Characterization of Binding of Human Lactoferrin to ... · Anti-PspA family 2 antiserum was...

INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.5.3372–3381.2001

May 2001, p. 3372–3381 Vol. 69, No. 5

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Characterization of Binding of Human Lactoferrin toPneumococcal Surface Protein A

ANDERS HAKANSSON,1,2* HAZELINE ROCHE,1 SHAPER MIRZA,1 LARRY S. MCDANIEL,3

ALEXIS BROOKS-WALTER,1† AND DAVID E. BRILES1

Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama1; Section of Microbiology,Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden2; and Department of

Microbiology and Surgery, The University of Mississippi Medical Center, Jackson, Mississippi3

Received 12 December 2000/Returned for modification 8 January 2001/Accepted 29 January 2001

Human lactoferrin is an iron-binding glycoprotein that is particularly prominent in exocrine secretions andleukocytes and is also found in serum, especially during inflammation. It is able to sequester iron frommicrobes and has immunomodulatory functions, including inhibition of both complement activation andcytokine production. This study used mutants lacking pneumococcal surface protein A (PspA) and PspC todemonstrate that the binding of human lactoferrin to the surface of Streptococcus pneumoniae was entirelydependent on PspA. Lactoferrin bound both family 1 and family 2 PspAs. Binding of lactoferrin to PspA wasshown by surface colocalization with PspA and was verified by the lack of binding to PspA-negative mutants.Lactoferrin was expressed on the body of the cells but was largely absent from the poles. PspC showed exactlythe same distribution on the pneumococcal surface as PspA but did not bind lactoferrin. PspA’s binding sitefor lactoferrin was mapped using recombinant fragments of PspA of families 1 and 2. Binding of humanlactoferrin was detected primarily in the C-terminal half of the a-helical domain of PspA (amino acids 167 to288 of PspA/Rx1), with no binding to the N-terminal 115 amino acids in either strain. The interaction washighly specific. As observed previously, bovine lactoferrin bound poorly to PspA. Human transferrin did notbind PspA at all. The binding of lactoferrin to S. pneumoniae might provide a way for the bacteria to interferewith host immune functions or to aid in the acquisition of iron at the site of infection.

Lactoferrin is an iron-binding glycoprotein present in milkand mucosal secretions. It is also released by specific granulesof polymorphonuclear leukocytes during inflammation (35,36). It is a member of the siderophilin family and is structurallyrelated to the more abundant serum protein transferrin (40).Lactoferrin has been ascribed many diverse biological func-tions, most of which are immunomodulatory or antibacterial(5, 7, 20–22, 37, 57, 58). It can inhibit cytokine activation,myelopoiesis, and complement activation (21, 34, 37, 54). Italso plays a role in host resistance by sequestering from bac-teria the free iron necessary for bacterial growth and by thebactericidal activity of an N-terminal fragment released afterpepsin digestion in the gut (22, 56, 58).

Streptococcus pneumoniae is an important cause of respira-tory tract infections, bacteremia, and meningitis. These infec-tions are especially common in young children and in theelderly (3, 26). Infection usually starts with asymptomatic car-riage in the nasopharynx. Bacteria can then, in some cases,spread to other locations such as the lungs, middle ear, andblood (4, 26, 55). To effectively infect the host, pneumococcihave to survive and evade the immune system in the nasophar-ynx as well as at other sites within the host. This may beaccomplished by binding immunomodulatory molecules, suchas lactoferrin, at the site of infection.

S. pneumoniae has been reported to bind lactoferrin (28).Using radiolabeled, milk-purified lactoferrin, Hammerschmidtet al. observed interaction of lactoferrin with 88% of the clin-ical S. pneumoniae isolates tested. The bacterial receptor waspurified by affinity chromatography and identified as pneumo-coccal surface protein A (PspA). This interaction with purifiedlactoferrin was further verified using purified PspA.

In the present study, we have more completely characterizedthe binding of lactoferrin to S. pneumoniae. To avoid the po-tential presence of other copurified proteins frequently asso-ciated with the purification of lactoferrin and other proteinsfrom milk, we used recombinant human lactoferrin in ourbinding studies. Also, we used fluorescence methodology toquantitate and microscopically visualize binding of lactoferrinto the bacterial surface, something that was not attempted inthe original study (28). By using recombinant lactoferrin andan isogenic pneumococcal strain lacking expression of PspA, ithas been shown for the first time that the binding of lactoferrinto the pneumococcal surface is dependent on PspA and thatPspC is not involved in lactoferrin binding. These studies havealso revealed localized surface distribution of PspA and iden-tified the region of PspA that binds to lactoferrin.

MATERIALS AND METHODS

Reagents. Protein markers were from Amersham Pharmacia Biotech (Pis-cataway, N.J.). NBT (nitroblue tetrazolium) and BCIP (5-bromo-4-chloro-3-indolylphosphate) were from Fisher Scientific (Atlanta, Ga.). Alkaline phos-phatase (AP)-conjugated streptavidin, biotin-conjugated goat anti-mouseimmunoglobulin (Ig), and biotin-conjugated goat anti-rabbit Ig antibodies werefrom Southern Biotechnology Associates (Birmingham, Ala.). Fluorescein iso-thiocyanate (FITC)-conjugated streptavidin, R-phycoerythrin (RPE)-conjugatedstreptavidin, FITC-conjugated rabbit anti-mouse Ig antibodies, and FITC-con-

* Corresponding author. Mailing address: Department of Microbi-ology, University of Alabama at Birmingham, BBRB-662 Box 10, 84519th Street South, Birmingham, AL 35294. Phone: (205) 934-8511.Fax: (205) 934-0605. E-mail: [email protected].

† Present address: Department of Biology, Florida A&M University,Tallahassee, FL 32307.

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jugated goat anti-rabbit Ig antibodies were from Dako A/S (Rothskild, Den-mark). Bacto-Todd Hewitt medium and yeast extract were from Difco Labora-tories (Detroit, Mich.). Human and bovine milk lactoferrin and humantransferrin were from Sigma Chemical Co (St. Louis, Mo.).

Recombinant human lactoferrin was kindly provided by Arne Forsgren, De-partment of Medical Microbiology, Lund University, University Hospital Malmo,Malmo, Sweden.

Monoclonal anti-PspA antibodies Xi126 and XiR278 have been previouslydescribed (38). Polyclonal anti-PspC serum was raised by immunization with atruncated PspC molecule from S. pneumoniae L81905 expressed in Escherichiacoli (19). Polyclonal anti-PspA family 1 antiserum was pooled from two rabbitsimmunized with recombinant PspA/L82016 (clade 1) or recombinant PspA/Rx1(clade 2) expressed in E. coli. Anti-PspA family 2 antiserum was produced bypooling serum from two rabbits immunized with either recombinant PspA/V-024(clade 3) or recombinant PspA/V-032 (clade 4) expressed in E. coli. To this pool,recombinant PspA/Rx1 was added to reduce its cross-reactivity with family 1(clade 1 and 2) PspAs. Approximately 10 mg of the purified PspA proteins wasinjected subcutaneously into a rabbit twice on consecutive weeks, and blood wascollected 10 days after the last injection. The primary immunization was givenwith Freund’s complete adjuvant, and the booster immunization was given insaline.

Bacteria. The strains and plasmids used in this study are described in Table 1.The pneumococcal strains were stored at 280°C in fetal calf serum, transferredto blood agar plates, and incubated at 37°C in a 5% CO2 atmosphere overnight.Colonies grown on blood agar were used to inoculate liquid growth medium(Todd-Hewitt medium containing 0.5% yeast extract [THY]). Upon reachinglate log phase, the bacteria were harvested by centrifugation at 1,500 3 g for 15min and suspended in 60 mM phosphate-buffered saline (PBS, pH 7.2). Thebacterial concentration was estimated by interference contrast microscopy (TELeitz Ortolux II microscope with interference contrast equipment; Leitz, Wetz-lar, Germany) using a Burker chamber and confirmed by counting viable cells.Appropriate dilutions of the bacteria were suspended in PBS.

The pspC gene was insertionally inactivated in S. pneumoniae D39. An internalfragment of pspC was amplified using PCR and cloned into pSF143 (51). Theligated plasmid was electroporated into E. coli DH5a, and clones were selectedfor tetracycline resistance. The plasmid, which contained the internal fragment ofpspC, was isolated from recombinant E. coli using standard procedures andtransformed into S. pneumoniae D39 (30). Tetracycline-resistant recombinants

were screened by both Southern hybridization and Western blotting to confirminactivation of pspC. Lysate from the strain containing the insertionally inacti-vated pspC (TRE118) was transformed into JY53 (erythromycin resistant, pspAnegative) to create a mutant that lacked both pspA and pspC (TRE121) (23, 59).

Binding of lactoferrin and transferrin to bacterial cells. Purified lactoferrinfrom human or bovine milk, recombinant human lactoferrin, and human trans-ferrin were biotinylated using the Roche biotin labeling kit according to themanufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, Ind.).

Bacteria were grown in THY medium (S. pneumoniae) or on chocolate agarplates (Moraxella catarrhalis) and suspended in PBS at a concentration of ap-proximately 5 3 108 bacteria/ml. The bacterial suspension (100 ml) was mixedwith 0.5 to 10 ml of biotinylated protein (2-mg/ml stock solution in PBS) for 30min at room temperature and washed by centrifugation at 1,500 3 g for 5 min inPBS. FITC-conjugated streptavidin (1:100 dilution in PBS) was added for anadditional 30 min at room temperature, and after a final wash in PBS, the cellswere inspected by epifluorescence and laser scanning confocal microscopy usingMRC-1024 confocal equipment (Bio-Rad Laboratories, Hemel-Hampstead,United Kingdom) attached to a Nikon Eclipse E800 upright microscope (Nikon,Tokyo, Japan). The binding was quantitated by flow cytometry using a FACS-Calibur flow cytometer (Becton Dickinson Biosciences, Rutherford, N.J.).

Antibody staining of S. pneumoniae. Studies of the colocalization of PspA andlactoferrin were performed using the monoclonal anti-PspA antibodies Xi126and XiR278, recognizing the N-terminal and the more distal part of the a-helicalregion of PspA, respectively (38). Bacteria were first incubated with 5 ml ofbiotinylated lactoferrin and washed in PBS, and the bacteria were then fixed for5 min in 4% formaldehyde in PBS. After the bacteria had been washed in PBS,monoclonal antibodies (undiluted hybridoma supernatant) were added for anadditional 30 min. After a third wash in PBS, the bacteria were incubated withRPE-conjugated streptavidin (1:100 in PBS) and FITC-conjugated rabbit anti-mouse Ig antibodies (1:100 in PBS) for 30 min at room temperature, and bindingwas inspected by epifluorescence and confocal microscopy. Controls treatedwithout the monoclonal anti-PspA antibody showed no staining with FITC.

Staining for PspC on the bacterial surface was done using anti-PspC antibodiesas described above. As the anti-PspC antibodies also recognize PspA (19),staining for PspC was performed using the PspA-negative mutant JY53.

Staining for the cell wall was achieved using anti-phosphoryl choline mono-clonal antibody 59.6C5 (13) using the technique described above.

TABLE 1. Bacterial strains and plasmids

Strain or plasmid Characteristics Source or reference

S. pneumoniaeD39 Type 2 encapsulated 6, 59Rx1 Nonencapsulated mutant of D39 48JY53 pspA mutant of D39 59TRE118 pspC mutant of D39 This studyTRE121 pspA pspC mutant of D39 This studyWU2 Type 3 encapsulated 17EF3296 Type 4 encapsulated 2, 59L81905 Type 4 encapsulated 19EF3030 Type 19F encapsulated 1CCUG10175 Type 19F encapsulated 2

M. catarrhalis Clinical isolate Clinical bacteriology lab, Lund, Sweden

E. coli M15 Qiagen

PlasmidspHR101 pQE40::BglII-HindIII fragment, encodes PspA/EF3296 (1–115) This studypHR102 pQE40::SalI-SphI fragment, encodes PspA/EF3296 (75–305) This studypHR105 pQE40::SalI-SphI fragment, encodes PspA/EF3296 (1–411) This studypHR107 pQE40::SalI-SphI fragment, encodes PspA/EF3296 (75–490) This studyJAS218 PQE40::BamHI-SalI fragment, encodes PspA/Rx1 (167–288) This studyPBAR4285 Encodes PspA/Rx1 (1–115) 38, 44, 59pBAR4310 Encodes PspA/Rx1 (1–192) 38, 44, 59pBAR501 Encodes PspA/Rx1 (288–563) 38, 44, 59pUAB55 Encodes PspA/Rx1 (1–370) 12, 19pUAB103 Encodes PspA/Rx1 (1–303) 19

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PspA and PspA fragments. Full-length PspA was purified from S. pneumoniaeRx1 and EF3296 as described (16, 60). PspA fragments BAR4285, BAR4310,and BAR501 from strain Rx1 were produced as described (38, 44). BAR4285 andBAR4310 are derived from pJY4285 and pJY4310, respectively, originally clonedinto the pUC18 vector, as described (59). The inserts were moved into pMal-p2and expressed as described (44). The predicted sizes of the expression constructswere 55.2 kDa (BAR4285), 63.6 kDa (BAR4310), and 72.8 kDa (BAR501). PspAfragments UAB055 and UAB103 from strain Rx1 were produced as described(12, 19). PspA fragments HR101 (primer pair ABW23 and LSM12), HR102(primer pair HR10 and HR11), and HR107 (primer pair HR10 and HR14) fromS. pneumoniae EF3296 pspA and JAS218 (primer pair LSM150 and LSM16)from S. pneumoniae Rx1 pspA were expressed in E. coli strain M15 using theexpression vector pQE40 (Qiagen Inc, Chatsworth, Calif.). The primers used forPCR are described in Table 2. PCR-amplified fragments of pspA were clonedinto SphI-SalI-, BglI-HindIII-, or BamHI-SalI-digested pQE40 vector (Table 1)and transformed into M15(pREP4), a K-12-derived E. coli strain containing aplasmid which carries a lac repressor, allowing control over expression. Clonescontaining the different pspA inserts were identified by Southern blot analysesusing digoxigenin-labeled pspA probes (38). Expression of PspA fragments frompositive clones was induced with 1 mM IPTG (isopropylthiogalactopyranoside)during growth at room temperature. The overexpressed protein fragments werepurified by affinity chromatography using a nickel resin according to the manu-facturer’s instructions. The different constructs encoded predicted 38.6-kDa(HR101), 52-kDa (HR102), 71.8-kDa (HR107), and 39.0-kDa (JAS218) PspAfragments, which were analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and quantified using the Bio-Rad DC protein assay (Bio-RadLaboratories, Hercules, Calif.). PspC was purified as described (19).

Dot blot. PspA, fragments of PspA, and PspC (10 mg/ml and 1:3 serial dilutionsof these stock solutions) were applied to a 0.45-mm nitrocellulose membrane(Millipore, Bedford, Mass.), and the membrane was allowed to dry. The mem-brane was blocked with 1% bovine serum albumin (BSA) in PBS for 45 min atroom temperature and washed three times with PBS containing 0.1% Tween 20(PBS-T). The membrane was overlaid with biotinylated recombinant and milk-purified human lactoferrin, bovine lactoferrin, or biotinylated human transferrin(1:500 dilution in PBS-T of the 2.0-mg/ml stock solution) for 45 min at roomtemperature and washed three times in PBS-T. After an additional incubationwith AP-conjugated streptavidin (1:500 dilution in PBS-T) for 45 min at roomtemperature, the membrane was developed using 1 mg of NBT and 5 mg of BCIPper 10 ml of 0.15 M Tris-HCl (pH 8.8).

Western blot. PspA, fragments of PspA, and PspC (0.5 mg) were run on 10%polyacrylamide gels (Bio-Rad Ready gels; Bio-Rad Laboratories); and the gelswere electroblotted to a 0.45-mm nitrocellulose membrane (Bio-Rad) in Tris-glycine buffer (20% methanol, 25 mM Tris, 192 mM glycine [pH 8.1 to 8.4]) at100 V for 1 h at 4°C. The blotted membrane was incubated with 1% BSA inPBS-T for 45 min at room temperature and washed three times (5 min each) withPBS-T. The membranes were overlaid with biotinylated human recombinant ormilk-purified lactoferrin (1:500 dilution in PBS-T of 2.0-mg/ml stock solution),with anti-PspA antibodies (monoclonal Xi126 or polyclonal anti-PspA family 1antiserum), or with polyclonal anti-PspA family 2 antiserum for 30 min at 37°Cand washed three times in PBS-T. The anti-PspA-exposed membrane was furtherincubated with a mix of biotinylated goat anti-mouse or goat anti-rabbit Igantibodies (1:1,000 in PBS-T) and AP-conjugated streptavidin (1:500 dilution inPBS-T) for 30 min at 37°C, and the lactoferrin-exposed membrane was incubated

with streptavidin only. After washing, the membrane was developed using 1 mgof NBT and 5 mg of BCIP per 10 ml of 0.15 M Tris-HCl (pH 8.8).

RESULTS

Binding of human lactoferrin to S. pneumoniae. S. pneu-moniae D39 was incubated with human recombinant lactofer-rin (Fig. 1A) or lactoferrin purified from human milk (data notshown), counterstained with FITC-conjugated streptavidin,and inspected by confocal or epifluorescence microscopy. Re-combinant lactoferrin and lactoferrin from human milkshowed identical binding patterns of strong binding to D39.This observation made it clear that lactoferrin, and not a con-taminating milk protein, was responsible for the binding to thepneumococcal surface. Similar binding was observed for S.pneumoniae EF3296 (data not shown).

The surface binding of recombinant lactoferrin was furtherquantitated using flow cytometry, resulting in a clear shift offluorescence intensity with increased concentrations of lacto-ferrin (Fig. 1C and D). At binding saturation (50 to 100 mg oflactoferrin per ml), 31.1 (range, 17.3 to 65.4) times higherfluorescence intensity was observed for S. pneumoniae D39compared with the streptavidin-treated control cells (Fig. 1Cand D). A similar fluorescence intensity shift was seen for thebinding of human lactoferrin to S. pneumoniae EF3296 (22.2times above the control [range, 15.3 to 27.6]).

Binding of human lactoferrin to PspA. The binding of lac-toferrin to S. pneumoniae has been reported to involve PspA.Recombinant PspA has been shown to inhibit the associationof radiolabeled lactoferrin with whole bacteria (28). However,the functional interaction of lactoferrin and PspA and thedependence on PspA for lactoferrin binding on the bacterialsurface were not previously addressed. We have investigatedthese questions in two ways: colocalization experiments of lac-toferrin and monoclonal anti-PspA antibodies on the bacterialsurface and experiments using mutants of S. pneumoniae lack-ing PspA surface expression. Binding was analyzed by confocalmicroscopy and flow cytometry.

Staining of the bacteria with lactoferrin and monoclonalanti-PspA antibodies showed a pattern of colocalization con-sistent with the binding of lactoferrin to PspA (Fig. 2A). Thepattern for lactoferrin binding and anti-PspA antibody stainingover the bacterial surface was not uniform but displayed local-ized binding with areas of higher and lower intensity. In mostcases PspA was only poorly expressed at the poles of the cells.Most of the localized staining was thus present along the bodyof the cell, although some examples of other staining patternscould be observed. On close examination, the most commonpattern of staining could be discerned based on diplococcalunits. Within a four-coccus chain, there was faint staining nearthe poles of the most recent cell division but none near thepoles of the preceding cell division (Fig. 1A and 2A). Thispattern of staining was seen consistently for all wild-typestrains used in the study regardless of capsular type (D39, type2; WU2, type 3; EF3296, type 4; L81905, type 4; EF3030, type19F; and CCUG10175, type 19) and was also present in pneu-mococci lacking a capsule (Rx1). The staining pattern of PspAwas different from the homogenous staining of the cell wall ofS. pneumoniae Rx1 using anti-phosphoryl choline antibodies(Fig. 1B). This suggests that the localized expression of PspA

TABLE 2. Primers used for expression of recombinantPspA fragmentsa

PrimerPosition insequence

(bp)Sequence

ABW23 400–417 59-TCTGATATTCAGCGTCAG-39HR10 428–447 59-GCATATAAAGAGTACCGAGA-39HR11 1103–1121 59-GATACTGCTGCTCTTCCAAA-39HR14 1656–1675 59-CTACTCAACCAGAAAAACCA-39LSM12 149–173 59-AGCGTCGCTATCTTAGGGGCTGGTT-39LSM150 577–591 59-CCAAGGATCCGTGGATGCTGAAGAA-39LSM16 928–942 59-CGCGTCGACACTCTCATTAACTGCTTT-39

a Primers LSM150 and LSM16 were designed from the pspA sequence ofS. pneumoniae Rx1, and the remaining primers were from the pspA sequence ofS. pneumoniae EF3296.

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away from the ends of the cells was not an artifact of theexperimental conditions.

The specificity of the binding of lactoferrin to PspA wasdemonstrated using PspA-negative mutants of S. pneumoniaeD39. The PspA-negative mutant did not express PspA on thesurface, as seen from the lack of staining with monoclonalanti-PspA antibodies, and did not bind milk-purified or recom-binant lactoferrin (Fig. 2B). The intensity of binding in flowcytometry was similar to that of the streptavidin-treated bac-teria, verifying the confocal microscopy results (Fig. 3B).

Lactoferrin does not bind to PspC. S. pneumoniae expressesa second choline-binding protein with high homology to PspA,designated PspC, SpsA, or CbpA (15, 29, 55). PspC has beensuggested to be involved in adherence to epithelial cells in therespiratory tract as well as in binding to the secretory compo-nent of IgA and complement factor C3 (19, 29, 45, 49). In theoriginal report, PspC was shown not to bind lactoferrin inWestern blot (28). This did not, however, exclude the involve-ment of PspC as a cofactor in lactoferrin binding. A complex oftwo proteins constituting the lactoferrin receptor is a commonfeature in gram-negative bacteria (9–11, 27, 46). To investigatethe potential role of PspC expressed on the surface of thebacteria for lactoferrin binding, we used a mutant of S. pneu-moniae D39 lacking PspC. This mutant still expressed PspA, asshown by staining with anti-PspA antibodies, and had an iden-tical lactoferrin binding pattern in confocal microscopy and thesame intensity of binding by flow cytometry as the wild-typecontrol (Fig. 2C and 3C).

Similarly, PspA-negative bacteria, which by fluorescencestaining still expressed PspC on the surface (data not shown),showed no residual binding of lactoferrin (Fig. 2B and 3B).Neither lactoferrin nor anti-PspA antibodies bound a doublemutant strain lacking both PspA and PspC (Fig. 2D and 3D).These results demonstrate that PspC is not able to bind lacto-ferrin to the pneumococcal surface and is not a necessarycofactor for PspA-dependent binding. Interestingly, the cellsurface distribution of PspC was identical to that seen forPspA, with areas of lower and higher intensity of expression(data not shown).

Finally, recombinant as well as milk-purified human lacto-ferrin was capable of detecting PspA but not PspC in dot blotand Western blot analyses (Fig. 4B and C). The total lack ofbinding to the PspA-negative bacteria further suggests thatlactoferrin does not bind to PspC or any other componentexpressed on the bacterial surface under the conditions used inthese experiments.

Mapping of the binding of lactoferrin to PspA. To identifythe general location of the binding site for lactoferrin on PspA,we compared the binding of lactoferrin to full-length PspAwith that of recombinant E. coli-expressed fragments consist-ing of various amino acids from the family 1 and family 2 PspAsequences (Fig. 4A). The PspA molecule is genetically variable

FIG. 1. Binding of lactoferrin to S. pneumoniae D39. Bacteria wereincubated with biotinylated recombinant lactoferrin, costained withFITC-conjugated streptavidin, inspected by confocal or epifluores-cence microscopy, and quantitated by flow cytometry. (A) Binding ofrecombinant human lactoferrin (hLF) to S. pneumoniae D39 (left), atransmission light detection image (middle) of the same bacteria, anda merged picture of the two (right). Binding was detected by confocalmicroscopy, and the staining showed a pattern with localized accumu-lation of binding to the sides of the bacteria, with less binding to thepoles and the interbacterial zones. (B) Staining of the cell wall of S.pneumoniae Rx1 with anti-phosphoryl choline (anti-PC) antibodies(left), a transmission light detection image (middle) of the same bac-teria, and a merged picture of the two (right). Binding was more or lesshomogenous around the bacterial surface. (C and D) Flow cytometryanalysis of recombinant lactoferrin binding to S. pneumoniae D39 (C)and EF3296 (D). Fluorescence detected from bacteria treated withbiotinylated lactoferrin. With lactoferrin at 10 mg/ml (arrow 10), bind-ing was 12.9 and 5.5 times above that of the controls for D39 andEF3296, respectively; at 40 mg/ml (arrow 40), binding was 27.6 and 9.9

times above that of the controls for D39 and EF3296, respectively; at100 mg/ml (arrow 100), binding was 31.1 and 22.2 times above that ofthe controls for D39 and EF3296, respectively; and at 150 mg/ml (arrow150), binding was 32.1 and 22.1 times above that of the controls forD39 and EF3296, respectively. Binding was compared to that withstreptavidin-alone-treated (SA) bacteria.



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FIG. 2. Colocalization of lactoferrin and PspA on isogenic mutants of D39. S. pneumoniae D39 and isogenic mutants lacking surface expressionof PspA, PspC, or both PspA and PspC were incubated with biotinylated recombinant lactoferrin, followed by incubation with anti-PspA antibodies(monoclonal Xi126), and counterstained with RPE-conjugated streptavidin and FITC-conjugated anti-mouse Ig antibodies. Binding was inspectedby confocal microscopy. Green fluorescence indicates staining with anti-PspA antibodies, red fluorescence indicates staining with lactoferrin, andyellow staining indicates colocalization of anti-PspA antibodies and lactoferrin. (A) D39 wild-type bacteria; (B) JY53 bacteria lacking PspA on thesurface; (C) TRE118 bacteria lacking PspC on the surface; (D) TRE121 bacteria lacking PspA and PspC on the surface. JY53, TRE118, andTRE121 are all specific mutants of strain D39. Bar, 2 mm.



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and has been classified into six clades by sequence homology(31). Five of these clades make up families 1 and 2 of PspAsequences and comprise over 90% of PspA in pneumococcalisolates. PspAs of families 1 and 2 are only partially cross-reactive in enzyme-linked immunosorbent assays with immunesera and display over 40% divergence at the level of nucleotidesequence. Full-length PspA from Rx1 (family 1) and EF3296(family 2) bound milk-purified and recombinant lactoferrin indot blot and Western blot analyses, suggesting that the bindingsite for lactoferrin was conserved between the two familiesdespite the variability in their amino acid sequences (Fig. 4Band C).

Lactoferrin failed to bind the most N-terminal region ofPspA, shown by the inability to bind Rx1 fragments BAR4285(amino acids 1 to 115) and BAR4310 (1 to 192) and EF3296fragment HR101 (1 to 115). On the other hand, lactoferrinbound significantly to the Rx1 fragments UAB055 (1 to 303),UAB103 (1 to 370), and JAS218 (167 to 288) and the EF3296fragments HR102 (75 to 305) and HR107 (75 to 490) (Fig. 4Band C). Lactoferrin appeared to show weak binding to frag-ment BAR501 (288 to 563), consisting of the proline-rich andcholine-binding domains. The binding results with these frag-ments suggest that the primary binding site of lactoferrin re-sides in the C-terminal part of the a-helical domain, with apossible secondary interaction with some of the more C-ter-minal elements of the molecule.

S. pneumoniae is bound more strongly by human lactoferrinthan by bovine lactoferrin. Among Moraxella spp., it has beenobserved that the strength of binding to lactoferrin differs forlactoferrins of different origins, with the strongest binding be-ing to lactoferrin of the host that it infects (10, 27). In additionto binding lactoferrin, most gram-negative bacteria can alsobind and utilize transferrin as an iron source, using a systemvery similar to that for acquisition of iron from lactoferrin (27).Hammerschmidt et al. demonstrated that bovine lactoferrincould not block binding of radiolabeled human lactoferrin topneumococcal cells (28). The direct binding of bovine lacto-ferrin to pneumococci was not attempted.

As S. pneumoniae is exclusively a human pathogen, we in-vestigated the difference in binding between lactoferrins ofhuman and bovine origin. Binding of lactoferrin to the surfaceof M. catarrhalis was used as a control, and binding of both thehuman and bovine forms of the protein was detected (data notshown), indicating that both mammalian proteins exhibit func-tional binding under the conditions used. Human lactoferrinbound much more strongly to S. pneumoniae D39 and EF3296than did bovine lactoferrin (Fig. 5A and B). The human pro-tein bound to the bacterial surface with an intensity of 32(D39) and 16 (EF3296) times that of the respective controlstreated with streptavidin alone. In contrast, bovine lactoferrinshowed only 2.1-fold (D39) or 1.3-fold (EF3296) greater bind-

FIG. 3. Flow cytometry analysis of lactoferrin binding to isogenicmutants of D39. S. pneumoniae D39 and isogenic mutants lackingsurface expression of PspA, PspC, or both PspA and PspC were incu-bated with biotinylated recombinant lactoferrin and counterstainedwith FITC-conjugated streptavidin, and binding was quantitated byflow cytometry. The white area indicates bacteria treated with strepta-vidin alone, and the gray area indicates binding of lactoferrin. (A) D39wild-type (wt) bacteria (binding 44.3 times the streptavidin control

value); (B) JY53 lacking PspA on the surface (binding 0.96 times thestreptavidin control value); (C) TRE118 lacking PspC on the surface(binding 40.4 times the streptavidin control value); (D) TRE121 lack-ing PspA and PspC on the surface (binding 0.96 times the streptavidincontrol value). The fluorescence intensity of the lactoferrin-treatedand streptavidin-treated PspA-negative cells revealed no residual bind-ing of lactoferrin to other components on the bacterial surface.



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ing compared to the respective controls. The interactions ofhuman and bovine lactoferrins with PspA were verified by dotblot analyses (Fig. 5C). These results indicate species specific-ity towards components present in the natural host.

Although human transferrin bound well to the surface of M.catarrhalis (binding intensity 30 times above the control; datanot shown), it failed to bind to the surface of S. pneumoniae,having a fluorescent signal intensity identical to that of thebacteria treated with streptavidin alone (Fig. 5A and B). Thissupported earlier studies showing no association of radiola-beled transferrin with the bacterial cells (28). The lack ofassociation with PspA was confirmed by dot blot analysis (Fig.5C). Thus, in contrast to strains of Neisseria and Moraxella, S.pneumoniae lacks the ability to bind human transferrin.

DISCUSSION

In the original demonstration of Hammerschmidt et al. thatlactoferrin binds to PspA (28), the lactoferrin used was purifiedfrom human milk. Because of the well-known difficulty of pu-rifying proteins from complex fluids such as milk, our resultswith recombinant human lactoferrin are an important confir-

mation of the earlier report that PspA binds lactoferrin. In ourfluorescence studies, lactoferrin binding was detected at con-centrations down to 1 mg/ml, which is the concentrationpresent in normal serum (8), but 50% saturation of binding tothe bacterial surface required approximately 40 mg/ml. Lacto-ferrin is a known acute-phase protein released in largeamounts during inflammation by polymorphonuclear cells.Thus, the concentrations used for half-saturation may very wellbe within the concentration range seen in an infected individ-ual or locally at a site of inflammation.

The binding of lactoferrin to the bacterial surface was totallydependent on the expression of PspA. Hammerschmidt et al.suggested that lactoferrin did not bind to PspC in Westernblots (28). The reactivity of lactoferrin with PspA but not PspCwas confirmed in this study using both Western and dot blotanalyses with recombinant PspA and PspC fragments purifiedfrom E. coli. To assess binding as it occurs under native con-ditions on the bacterial surface, we used mutants lacking ex-pression of PspA and/or PspC. Using these mutants, we foundthat lactoferrin binding to PspA and PspC mutants of pneu-mococci was dependent on the expression of PspA, but the

FIG. 4. Binding of human lactoferrin to PspA and recombinantPspA fragments in dot blot and Western blot analyses. (A) Map of therecombinant PspA/Rx1 (family 1) and PspA/EF3296 (family 2) frag-ments used in the study. (B) Dot blot. Full-length PspA and PspC andrecombinant fragments of PspA were dot blotted to a nitrocellulosemembrane. The membrane was blocked, overlaid with human recom-binant lactoferrin, and developed with NBT after incubation with AP-conjugated streptavidin. Binding of lactoferrin to both full-lengthPspA/Rx1 and PspA/EF3296 was detected but not to PspC. When thePspA fragments were investigated, human lactoferrin was shown tobind to all Rx1 fragments except BAR4285, consisting of amino acids1 to 115, and BAR4310, consisting of amino acids 1 to 192. The samewas seen for fragments of PspA/EF3296, where lactoferrin was shownto bind to all fragments except HR101, consisting of amino acids 1 to115. (C) Western blot. Full-length PspA, recombinant fragments ofPspA, and PspC were run on 10% gels and electroblotted to a nitro-cellulose membrane. The membrane was blocked, overlaid with humanrecombinant lactoferrin, and stained with NBT after incubation withAP-conjugated streptavidin. No binding was detected for BAR4285,BAR4310, HR101, or PspC. These results confirmed the dot blotresults. The positions of size markers are shown on the left (in kilo-daltons).



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presence of PspC was neither sufficient nor necessary for lac-toferrin binding. Hammerschmidt et al. also proposed a secondlow-affinity interaction of lactoferrin with pneumococci (28):evidence for this was not observed in our study.

The binding of lactoferrin and of the antibodies to PspA andPspC indicated that the expression of both proteins was, incontrast to phosphoryl choline, not uniform over the pneumo-

coccal surface. The most common staining pattern showed thatPspA was localized mainly along the lateral body of the bac-teria, with little binding at the poles or between bacterial cells.Faint staining was observed near the poles of the most recentcell division, but not near the poles of the preceding cell divi-sion. A minority of cells displayed staining between cells or atthe poles. On close examination, these cells were irregular inshape and appeared to be in an early stage of cell division. Thelocalized staining may indicate either a loss of surface-ex-pressed PspA over time or the fact that proteins like PspA andPspC may be secreted to the surface in a localized manner. Itis interesting that production of the cell wall of pneumococciwas limited to a thin growing zone in the lateral body of thebacterial cell (18). Whether PspA and PspC are secreted inconjunction with the production of cell wall remains to bedetermined.

Sequence homology data (31) and serologic cross-reactivity(41; M. C. V. Coral, N. Fonseca, E. Castaneda, J. L. Di Fabio,S. Hollingshead, and D. E. Briles, submitted for publication)have led to the classification of PspAs into different familiesand clades. Although PspA is one of the more variable geneproducts in pneumococci, genetically variable PspAs from dif-ferent strains can still display some cross-reactivity and canelicit broadly cross-protective antibodies (14, 39, 41, 52). In thisreport, we show that lactoferrin binds to PspAs from both ofthe major PspA families. Thus, although PspA is highly vari-able between strains, there are apparently conformationallyconserved regions of the molecule that are responsible forlactoferrin binding. One interpretation of the conservation oflactoferrin binding among strains expressing very variablePspAs sequences is that lactoferrin binding is important andbeneficial for the bacteria.

The PspA molecule has been divided into three distinctregions based on its sequence. It has an N-terminal a-helix-richdomain, which is suggested to form a coiled-coil structuresimilar to that of many gram-positive fibrillar surface proteins.This is the most variable domain of the protein and is exposedon the surface of the cell (31, 38). C-terminal to the a-helicaldomain is the proline-rich domain, which is known to span thecell wall of pneumococci (32). C-terminal to the proline-richregion is the repeat region that forms a choline-binding sitethat anchors PspA to the cell wall. Using recombinant frag-ments of family 1 and family 2 PspAs, we were able to showthat lactoferrin binds to the carboxy end of PspA’s a-helicalregion. Lactoferrin bound to full-length PspA from bothstrains in dot blot and Western blot analyses, consistent withthe results using whole bacteria. When investigating binding tothe different fragments, we observed that no binding could bedetected to fragments constituting the first 115 amino acids ofthe N-terminal region. Thus, lactoferrin binds to the samegeneral region of PspA that has been found to be most impor-tant in eliciting cross-protective immune responses (38, 52).

S. pneumoniae was shown to bind human lactoferrin withhigher intensity than the bovine protein. This confirmed theresults by Hammerschmidt et al. (28). Although this earlierstudy did not investigate direct binding of bovine lactoferrin tothe pneumomcoccal surface or to purfied PspA, it did showthat bovine lactoferrin could not inhibit binding of humanlactoferrin to whole S. pneumoniae. A similar situation existswith M. catarrhalis and Moraxella bovis, which have the highest

FIG. 5. Binding of human and bovine lactoferrin. S. pneumoniaeD39 and EF3296 were incubated with biotinylated recombinant human(hLF) or bovine (bLF) lactoferrin or with biotinylated human trans-ferrin (hTF) and counterstained with FITC-conjugated streptavidin.Binding was quantitated by cell sorting (A and B) or visualized by dotblotting (C). (A and B) Binding of human and bovine lactoferrin andhuman transferrin to S. pneumoniae D39 and EF3296. Human recom-binant lactoferrin (hLF) had a binding intensity 35.4 (D39) and 22.2(EF3296) times that of the streptavidin control. Bovine lactoferrin(bLF) only bound with intensities of 2.1 (D39) and 1.3 (EF3296) timesthat of their streptavidin-treated controls. Human transferrin (hTF)showed no greater binding than the streptavidin-only controls. (C) Dotblot. Full-length PspA was dot blotted in a 1:3 serial dilution series toa nitrocellulose membrane. The membrane was blocked, overlaid withrecombinant human or bovine lactoferrin (LF) or human transferrin(TF), and developed with NBT after incubation with AP-conjugatedstreptavidin. Only human lactoferrin showed significant binding toPspA.



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affinity for the lactoferrin of their natural host (10, 27). Thefact that each of these three bacterial species recognizes thelactoferrin of its host more strongly than that of an unrelatedmammal argues that the ability of each species to bind lacto-ferrin is important to its ability to colonize or infect its hosts.

Most infections start at the mucosal surface and require thatthe infecting pathogens have the ability to assimilate nutrientsfor survival and growth at the site of infection and that theyalso have ways to effectively evade the host immune system.Binding of lactoferrin may serve both these purposes. Lacto-ferrin binding has been documented for numerous bacterialspecies as a way to acquire iron at the site of infection (9, 10,24, 25, 42, 47). Although iron utilization by S. pneumoniae hasnot been extensively studied, it is known that pneumococci donot produce siderophores during invasive infection but canutilize hemin and hemoglobin as iron sources in the circulation(50). The means by which the pneumococcus acquires iron atthe mucosal surface is less well understood, but there is evi-dence that it cannot use either lactoferrin or transferrin as aniron source (50). If pneumococci do not use lactoferrin toacquire iron, it must play some other role in human infections.

Lactoferrin has also been shown to inhibit complement ac-tivation and to depress immune activity (21, 33, 34, 37, 54).Human tear lactoferrin was shown to block the assembly of theC3 convertase of the classical pathway, probably through in-teractions with complement factor C2 (34, 54). The results ofcomplement inhibition are, however, conflicting, as there arealso reports claiming that lactoferrin binding to bacterial sur-faces will enhance complement activation. The modulatoryeffects of lactoferrin may thus depend on the bacterial surface,the way it is bound, and the environment where activationoccurs (43). PspA has been shown to inhibit complement ac-tivation in vivo (53). Infection with PspA-negative S. pneu-moniae caused higher levels of complement activation in theserum of mice than infection with bacteria carrying PspAon the surface. Moreover, PspA-negative pneumococci arecleared more rapidly from the circulation of mice than thoseexpressing PspA. Binding of lactoferrin by PspA may be amechanism for pneumococci to inhibit complement activation.It may also be a way to subdue the immune system through theimmune-suppressive effects inherent in the lactoferrin mole-cule (21, 37).

Finally, lactoferrin receptors are known to exist on host cellsand may play a role in pneumococcal adherence by allowinglactoferrin to form a bridge between the bacteria and hostcells. A similar situation has recently been described for com-plement protein C3 and its binding to PspC on the pneumo-coccal surface. This interaction caused increased binding tohost epithelial cells (49). Further studies aim at understandingthe significance of lactoferrin binding to PspA and its overallrole in S. pneumoniae infections.

ACKNOWLEDGMENTS

We acknowledge Catharina Svanborg for her input, interest, andsupport of these studies; Susan Hollingshead for input in the study andgenerously providing us with some of the cloned PspA fragments; BethRalph and Xinping Wu for help with producing some of the clonedPspA fragments; and Jason Caldwell for help with producing fragmentJAS218. We also acknowledge William Benjamin for input in thestudy, Janet Yother for sharing her earlier experiences looking at PspA

with fluorescent techniques, and Flora Gathof, whose handling of theadministrative details greatly facilitated this study.

This study was supported by the Swedish Cancer Society (A.H.) andgrants AI21548 and Hl54818 (D.E.B.) and AI43653 (L.S.M.) from theNational Institutes of Health.

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