluated by sodium dodecyl sulfate (SDS)–12.5% polyacrylamide gel elec-trophoresis (PAGE) analysis of culture supernatants obtained at various stagesduring culturing (mid-log, early stationary, and late stationary growth phases).Electrophoresis was carried out by the procedure of Laemmli (22), and gels werestained for proteins with Coomassie brilliant blue.

Detection of siderophore activity. The universal siderophore assay of Schwynand Neilands (28) was used to evaluate the production of siderophores byP. nigrescens ATCC 33563 and P. intermedia ATCC 25611. Culture supernatantsfrom bacteria grown (36 h) in iron-chelated MBB-glucose medium (hemin free;third subculture) were mixed with Chrome Azurol S solution, and the absorbanceat 630 nm was measured. Culture supernatants concentrated 10-fold by freeze-drying were also tested. Serial dilutions (1:2) of ferrichrome (100 mg/ml inMBB-glucose; Sigma), a siderophore produced by Ustilago sphaerogena (11),were used to establish the sensitivity of the colorimetric method.

Determination of ferric reductase activity. The procedure of Morrissey et al.(25) was used to detect ferric reductase activity in supernatants and whole cellsof P. nigrescens ATCC 33563 and P. intermedia ATCC 25611 from cultures grown(36 h) in iron-chelated MBB-glucose medium (hemin free; third subculture). Analiquot of 500 ml of a culture was centrifuged, and cells were suspended in 1 mlof assay buffer (50 mM sodium citrate [pH 6.5], 5% glucose) containing 1 mMferric chloride and 1 mM bathophenanthroline disulfonate. The supernatant (pHadjusted to 6.5) of the culture was mixed with 500 ml of assay buffer (twofoldconcentrated). Samples were incubated at 30°C for 60 min in the dark, and theabsorbance at 520 nm of the assay mixture supernatant was measured. Whencells were tested, the assay mixture was centrifuged prior to measurement of theabsorbance. The level of ferrous ions produced was estimated from a calibrationcurve constructed from a solution of known ion concentrations. An uninoculatedculture medium served as a negative control. Cells of Candida albicans LAM-1were previously reported as possessing ferric reductase activity (25) and wereused as a positive control.

Determination of transferrin-binding activity. The binding of human trans-ferrin by whole cells of the strains listed above was evaluated by a solid-phase dotblot enzyme procedure. A nitrocellulose membrane was spotted with 5 ml of acell suspension (optical density at 660 nm in 50 mM phosphate-buffered saline[pH 7.2] [PBS], 1.0) of bacteria grown (36 h) in iron-chelated MBB-glucosemedium (hemin free; third subculture). This quantity corresponded to the ap-plication of approximately 5 3 106 to 15 3 106 cells, as determined with aPetroff-Hausser counting chamber. The membrane was incubated in 20 mM Trisbuffer (pH 7.5)–0.5 M NaCl (TBS) supplemented with 3% bovine serum albuminfor 1 h at room temperature with shaking. The membrane was transferred to TBScontaining 1.5% bovine serum albumin and 1 mg of horseradish peroxidase(HRP)-conjugated human transferrin (Bio/Can Scientific, Mississauga, Ontario,Canada) per ml and incubated for 4 h at room temperature with shaking. Themembrane was washed (four times for 15 min each time) in TBS containing0.05% Tween 20 and stained with a color development kit (Bio-Rad Laborato-ries, Mississauga, Ontario, Canada) in accordance with the manufacturer’s in-structions. Positive transferrin-binding activity was indicated by appearance of apurple spot. Cells of Moraxella catarrhalis PD were used as a positive control(34).

Effects of growth conditions and treatments on transferrin-binding activity.The effects of growth conditions on the transferrin-binding activity of P. nigres-cens ATCC 33563 were investigated by comparing in the solid-phase dot blotenzyme assay the activities of cells obtained after growth in (i) iron-chelatedMBB-glucose medium and (ii) iron-chelated MBB-glucose medium supple-mented with 10 mM hemin. In order to determine the nature of the moleculesinvolved in the transferrin-binding activity, cells of P. nigrescens ATCC 33563were submitted to various treatments prior to the dot blot enzyme assay. Theheat stability of the transferrin-binding activity was tested by incubation (10 min)of whole cells at 60, 70, or 80°C. Bacteria were also incubated for 4 h at 37°C witheither pancreatic trypsin, pancreatic chymotrypsin, or proteinase K at a finalconcentration of 1.0 mg/ml. Lastly, the effects of putative inhibitors of trans-ferrin-binding activity were evaluated by preincubating the cells in the presenceof selected substances. The molecules included in this experiment were hemin,human holotransferrin, human apotransferrin, human hemoglobin, bovine lac-toferrin, human fibrinogen, human immunoglobulin G, and human laminin, all at1.0 mg/ml.

Identification of transferrin-binding proteins. Transferrin-binding proteins ofP. nigrescens ATCC 33563 were extracted by suspending whole cells from a500-ml culture (36 h; third subculture in iron-chelated hemin-free MBB-glucose

medium) in 40 ml of 50 mM PBS (pH 8.0) containing 10 mM EDTA and 0.5%Zwittergent 3-14 (Calbiochem, La Jolla, Calif.). After gentle shaking for 18 h at4°C, the suspension was centrifuged (10,000 3 g for 30 min), and the supernatantwas collected. This extract was submitted to affinity chromatography to isolatemolecules with transferrin-binding activity as described by Ferron et al. (12).Briefly, holotransferrin in PBS (pH 8.0) (50 mg in 10 ml) was incubated withagarose beads (Affi-Gel 15; Bio-Rad) for 18 h at 4°C. Thereafter, the beads wereharvested and further incubated (1 h at 4°C) in PBS (pH 8.0) containing 1 Mglycine to block the nonreactive ester groups. After the samples were washed inPBS (pH 8.0), the agarose-transferrin was placed in a column (0.7 by 12 cm)equilibrated with PBS (pH 8.0) containing 0.5% Zwittergent 3-14. The bacterialextract was loaded, and the column was washed with PBS. Bound proteins wereeluted with 100 mM glycine-HCl (pH 3.2), and the fractions (2 ml) obtained werebrought to neutrality with 1 N NaOH. The fractions were analyzed for trans-ferrin-binding activity with the solid-phase dot blot enzyme assay. Fractionsshowing activity were pooled and concentrated (10-fold) by ultrafiltrationthrough a 10,000-Da (nominal molecular mass cutoff) membrane filter. Proteinscontained in this final fraction were separated by SDS–12% PAGE by theprocedure of Laemmli (22). After electrophoresis, proteins were visualized bystaining with silver nitrate. The fraction was also electrophoretically transferredto a nitrocellulose membrane at a constant voltage of 60 V for 2 h. The presenceof protein bands with transferrin-binding activity was determined with HRP-conjugated transferrin as described for the dot blot enzyme assay.

RESULTS

In the first part of this study, the growth of P. nigrescensATCC 33563 and P. intermedia ATCC 25611 was evaluated byuse of an iron-chelated MBB-glucose medium supplementedwith different sources of iron. The results obtained are sum-marized in Table 1. Although the bacteria could grow signifi-cantly for the first three subcultures, the iron-chelated mediumwithout supplements was unable to support the long-termgrowth of either species. The initial growth obtained in theabsence of a source of iron was likely related to cellular re-serves of hemin or to a carryover of iron during inoculation.Supplementing the medium with either hemin, lactoferrin, orholotransferrin allowed growth for at least 10 successive sub-cultures. The minimal amount of holotransferrin required tosupport bacterial growth was found to be 2.5 mM. On the otherhand, ferric chloride and apotransferrin, the iron-free form oftransferrin, were not able to support the long-term growth ofeither species. When human serum was used as a growth me-dium, P. nigrescens could be cultivated for 10 subcultures,whereas P. intermedia did not grow for more than 1 subculture.Comparison of the growth of P. nigrescens in a medium con-taining either holotransferrin or hemin (10 mM) as the sourceof iron revealed similar growth rates (doubling time, 5.75 h).However, the final optical density at 660 nm obtained withhemin was higher than that obtained with holotransferrin (1.12compared to 0.82, respectively).

SDS-PAGE analysis of supernatants obtained at variousstages during the growth of P. nigrescens revealed partial pro-teolytic degradation of transferrin (Fig. 1). Most of the degra-dation seemed to occur once the culture had reached thestationary growth phase. Initial cleavage of the transferrin mol-

TABLE 1. Growth responses of P. nigrescens and P. intermediaunder different iron conditions

StrainGrowth response under the following iron conditiona:

None Hemin FeCl3 Lactoferrin Apo-Tf Holo-Tf Serum

P. nigrescensATCC 33563

2 1 2 1 2 1 1

P. intermediaATCC 25611

2 1 2 1 2 1 2

a Hemin, FeCl3, lactoferrin, apotransferrin (Apo-Tf), and holotransferrin(Holo-Tf) were added at 10 mM in iron-chelated MBB-glucose medium. None,no iron was added. 1, growth was obtained for at least 10 successive subcultures;2, no long-term growth.

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ecule was associated with the generation of a fragment whichhad a molecular mass of approximately 40 kDa and which wasfurther degraded into peptides too small (,15 kDa) to bedetected by the electrophoretic procedure used. The possibilitythat the binding of transferrin to bacterial cells also might havebeen partly responsible for the decreased intensity of the trans-ferrin band in the culture supernatant should not be excluded.Similar results for the proteolytic degradation of transferrin byP. intermedia were also obtained (data not shown).

Siderophore activity in the culture supernatants of P. nigres-cens ATCC 33563 and P. intermedia ATCC 25611 grown underiron-restricted conditions could not be detected by the univer-sal Chrome Azurol S assay (28). Concentrated culture super-natants (10-fold) were also devoid of activity. The minimalconcentration of ferrichrome required to yield a positive reac-tion was 5 mg/ml. Ferric reductase activity could not be de-tected in the supernatants of P. nigrescens and P. intermediagrown in the iron-restricted culture medium. Bacterial cellswere also devoid of ferric reductase activity. The reduction ofFe31 was observed with cells of C. albicans, which served as thepositive control.

Transferrin-binding activity was tested by a solid-phase dotblot enzyme assay in which whole cells were immobilized on anitrocellulose membrane, which was then probed with HRP-conjugated human transferrin (Fig. 2). With this binding assay,a positive reaction was obtained with M. catarrhalis, P. nigres-cens ATCC 33563, and P. intermedia ATCC 25611. Additional

strains of P. nigrescens and P. intermedia as well as a number ofgram-positive and gram-negative bacterial species (A. viscosus,S. mutans, A. actinomycetemcomitans, C. ochracea, F. nuclea-tum, P. micros, and T. denticola) were tested in this assay. Allstrains of P. nigrescens and P. intermedia were found to bindtransferrin to various extents, whereas the other bacterial spe-cies under investigation did not show any transferrin-bindingactivity. Strains of P. nigrescens and P. intermedia could becategorized as reacting strongly (ATCC 33563, ATCC25611, G8-9K-3, T2, NY363, R102, BH20/30, and Cg1265) orweakly (5W2, YD22-4, BMH, and A5.4/6) in the assay.

The transferrin-binding activity of P. nigrescens ATCC 33563was further investigated. Growth conditions appeared to mod-ulate the level of transferrin-binding activity (Fig. 3). Cellscultivated in the presence of hemin showed much less trans-ferrin-binding activity than cells grown in the hemin-free iron-chelated MBB-glucose medium. The effects of various treat-ments on the binding of transferrin by P. nigrescens ATCC33563 are shown in Fig. 3. Heat treatment (10 min) of wholecells at 70°C completely inhibited the binding of transferrin,whereas no effect was observed after treatment at 60°C. Treat-ment of cells with proteolytic enzymes (trypsin, chymotrypsin,or proteinase K) was associated with a strong decrease intransferrin-binding activity. The effect of putative inhibitors onthe transferrin-binding activity of P. nigrescens was also inves-tigated by the dot blot enzyme assay. Preincubation of bacteriawith hemin, hemoglobin, lactoferrin, fibrinogen, immunoglob-ulin G, or laminin did not affect transferrin-binding activity,whereas complete inhibition was obtained when cells werepreincubated with either the iron-free or the iron-saturatedform of transferrin.

Cells of P. nigrescens ATCC 33563 were treated with a zwit-terionic detergent (Zwittergent 3-14) to solubilize transferrin-binding proteins from the outer cell envelope. This extractdemonstrated strong transferrin-binding activity, as revealedby the solid-phase dot blot enzyme assay (data not shown). Inorder to identify the transferrin-binding proteins of P. nigres-cens, the extract was submitted to affinity chromatography onan agarose-transferrin column. The fractions showing trans-ferrin-binding activity were pooled and analyzed by SDS-PAGE (Fig. 4A). Two bands, 37 and 80 kDa, were visualizedby silver nitrate staining. The 80-kDa band was found to reactwith an antitransferrin antibody and likely represented trans-ferrin molecules that got loose from the agarose beads (datanot shown). The pooled fractions were also analyzed by SDS-PAGE, Western blotting, and reactivity with HRP-conjugatedtransferrin (Fig. 4B). These procedures revealed that only the37-kDa band possessed transferrin-binding activity.

FIG. 1. SDS-PAGE analysis of transferrin in the culture supernatant of P. ni-grescens ATCC 33563 grown in iron-chelated MBB-glucose medium (hemin-free) supplemented with holotransferrin. Lane 1, uninoculated culture medium;lane 2, mid-log growth phase (12-h culture); lane 3, early stationary growth phase(24-h culture); lane 4, late stationary growth phase (36-h culture); and lane 5, latestationary growth phase (48-h culture). Molecular mass markers were, from topto bottom, phosphorylase b (97.4 kDa), bovine serum albumin (68 kDa), ovalbu-min (43 kDa), carbonic anhydrase (29 kDa), and b-lactoglobulin (19 kDa).

FIG. 2. Demonstration of transferrin-binding activity of oral bacteria by asolid-phase dot blot enzyme assay. (A) M. catarrhalis PD. (B) P. nigrescens ATCC33563. (C) P. intermedia ATCC 25611. (D) P. nigrescens 5W2. (E) P. intermediaBMH. (F) A. actinomycetemcomitans ATCC 29522.

FIG. 3. Effect of various growth conditions or treatments on transferrin-binding activity of P. nigrescens ATCC 33563, as determined by the solid-phasedot blot enzyme assay. (A) Cells grown in the presence of hemin. (B) Cells grownin the iron-chelated medium. (C) Cells treated with trypsin. (D) Cells treated at70°C. (E) Cells preincubated with immunoglobulin G. (F) Cells preincubatedwith iron-free transferrin.

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DISCUSSION

Since iron plays significant roles in metabolic reactions, theability of bacterial pathogens to obtain this growth-essentialnutrient from their hosts is a major virulence determinant.Transferrin, whose major physiological role is the solubiliza-tion of Fe31 and its delivery from sites of absorption andstorage to sites of utilization, plays an important role in hostdefense by rendering the iron nonavailable for microorgan-isms. Several different mechanisms for iron acquisition fromtransferrin have been demonstrated for pathogenic bacteria(15, 24, 26, 32, 33).

In this study, we showed the capacity of P. nigrescens andP. intermedia to grow in the presence of holotransferrin but notapotransferrin. This finding suggests that both bacterial speciescan obtain iron from the iron-loaded form of the plasma pro-tein transferrin. The absence of growth in the presence ofapotransferrin, in addition to being related to the lack of iron,may have resulted from the antibacterial activity of the mole-cule. Indeed, Ellison et al. (10) previously reported that iron-binding proteins (lactoferrin and transferrin) can alter theouter membrane permeability of Gram-negative bacteria. Thisalteration was thought to be related to the chelating propertyof the molecules. We also found that P. nigrescens could growin human serum, a condition that more closely mimics an invivo situation. This result indicates that P. nigrescens is resistantto serum bactericidal activity and supports the idea that thisbacterial species may be able to utilize iron-bound transferrinpresent in the GCF found in periodontal pockets.

Neither siderophore activity nor ferric reductase activitycould be detected in P. nigrescens and P. intermedia. However,

both species showed transferrin-binding activity as well as pro-teolytic activity toward transferrin. The transferrin-binding ca-pacity appeared not to be a characteristic common in oralbacteria, since among the nine bacterial species tested, onlystrains of P. nigrescens and P. intermedia were found to attachto transferrin. The transferrin-binding activity was decreasedwhen cells were grown under hemin-plentiful conditions com-pared to iron-restricted conditions. Since preincubation of bac-terial cells with hemin did not show any inhibitory effect on thetransferrin-binding activity (in the dot blot enzyme assay), theactivity may be hemin regulated. Regulation of transferrinreceptor expression by hemin has been reported for Haemophi-lus influenzae (17).

The binding of transferrin to P. nigrescens did not involveelectrostatic interactions, since sodium chloride was includedat 0.5 M during the incubation with HRP-conjugated trans-ferrin. Preincubation of cells with either iron-free or iron-saturated transferrin prevented the binding of HRP-conju-gated transferrin, indicating that the level of iron saturation ofthe transferrin molecule had no effect on the binding to bac-teria. Proteins with transferrin-binding activity could be ex-tracted from the cell surface of P. nigrescens by treatment witha zwitterionic detergent. Affinity chromatography on a trans-ferrin-agarose column allowed the isolation of a transferrin-binding protein with an estimated molecular mass of 37 kDa.

Receptors for human transferrin have been demonstratedfor a variety of pathogenic bacteria (13, 14, 32, 33). Theirmolecular masses appear to be variable, as a receptor of ap-proximately 102 kDa has been reported for Neisseria gonor-rhoeae (13), whereas in Borrelia burgdorferi, the binding oftransferrin involves a protein of 28 kDa (3). The best-charac-terized mechanism of iron acquisition involving cell surfacetransferrin-binding proteins concerns N. gonorrhoeae (4, 6, 13,24). It has been demonstrated that two proteins (TbpA [;102kDa] and TbpB [;85 kDa]) of the outer membrane are in-volved in the binding of transferrin and that the iron is re-moved from the transferrin in an energy-dependent process. Athird protein (FbpA [;33.5 kDa]) acts as a shuttle vector,transporting the iron through the periplasm to the cytoplasmicmembrane. Transferrin-binding activity has been also observedfor the oral bacterial species P. gingivalis (31) and Streptococcusoralis (1). Tazaki et al. (31) reported the capacity of P. inter-media to bind human transferrin but did not investigate furtherthe activity.

We observed that P. nigrescens and P. intermedia could de-grade human transferrin. This finding is in agreement with aprevious study by Jansen et al. (18), who reported the degra-dation of various serum proteins, including transferrin, hapto-globin, albumin, and immunoglobulins. Moreover, P. interme-dia has been shown to possess on its cell envelope a 31-kDaserine protease with elastase-like activity (29). Since the deg-radation of transferrin by P. nigrescens and P. intermedia oc-curred mostly at the end of growth (stationary growth phase),it is likely that this activity plays only a minor role in the abilityof the bacteria to grow in the presence of this plasma proteinas the source of iron.

Our study also revealed that lactoferrin could serve as asource of iron for P. nigrescens and P. intermedia. Lactoferrin-binding activity was previously demonstrated for both species(8, 9, 20). However, the relationship between the presence ofthis activity in the bacteria and their ability to grow in thepresence of lactoferrin as a source of iron has not been estab-lished. The lactoferrin-binding protein present in the outermembrane of P. nigrescens was reported to have a molecularmass of 40 kDa (9), close to the 37-kDa transferrin-bindingprotein demonstrated in the present study. We found that no

FIG. 4. SDS-PAGE analysis of transferrin-binding proteins obtained by af-finity chromatography of a zwitterionic detergent extract from P. nigrescensATCC 33563. (Left gel) Silver nitrate staining. Lane 1, molecular mass standards(from top to bottom) phosphorylase b (97.4 kDa), bovine serum albumin (68kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and b-lactoglobulin (19kDa); lane 2, zwitterionic detergent extract (initial material); lane 3, pooledfractions, which showed transferrin-binding activity in the solid-phase dot blotenzyme assay. (Right gel) Western blot showing reactivity with HRP-conjugatedtransferrin. Pooled fractions which showed transferrin-binding activity in thesolid-phase dot blot enzyme assay were tested.

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inhibition of transferrin-binding activity occurred in the pres-ence of lactoferrin, suggesting the involvement of two differentproteins. P. nigrescens is also known to possess receptors forimmunoglobulin G, laminin, and fibrinogen (19, 21, 23). Onceagain, no inhibition of binding of transferrin was observedwhen bacteria were preincubated with these molecules, sug-gesting that different receptors participate in binding.

In summary, we showed that P. nigrescens and P. intermediahave the capacity to use human transferrin as a source of ironand possess transferrin-binding activity. A 37-kDa protein withtransferrin-binding activity was identified on the surface ofP. nigrescens. However, further studies are required to demon-strate that this protein is a receptor specific for human trans-ferrin. P. nigrescens and P. intermedia also demonstrated thecapacity to proteolytically cleave transferrin. Both activitiesmay permit the bacteria to obtain iron for their survival andgrowth in periodontal pockets.

ACKNOWLEDGMENTS

This work was supported by the Fonds FCAR, the Reseau de Re-cherche en Sante Bucco-Dentaire du FRSQ, and the Laboratoire deControle Microbiologique.

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