of June 13, 2013.This information is current as

Serum ResistanceE Binds Vitronectin and Is Important for Nontypeable Haemophilus influenzae Protein

Kristian RiesbeckTeresia Hallström, Anna M. Blom, Peter F. Zipfel and

http://www.jimmunol.org/content/183/4/2593doi: 10.4049/jimmunol.0803226July 2009;

2009; 183:2593-2601; Prepublished online 27J Immunol 

Referenceshttp://www.jimmunol.org/content/183/4/2593.full#ref-list-1

, 16 of which you can access for free at: cites 43 articlesThis article

Subscriptionshttp://jimmunol.org/subscriptions

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on June 13, 2013http://w

ww

.jimm

unol.org/D

ownloaded from

Nontypeable Haemophilus influenzae Protein E BindsVitronectin and Is Important for Serum Resistance1

Teresia Hallstrom,* Anna M. Blom,† Peter F. Zipfel,‡ and Kristian Riesbeck2

Nontypeable Haemophilus influenzae (NTHi) commonly causes local disease in the upper and lower respiratory tract and hasrecently been shown to interfere with both the classical and alternative pathways of complement activation. The terminal pathwayof the complement system is regulated by vitronectin that is a component of both plasma and the extracellular matrix. In thisstudy, we identify protein E (PE; 16 kDa), which is a recently characterized ubiquitous outer membrane protein, as a vitronectin-binding protein of NTHi. A PE-deficient NTHi mutant had a markedly reduced survival in serum compared with the PE-expressing isogenic NTHi wild type. Moreover, the PE-deficient mutant showed a significantly decreased binding to both solubleand immobilized vitronectin. In parallel, PE-expressing Escherichia coli bound soluble vitronectin and adhered to immobilizedvitronectin compared with controls. Surface plasmon resonance technology revealed a KD of 0.4 �� for the interaction betweenrecombinant PE and immobilized vitronectin. Moreover, the PE-dependent vitronectin-binding site was located at the heparin-binding domains of vitronectin and the major vitronectin-binding domain was found in the central core of PE (aa 84–108).Importantly, vitronectin bound to the surface of NTHi 3655 reduced membrane attack complex-induced hemolysis. In contrast toincubation with normal human serum, NTHi 3655 showed a reduced survival in vitronectin-depleted human serum, thus dem-onstrating that vitronectin mediates a protective role at the bacterial surface. Our findings show that PE, by binding vitronectin,may play an important role in NTHi pathogenesis. The Journal of Immunology, 2009, 183: 2593–2601.

T he complement system is the first line of innate defenseagainst pathogenic microorganisms. Activation of thissystem leads to a deposition of complement proteins on

the bacterial surface, which results in opsonization of pathogens.This facilitates phagocytosis, release of chemoattractants as well asformation of the membrane attack complex (MAC).3 Formation ofMAC is under control of two inhibitors, the membrane proteinCD59 and the multifunctional glycoprotein vitronectin that isfound both in plasma and in the extracellular matrix (ECM) (1). Inhuman plasma, vitronectin exists in three forms, a single chain (75kDa) and two truncated variants of 65 and 10 kDa. Vitronectinbinds the C5b-7 complex at its membrane-binding site and therebyinhibits the insertion of the complex into the cell membrane.Therefore, formation of cytolytic MAC is inhibited and lysis of thecell prevented (2). In the presence of vitronectin, the C5b-7 com-

plex is still able to bind C8 and C9 to form C5b-8 and C5b-9complexes but the latter is nonlytic. Furthermore, vitronectinblocks pore-forming polymerization of C9 by binding C5b-9 (3,4). Vitronectin is also involved in cell adhesion, spreading andmigration, coagulation, fibrinolysis, and finally as a 75-kDa build-ing block in the ECM (1, 2).

Haemophilus influenzae is an important human-specific patho-gen that can be classified according to the presence of a polysac-charide capsule (5). The encapsulated strains cause invasive dis-eases, whereas the unencapsulated and hence nontypeable H.influenzae (NTHi) are mainly found in local upper and lower re-spiratory tract infections (6, 7). NTHi is, after Streptococcus pneu-moniae, the most common cause of acute otitis media in childrenand is the main cause of exacerbations in patients suffering fromchronic obstructive pulmonary disease and bronchiectasis (8, 9).

The pathogenesis of many microorganisms relies on their ca-pacity to avoid, resist, or neutralize the host defense including thecomplement system. Therefore, many pathogens have evolved dif-ferent mechanisms to avoid complement-mediated killing. Somepathogens are able to inactivate complement components, whereasothers express proteins that mimic the complement inhibitors ofthe host (10). Another frequent strategy used by some pathogens isbinding of complement inhibitors such as vitronectin, C4b-bindingprotein (C4BP), and factor H, which all protect from the comple-ment-mediated attack. These inhibitors are captured on the bacte-rial surface in such a way that they are still functionally active. Theexact mechanism of how H. influenzae evades the host comple-ment attack is unclear. In a previous study, we showed that NTHibinds C4BP and that this interaction significantly contributes tobacterial serum resistance (11). Both H. influenzae type b (Hib)and NTHi bind surface-associated vitronectin equally well, and ithas been suggested that adhesins are involved in the binding (12).We have shown in a previous study that H. influenzae surfacefibrils (Hsf), which solely can be found in Hib, interacts with vi-tronectin and thereby prevents complement-induced lysis, result-ing in increased bacterial survival in normal human serum (NHS)

*Medical Microbiology and †Medical Protein Chemistry, Department of LaboratoryMedicine, Lund University, University Hospital Malmo, Malmo, Sweden; ‡LeibnizInstitute for Natural Product Research and Infection Biology, Hans Knoell Institute,Friedrich Schiller University, Jena, Germany

Received for publication September 26, 2008. Accepted for publication June 7, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Alfred Osterlund, the Anna and EdwinBerger, the Crafoord, the Marianne and Marcus Wallenberg, and the Greta and JohanKock Foundations, the Swedish Medical Research Coucnil, the Swedish Society ofMedicine, the Cancer Foundation at the University Hospital in Malmo, and the SkaneCounty Council’s Research and Development Foundation.2 Address correspondence and reprint requests to Dr. Kristian Riesbeck, Medical Mi-crobiology, Department of Laboratory Medicine, University Hospital Malmo, LundUniversity, SE-205 02 Malmo, Sweden. E-mail address: kristian.riesbeck@med.lu.se3 Abbreviations used in this paper: MAC, membrane attack complex; C4BP, C4b-binding protein; ECM, extracellular matrix; NTHi, nontypeable Haemophilus influ-enzae; Hib, H. influenzae type b; Hsf, H. influenzae surface fibril; NHS, normalhuman serum; OMP, outer membrane protein; pAb, polyclonal antibody; PE, proteinE; MID, Moraxella IgD-binding protein.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803226

by guest on June 13, 2013http://w

ww

.jimm

unol.org/D

ownloaded from

(13). In addition, both NTHi and encapsulated H. influenzae bindthe alternative pathway inhibitor factor H, which is able to inhibitlysis of H. influenzae (14).

An initial step in the H. influenzae pathogenesis is adherence tothe mucosa in the respiratory tract (15). Thereafter, adherence tothe epithelial cells is a mechanism to circumvent the mucociliaryclearance. Penetration of bacteria between host cells may also fa-cilitate evasion from the immune system. Moreover, bacteria canreach the basement membrane and the ECM and may penetrateinto deeper tissue layers and consequently into the circulation. Infact, H. influenzae expresses a number of surface structures thatinfluence the process of adherence and colonization. Pili have beenshown to exhibit adherence to human oropharyngeal epithelialcells, fibronectin, and other ECM proteins (16, 17). Haemophilusadhesion and penetration protein is another adhesin that has beenreported to bind fibronectin, laminin, and collagen IV (18). Hsf isthe major nonpilus adhesin found in encapsulated H. influenzaeand this large outer membrane protein interacts with epithelialcells in addition to vitronectin (13, 19).

The recently discovered H. influenzae protein E (PE) is a low-molecular-mass (16 kDa) outer membrane lipoprotein with adhe-sive properties (20, 21). PE-expressing H. influenzae in addition tosoluble recombinant PE(22–160) without a lipid moiety induces aproinflammatory epithelial cell response, resulting in an increasedIL-8 secretion and ICAM-1 up-regulation that leads to an en-hanced neutrophil adherence to epithelial cells. The adhesive PEdomain is located within the central part of the molecule (aa 84–108) and preincubation of epithelial cells with this peptide blocksadhesion of several clinical NTHi isolates. In the present study, wedemonstrate that H. influenzae PE binds both immobilized andsoluble vitronectin and that this interaction contributes to bacterialserum resistance. Importantly, vitronectin bound to the surface ofNTHi 3655 was functionally active and reduced MAC-inducedhemolysis, and when NTHi 3655 was exposed to vitronectin-de-pleted serum a significantly decreased bacterial survival was seen.

Materials and MethodsBacterial strains and culture conditions

The NTHi 3655 was a gift from R. Munson (The Ohio State University,Columbus, OH) (22) and the NTHi 3655�pe mutant was previously de-scribed (21). NTHi, wild-type, and mutant were routinely cultured in brain-heart infusion liquid broth supplemented with NAD and hemin (both at 10�g/ml) or on chocolate agar plates at 37°C in a humid atmosphere con-taining 5% CO2. The PE-deficient mutant was cultured in the presence of17 �g/ml kanamycin (Merck). Escherichia coli BL21 (DE3) and DH5�were grown in Luria-Bertani liquid broth, whereas PE transformants werecultured with 50 �g/ml ampicillin (Sigma-Aldrich).

Antibodies

Rabbits were immunized i.m. with 200 �g of recombinant PE(22–160)emulsified in CFA (Difco and BD Biosciences) and boosted on days 18 and36 with the same dose of protein in IFA. Blood was drawn 2–3 wk later.To increase the specificity, the PE antiserum was affinity purified withSepharose-conjugated recombinant PE(22–160) (23). To ensure that thepolyclonal Ab (pAb) reacted with recombinant PE, the pAb was analyzedin ELISA. PE (100 ng/well) was immobilized overnight in microtiter plates(F96 Maxisorb; Nunc-Immuno Module) and incubated with increasingconcentrations of the rabbit anti-PE pAb followed by HRP-conjugated goatanti-rabbit pAb (Dakopatts). The goat anti-human vitronectin and theFITC-conjugated donkey anti-goat pAb were purchased from Serotec. TheHRP-conjugated rabbit anti-goat pAb was from Dakopatts.

DNA cloning and protein expression

The full-length PE(22–160) was expressed in E. coli as described previ-ously (21). Briefly, the PE DNA constructs were amplified by PCR intro-ducing the restriction enzyme sites BamHI and HindIII. Genomic DNAfrom NTHi 772 (HI0175–HI0178) was used as template (21). The homol-ogy between PE in NTHi 772 and NTHi 3655 is �98%. The sequence

coding for the signal peptide was excluded. The PCR products were clonedinto both pET26� and pET16. The resulting plasmids were transformedinto E. coli DH5� and thereafter into the expressing host BL21(DE3). Theconstructs were verified by sequencing using the BigDye Terminator CycleSequencing version 3.1 Ready Reaction Kit (Applied Biosystems). To pro-duce recombinant PE(22–160) or expressing PE on the E. coli surface,bacteria were grown to mid-log phase (OD600, 0.6–0.8) followed by1–3.5 h of induction with 1 mM isopropyl-1-thio-�-D-galactoside. Bacteriawere sonicated and the recombinant proteins were purified using columnscontaining a nickel resin (Novagen and VWR International) according tothe manufacturers’ instructions for native conditions.

Serum bactericidal assay

NHS was prepared from pooled blood obtained from healthy volunteerswith informed consent. Human serum was depleted from C7 or vitronectinby affinity chromatography using goat anti-human C7 pAb or goat anti-human vitronectin pAb coupled to a mix of protein A-Sepharose and pro-tein G- Sepharose (GE Healthcare) overnight at 4°C. NHS was incubatedwith the Ab-coated Sepharose for 1 h at 4°C, followed by centrifugation tocollect the depleted serum. The C7-depleted NHS was analyzed for activityin a hemolytic assay using rabbit erythrocytes. The vitronectin-depletedserum was analyzed for the presence of vitronectin and for complementactivity by Western blot and hemolytic assay, respectively. The NTHi 3655wild-type (104) and the corresponding PE-deficient mutant (104) were di-luted in DGVB�� (2.5 mM veronal buffer (pH 7.3) containing 0.1% (w/v)gelatin, 1 mM MgCl2 and 0.15 mM CaCl2) and incubated in 10% NHS,10% heat-inactivated NHS (30 min at 56°C), and increasing concentrationsof NHS (5–20%) in a final volume of 100 �l at 37°C. To analyze the effectof MAC-deficient NHS or the effect of vitronectin, the NTHi 3655 wasincubated with 10% C7-depleted NHS or 10% vitronectin-depleted NHS.At different time points, 10-�l aliquots were removed and spread ontochocolate agar plates. After 18 h of incubation at 37°C, CFU weredetermined.

Protein labeling and direct binding assay using 125I-labeledvitronectin

Vitronectin (Sigma-Aldrich) from human plasma was labeled with 0.05mol of iodine (GE Healthcare) per mol protein using the chloramine-Tmethod (24). The NTHi 3655 wild-type and PE-deficient mutant strainswere grown in brain-heart infusion liquid broth until mid-log phase andwashed in PBS-1% BSA (Sigma-Aldrich). Bacteria (2 � 107) were incu-bated with increasing concentrations (0.6–60 ng/ml) of 125I-labeled vitro-nectin at 37°C for 1 h. After incubation, the bacteria were centrifuged(10,000 � g) through a 20% sucrose column. The tubes were frozen andcut, and radioactivity in the pellet and supernatant was measured in agamma counter. Binding was calculated as amount of bound radioactivity(pellet) vs total radioactivity (pellet plus supernatant). In the competitionassay, the NTHi 3655 wild-type was preincubated with the goat anti-vitronectin pAb (5 �g/ml) or increasing concentrations of the rabbit anti-PE pAb (0–30 �l) followed by addition of 125I-labeled vitronectin.

Flow cytometry analysis

The capacity for NTHi to bind vitronectin and the PE protein expressionwere analyzed by flow cytometry. The NTHi 3655 strain and the PE-de-ficient mutant from overnight cultures were grown in broth until mid-logphase and washed once in PBS containing 2% BSA (PBS-2% BSA). Bac-teria (108) were incubated with increasing concentrations of vitronectin(0.1–10 �g/ml) or NHS (0.1–10%) for 1 h at 37°C. After washings, bac-teria were incubated with goat anti-vitronectin pAb for 30 min on ice,followed by incubation with the FITC-conjugated donkey anti-goat pAb.After three additional washes, bacteria were analyzed in a flow cytometer(EPICS XL-MCL; Beckman Coulter). To analyze the expression of PE,bacteria were incubated with rabbit anti-PE pAb followed by washing withPBS-2% BSA and incubation for 30 min on ice with FITC-conjugated goatanti-rabbit pAb (Dakopatts) diluted according to the manufacturer’s in-structions. After additional washes, bacteria were analyzed by flow cytom-etry. All incubations were done in a final volume of 100 �l of PBS-2%BSA, and the washings were done with the same buffer. Secondary pAbwere added separately as negative controls for each strain analyzed. PE-expressing E. coli was analyzed for PE expression and vitronectin bindingas described above. In the competition assay, the NTHi 3655 wild type wasincubated with increasing concentrations of heparin (Leo, Lovens KemiskeFabrik), NaCl, PE(84–108) (PKRYARSVRQYKILNCANYHLTQVR)(Innovagen), PE(140–160) (LYNAAQIICANYGKAFSVDKK) (Innova-gen), or BSA (100 �g/ml) followed by addition of 2 �g/ml vitronectin.

2594 PE INTERACTS WITH VITRONECTIN

by guest on June 13, 2013http://w

ww

.jimm

unol.org/D

ownloaded from

Binding of H. influenzae to immobilized vitronectin

Glass slides were coated with 2 �g of human plasma vitronectin or 2 �gof BSA, air dried at room temperature, and then washed twice with PBS(13). The slides were incubated with bacteria harvested at the late expo-nential phase (OD600 � 0.9) for 2 h at room temperature, washed twicewith PBS followed by standard Gram staining.

ELISA

Microtiter plates (F96 Polysorb; Nunc-Immuno Module) were coatedwith peptide fragments of PE (Innovagen), full-length PE (10 �g/ml),or BSA (10 �g/ml) in 0.1 M Tris-HCl (pH 9.0) overnight at 4°C. Plateswere washed with PBS-0.05% Tween 20 and blocked for 1 h at roomtemperature with PBS-2% BSA. After washings, vitronectin (5 �g/ml)in PBS-2% BSA was added and incubated for 1 h at room temperature.Thereafter, the plates were washed and incubated with goat anti-humanvitronectin pAb for 1 h. After additional washings, HRP-conjugatedrabbit anti-goat pAb was added and incubated at room temperature for40 min. The wells were washed, developed with 20 mM tetramethyl-benzidine and 0.1 M potassium citrate, and finally the absorbance wasmeasured at 450 nm with a MRX Microplate reader (DynatechLaboratories).

Surface plasmon resonance

The interaction between PE(22–160) or PE(84–108) and vitronectin wasfurther analyzed using surface plasmon resonance (Biacore 2000; Biacoreand GE Healthcare). The individual flow cells of a CM5 sensor chip wereactivated, each with 20 �l of a mixture of 0.2 M 1-ethyl-3-(3- dimethyl-aminopropyl)carbodiimide and 0.05 M N-hydroxy-sulfosuccinimide at aflow rate of 5 �l/min, after which vitronectin diluted to 20 �g/ml in 10 mMsodium acetate buffer (pH 4.0) was injected over individual flow cells toreach 2000 resonance units. Not reacted groups were blocked with 20 �l of1 M ethanolamine (pH 8.5). A negative control was prepared for each chipby activating and subsequently blocking the surface of flow cell one. Theassociation kinetics was studied for various concentrations of purifiedPE(22–160) (20–1200 nM) and the synthetic peptide PE(84–108) (30–1700 nM), or the negative controls Moraxella IgD-binding protein (MID)(1000–1200) (25) and MID(1841–1868) (26) in 10 mM HEPES (pH 7.5)supplemented with 150 mM NaCl, 3 mM EDTA, and 0.005% Tween 20.Protein solutions were injected for 120 s during the association phase ata constant flow rate of 30 �l/min. The sample was injected first over thenegative control surface and then over immobilized vitronectin or re-combinant MID(1000 –1200). Signal from the control surface was sub-tracted. The dissociation was followed for 120 s at the same flow rate.In all experiments, two 10-�l injections of 2 M NaCl were used toremove bound ligands during a regeneration step. The BiaEvaluation3.0 software (Biacore) was used to analyze sensorgrams obtained. Re-sponse units obtained at the plateau of sensorgrams were plotted againstconcentrations of injected protein and used for calculation of equilib-rium affinity constants.

Hemolytic assay

Hemolytic assays with rabbit erythrocytes were used to determine the com-plement activity of the C7-depleted NHS and the vitronectin-depletedNHS. Rabbit erythrocytes (2 � 108 cells/ml; Rockland and BioTrendChemikalien) were incubated with 10% C7- or vitronectin-depleted NHSin Mg-EGTA buffer (20 mM HEPES, 144 mM NaCl, 7 mM MgCl2, and 10mM EGTA, pH 7.4). After an incubation for 15 min at 37°C, the eryth-rocytes were centrifuged and the amount of lysed erythrocytes was mea-sured at 414 nm. To analyze the MAC regulatory effect of PE-bound orNTHi 3655-bound vitronectin, hemolytic assays using sheep erythrocytes(Rockland) were performed. Sheep erythrocytes (30 �l; 2 � 108 cells/ml)were preincubated with 1 �g/ml C5b-6 (Complement Technology) in Mg-EGTA buffer for 10 min at room temperature. NTHi 3655 (108) was pre-incubated with vitronectin (20 �g/ml) for 45 min at 37°C, followed byextensive washings. C7 (1 �g/ml; Complement Technology), C8 (0.2 �g/ml; Complement Technology), and C9 (1 �g/ml; Complement Technol-ogy) were preincubated with or without factor H (Complement Technol-ogy) with increasing concentrations (20–100 �g/ml) of PE with or withoutvitronectin (50 �g/ml) or vitronectin-coated NTHi 3655 for 5 min at roomtemperature in a total volume of 30 �l. After preincubation, the C5b-6-coated sheep erythrocytes were added to the mixture. After 30 min at 37°C,the erythrocytes were centrifuged and the amount of lysed erythrocytes wasmeasured at 414 nm.

Statistics

Results were assessed by Student’s t test for paired data. A value of p �0.05 was considered to be statistically significant (�, p � 0.05; ��, p �0.01; ���, p � 0.001).

ResultsPE is important for NTHi survival in human serum

To analyze whether the outer membrane protein PE may have a rolein NTHi survival upon exposure to NHS, the wild-type strain NTHi3655 was mutated by introduction of a kanamycin resistance genecassette in the gene encoding for PE (21). The wild-type NTHi 3655and the PE-deficient mutant (NTHi 3655�pe) were thereafter tested ina serum bactericidal assay. Interestingly, the mutant devoid of PE wassignificantly more sensitive to NHS compared with the isogenic wild-type strain (Fig. 1A). After 10 min of incubation with 10% of NHS,�60% of the NTHi 3655�pe was killed compared with none of thewild type. Both the wild-type strain and the mutant were resistant toheat-inactivated serum (Fig. 1B). Moreover, when a titration of theserum concentration was performed, the NTHi 3655�pe was moresensitive to all concentrations of serum compared with the wild-typestrain (Fig. 1C). Thus, these experiments suggested that PE playsa significant role in NTHi serum resistance. To investigate that theterminal pathway and MAC were responsible for the killing of NTHi3655, the survival of NTHi 3655 in C7-depleted NHS, i.e., terminalpathway-deficient serum, was analyzed. NTHi 3655 was resistant toC7-depleted NHS, suggesting that the MAC is responsible for thekilling of NTHi 3655 in NHS (Fig. 1D).

FIGURE 1. NTHi equipped with PE has a significantly better survivalin human serum. NTHi 3655 and the NTHi 3655�pe mutant were incu-bated with 10% NHS (A), 10% heat-inactivated serum (B), or increasingconcentrations of NHS (5–20%) (C). D, NTHi 3655 survived in C7-de-pleted human serum. The NTHi 3655 was incubated in the presence of 10%NHS or 10% C7-depleted serum both diluted in DGVB��. At the indicatedtime points, bacteria were collected and spread on chocolate agar plates toallow determination of surviving bacteria. The Number of bacteria (CFU)at the initiation of the experiment was defined as 100%. The mean valuesfrom three independent experiments are shown with error bars indicatingSD. �, p � 0.05 and ��, p � 0.01.

2595The Journal of Immunology

by guest on June 13, 2013http://w

ww

.jimm

unol.org/D

ownloaded from

PE from H. influenzae binds human vitronectin

To determine whether H. influenzae PE is important for vitronec-tin-binding, NTHi 3655 and the PE mutant were incubated withsoluble I125-labeled vitronectin at increasing concentrations using

a direct binding assay (Fig. 2A). The NTHi 3655 bound vitronectinin a dose-dependent and saturable manner. In contrast, a signifi-cantly decreased vitronectin binding was observed with NTHi3655�pe compared with the isogenic NTHi3655 wild-type strain.However, when NTHi 3655 and the PE-deficient mutant were in-cubated with higher concentrations of vitronectin (1–10 �g/ml)and the binding was analyzed by flow cytometry, we found that thePE-deficient NTHi 3655�pe mutant also bound vitronectin (Fig.2B), although to a significantly lower level than the wild type.These results suggested existence of an additional NTHi protein,which mediates bacterial vitronectin binding. Similar results wereobtained by flow cytometry when NHS was used as a source ofvitronectin (Fig. 2C). Flow cytometry analysis with an affinity-purified anti-PE pAb confirmed that the H. influenzae PE mutant(NTHi 3655�pe) was PE deficient (Fig. 2D) (20). Moreover, apAb against human vitronectin inhibited the binding of vitronectinto NTHi 3655 as examined in a direct binding assay (Fig. 2E). Tofurther show that PE was involved in the interaction between vi-tronectin and NTHi 3655, the pAb against PE was used for block-ing. This Ab inhibited �50% of the binding (Fig. 2F). Thus, PE isimportant for NTHi-dependent vitronectin binding, but most likelyalso another vitronectin-binding outer membrane protein exists.

To further demonstrate that PE interacted with soluble vitronec-tin, E. coli-expressing PE at the surface was included in our studyand analyzed by the direct binding assay (Fig. 3A) and flow cy-tometry (Fig. 3B). E. coli-expressing PE bound vitronectin �4-fold better than the E. coli control that was transformed with anempty vector (Fig. 3A). When analyzed by flow cytometry, E.coli-PE bound vitronectin (10–100 �g/ml) in a dose-dependent

FIGURE 2. PE-expressing NTHi binds vitronectin. A, The NTHi 3655wild type (wt) bound vitronectin in a dose-dependent manner. In contrast,the NTHi 3655�pe mutant displayed a much weaker binding to variousconcentrations of vitronectin in a direct binding assay. B and C, NTHi 3655wild type bound purified vitronectin and vitronectin from NHS in a dose-dependent manner as analyzed by flow cytometry, whereas NTHi 3655�pedisplayed a reduced binding to lower concentrations of vitronectin whencompared with the wild type. D, Expression of PE on the NTHi strains usedin this study was determined by flow cytometry. E, An anti-vitronectin pAbinhibited the binding of 125I-labeled vitronectin to NTHi 3655. F, Anti-PEpAb inhibited �50% of the binding of 125I-labeled vitronectin to NTHi3655. In A, the wild type and isogenic mutant were incubated with 125I-labeled vitronectin at increasing concentrations (0.6–60 ng/ml) and in Dand E, the wild type was incubated with 125I-labeled vitronectin and anti-vitronectin pAb or anti-PE pAb. Binding was determined as percentage ofbound radioactivity vs added radioactivity measured after separation offree and bound 125I-labeled vitronectin over a sucrose column. In B and C,bacteria were incubated with increasing concentrations of vitronectin (B) orincreasing concentrations of NHS (C) as indicated, followed by an anti-vitronectin pAb. A FITC-conjugated anti-goat pAb was added subse-quently followed by flow cytometry analysis. In D, the NTHi 3655 wildtype and PE-deficient mutant were incubated with a rabbit anti-PE anti-serum and finally a FITC-conjugated goat anti-rabbit antiserum. In E andF, the vitronectin binding of NTHi in the absence of Abs was defined as100%. The mean values of three experiments are shown with error barscorresponding to SD. ��, p � 0.01 and ���, p � 0.001.

FIGURE 3. PE expressed on the surface of E. coli binds vitronectin. A,Bacteria were incubated with 125I-labeled vitronectin. Binding was deter-mined as percentage of bound radioactivity vs added radioactivity mea-sured after separation of free and bound 125I-labeled vitronectin over asucrose column. B, Bacteria were incubated with increasing concentrationsof vitronectin followed by flow cytometry analysis using an anti-vitronectin pAb and a secondary FITC-conjugated anti-goat pAb. C, Typ-ical flow cytometry profiles showing the binding of 100 �g/ml vitronectinto E. coli-PE and the E. coli control without the PE insert. The mean valuesof three experiments are shown in A and B. Error bars indicate SD. �, p �

0.05; ��, p � 0.01; and ���, p � 0.001.

2596 PE INTERACTS WITH VITRONECTIN

by guest on June 13, 2013http://w

ww

.jimm

unol.org/D

ownloaded from

manner, whereas a significantly decreased binding was detected inthe control (Fig. 3B). When flow cytometry mean fluorescenceintensity values were compared, E. coli-PE bound vitronectin�15-fold better than the E. coli control bacteria when incubatedwith the highest concentration of vitronectin (100 �g/ml; Fig. 3C).

Since vitronectin has been suggested to function as a receptorfor bacteria (27), attachment of bacteria to immobilized vitronectinwas investigated. NTHi 3655 and the isogenic PE mutant wereapplied to vitronectin-coated glass slides followed by standardGram staining. NTHi 3655 wild type adhered to the immobilizedvitronectin (Fig. 4A), whereas the NTHi 3655�pe mutant barelybound (Fig. 4B). To further prove that PE interacted with immo-bilized vitronectin, the vitronectin-binding capacity of PE-express-ing E. coli was analyzed. In similarity to PE expressing H. influ-enzae, E. coli-PE adhered to the vitronectin-coated glass slides(Fig. 4C), whereas only a few bacteria were detected when the E.

coli control was analyzed (Fig. 4D). Neither NTHi nor E. coliadhered to the BSA-coated glass slides that were included as anegative control (Fig. 4E, NTHi, and data not shown). The numberof bacteria (bacteria/low-power field) was determined by a blindedobserver and 10 low-power fields were counted to calculate means,which are shown in Fig. 4F. Taken together, PE is important forbinding to both immobilized and soluble vitronectin as shown withPE-expressing NTHi and E. coli.

The interaction between PE and vitronectin is based uponnonionic interactions and is inhibited by heparin

The vitronectin molecule harbors several different functional re-gions that are involved in cell attachment, collagen, and glycos-aminoglycan binding. Vitronectin contains three heparin-bindingdomains (1, 28). To further investigate the nature of the interactionof vitronectin to NTHi, competition experiments were performed.The NTHi 3655 wild type was preincubated with heparin at in-creasing concentrations (0.01–5 �g/ml) followed by addition ofvitronectin (2 �g/ml) and analysis by flow cytometry. Heparininhibited the binding of vitronectin to NTHi in a dose-dependentmanner (Fig. 5A). The inhibitory concentration of heparin requiredto reduce the binding by 50% (IC50) was 0.1 �g/ml. To testwhether an enhanced ionic strength could inhibit the interactionbetween NTHi 3655 and vitronectin, increasing concentrations ofNaCl (0.01–1 M) were added to the reaction mixture followed byanalysis of vitronectin binding by flow cytometry. However, thevitronectin binding to NTHi 3655 was not based on ionic interac-tions since the interaction was not inhibited even at the highest

FIGURE 4. PE is important for adhesion to immobilized vitronectin. A,The H. influenzae wild type adhered at a high density to vitronectin-coatedglass slides. B, The NTHi 3655�pe mutant adhered poorly to vitronectin.C, E. coli expressing PE at the bacterial cell surface adhered significantlyto vitronectin. D, Wild-type E. coli adhered poorly to immobilized vitro-nectin. E, NTHi 3655 did not bind to BSA that was included as a negativecontrol. F, The mean values of bacteria from 10 low-power fields areshown with error bars indicating SD. Glass slides were coated with vitro-nectin or BSA and incubated with the bacteria. After several washes, bac-teria were Gram stained. ��, p � 0.01 and ���, p � 0.001.

FIGURE 5. The effect of heparin and ionic strength on the binding of vi-tronectin to NTHi 3655. Bacteria were incubated with vitronectin (2 �g/ml)and increasing concentrations of heparin, NaCl, or BSA followed by an anti-vitronectin pAb. A, The interaction between NTHi and vitronectin is inhibitedby increasing concentrations of heparin. The vitronectin binding of NTHi inthe absence of heparin, NaCl, or BSA was defined as 100%. B, The interactionbetween NTHi and vitronectin is not based on ionic interactions. C, BSA didnot affect the binding of vitronectin to NTHi 3655. A FITC-conjugated anti-goat pAb was subsequently added as a secondary layer followed by flow cy-tometry analysis. The mean values of three experiments are shown with errorbars indicating SD. ��, p � 0.01; and ���, p � 0.001.

2597The Journal of Immunology

by guest on June 13, 2013http://w

ww

.jimm

unol.org/D

ownloaded from

NaCl (1 M) concentration (Fig. 5B). BSA, that was included as anegative control, did not interfere with the binding (Fig. 5C). Theaffinity of the interaction between PE(22–160) and vitronectin wasestimated using surface plasmon resonance technology. The equi-librium affinity constant (KD) was found to be 0.4 �M (Fig. 6). Nointeraction with vitronectin was seen upon injection of the negativecontrols recombinant MID(1000–1200) or MID(1841–1868) atany concentration tested (data not shown).

The major vitronectin binding region is located in the centralpart of PE (aa 84–108)

To define the vitronectin-binding domain of PE, a series of pep-tides (with an overlap of four amino acids) spanning the entirePE(22–160) molecule was synthesized. The PE fragments wereimmobilized onto microtiter plates and vitronectin-binding wasquantified by ELISA. One major vitronectin-binding domain was

found within amino acids PE(84–108), which is located in themiddle part of the molecule (Fig. 7). However, two additional vi-tronectin binding sites, i.e., PE(41–68) and PE(64–88), were mostlikely involved in the PE-dependent vitronectin binding, albeitthese sequences had a lower binding capacity compared withPE(84–108). We could also detect a clear binding of PE(84–108)to vitronectin immobilized on the surface of a Biacore chip, but thenature of the obtained sensorgrams did not allow calculation of anaccurate KD value (data not shown).

The interaction between vitronectin and the most efficient bind-ing domain (PE(84–108)) was further confirmed using ELISA anda competition assay. Saturated conditions of the interaction be-tween vitronectin and PE(84–108) were first defined in the ELISA(Fig. 8A). Thereafter, NTHi 3655 was incubated with vitronectin inthe presence of increasing concentrations of PE(84–108) orPE(140–160) followed by flow cytometry analyses. PE(84–108)specifically inhibited the binding between NTHi 3655 and vitro-nectin (Fig. 8B). A total of 50 �g/ml PE(84–108) was required toblock the NTHi 3655-vitronectin interaction by 75%. PE(140–160) was used as a negative control and this peptide did not blockthe binding between NTHi 3655 and vitronectin (Fig. 8B). Takentogether, the interaction between PE and vitronectin was depend-ing on the middle part of the PE molecule and was specific asrevealed by the competition experiment.

Vitronectin bound to the surface of NTHi 3655 is still active andable to inhibit the MAC formation

Vitronectin regulates the terminal pathway by inhibiting mem-brane binding of C5b-7 and therefore inhibits the MAC assemblyon the cell surface and protects it from lysis (1, 2). To analyzewhether vitronectin in the presence of PE still inhibits the cytolyticactivity of MAC, a hemolytic assay with the purified terminal com-plement proteins C5b-6, C7, C8, and C9 was performed. In thefluid phase, PE-bound vitronectin inhibited the cytolytic activity ofC5b-9/MAC in a concentration-dependent manner (Fig. 9A). Incontrast, PE(22–160) alone did not inhibit the cytolytic activity ofMAC. Vitronectin, used as a positive control, blocked lysis,whereas factor H, used as a negative control, did not inhibit thelysis. Furthermore, to determine whether surface-bound vitronec-tin was still active and able to inhibit MAC, NTHi 3655 was pre-incubated with vitronectin and the capacity to inhibit MAC was

FIGURE 6. PE(22–160) bound vitronectin as shown by surface plasmonresonance. PE was injected at least twice at increasing concentrations (20-1200 nM) over a CM5 chip with bound vitronectin and in parallel over acontrol flow cell that was used to subtract nonspecific signal. The amount ofbound PE was measured in arbitrary response units (RU, inset). The responseobtained for each concentration of PE at equilibrium (Req) was plotted againstthe concentration of PE, which allowed for estimation of the KD.

FIGURE 7. The active vitronectin-binding domain of PE is locatedwithin PE(84–108). Peptides spanning the entire PE molecule are shown.All fragments were tested for binding to vitronectin by ELISA. The pep-tides were coated on microtiter plates and incubated with 5 �g/ml vitro-nectin. Bound vitronectin was detected with a goat anti-human vitronectinpAb followed by HRP-conjugated anti-goat pAb. Results are the meanvalues of three experiments and error bars indicate SD. The significancevalues were calculated using PE(140–160) as a reference for a nonbindingfragment. �, p � 0.05 and ���, p � 0.001.

FIGURE 8. The binding between PE(84–108) and vitronectin is spe-cific. A, To define saturating conditions, PE(84–108) (5 �g/ml) coated inmicrotiter plates was incubated with increasing concentrations of vitronec-tin as indicated. B, Bacteria were incubated with vitronectin (2 �g/ml) andincreasing concentrations of PE(84–108) or PE(140–160) as indicated fol-lowed by a goat anti-human vitronectin pAb. FITC-conjugated anti-goatpAb was subsequently added followed by flow cytometry analysis. Thevitronectin binding of NTHi in the absence of competitor was defined as100%. The mean values of three experiments are shown with error barsindicating SD. �, p � 0.05; ��, p � 0.01; and ���, p � 0.001.

2598 PE INTERACTS WITH VITRONECTIN

by guest on June 13, 2013http://w

ww

.jimm

unol.org/D

ownloaded from

analyzed in the hemolytic assay using purified terminal comple-ment components. Vitronectin bound to the surface of NTHi 3655reduced the cytolytic activity of MAC significantly compared withthe untreated NTHi 3655 (Fig. 9B), demonstrating that vitronectinwas still active and regulated the terminal pathway of complement.To further confirm that NTHi-bound vitronectin had an effect onthe bacterial serum resistance, bacteria were incubated in vitronec-tin-depleted human serum, which retained full complement activ-ity. NTHi 3655 showed reduced survival in the vitronectin-de-pleted serum (Fig. 9C), suggesting that an interaction withvitronectin was important for bacterial survival.

DiscussionNTHi is a frequent colonizer of the healthy upper respiratory tract andis rarely associated with invasive disease (29). However, this patho-gen is one of the most common causes of exacerbations in patients

that suffer from chronic obstructive pulmonary disease (30–34). Inaddition, NTHi is a frequent cause of acute otitis media in childrenand frequently colonizes patients with bronchiectasis (35). In thisstudy, we describe for the first time a direct interaction between vi-tronectin, which is a regulator of the terminal complement pathway,and the outer membrane protein PE from NTHi. This previously un-known interaction is of importance for NTHi survival in contact withthe complement system. A generally important feature for pathogensthat allows them to cause disease is their ability to avoid and resist thebactericidal activity of the complement system. A pathogen havingthe capability to survive in human blood has the potential to spread toother sites of the body. NTHi is a pathogen that only rarely is asso-ciated with bacteremia. Hence, the binding of vitronectin most likelyoccurs at the mucosal surface. Reports of the presence of complementcomponents in the respiratory tract of healthy individuals are scarce.However, several studies indicate the importance of complement inthe respiratory tract during infections. During inflammation, the per-meability of the mucosa increases and plasma, including complementproteins and immunoglobulins, enter the airway lumen (36–38). Thisprocess designated plasma exudation is likely the first line of the mu-cosal defense. In patients with chronic otitis medium with effusion,local complement activation in the middle ear mucosa has been ob-served, including an intense deposition of C3 (39). In addition, factorH, factor H-like protein 1, and factor H-related protein 1/2/3/4/5 arecomplement components found in middle ear effusions of patientswith otitis media (40). Moreover, complement activity can also bedetected in the ECM during inflammation (37).

Numerous microbes interact with the complement system inseveral ways to protect themselves from complement attacks. Oneefficient strategy used by several pathogens is binding of comple-ment inhibitors, including vitronectin, factor H, factor H-like pro-tein 1, and C4BP (41, 42). We have previously demonstrated thatHib binds vitronectin (13). This interaction contributed to serumresistance of Hib strains and was linked to Hsf protein expression.Moraxella catarrhalis ubiquitous surface protein A2 is anothersurface molecule that has been found to bind vitronectin and sig-nificantly contributes to serum resistance (43).

In this study, we demonstrate that NTHi devoid of PE had amarkedly reduced survival in NHS compared with the wild type.The initial increase in the number of wild-type bacteria after 5 minsuggests that NTHi uses NHS as a substrate for growth. Interest-ingly, this phenomenon has been seen before with other NTHistrains. One example is the C4BP binding strain NTHi 506 whichalso initially grew better after incubation with human serum (11).In parallel, in a study performed by Hood et al. (44), a similarincrease in the number of bacteria was observed when low con-centrations of NHS (1.25–5%) were added to a NTHi strain. In ourexperimental set up, upon prolonged incubation (�40 min), theNTHi were killed by the complement system in NHS. However,even if the majority of the bacteria is sensitive to human serum, ifjust a few virulent bacteria survive, these might be able to multiplyand initiate an infection. When bacteria were incubated in C7-depleted NHS, which is deficient in the terminal pathway, theNTHi 3655 strain survived, confirming that activation of the ter-minal pathway and consequently MAC formation is responsiblefor the killing of NTHi.

Furthermore, a direct binding assay showed that PE was impor-tant for binding of soluble vitronectin. This finding is in contrast toa previous study, in which no binding of soluble vitronectin to H.influenzae was found (12). The reason for this discrepancy is atpresent unclear. However, in parallel to NTHi, Hib also binds sol-uble vitronectin (13). In addition to the direct binding assay, flowcytometry experiments showed a significantly decreased vitronec-tin binding to the NTHi 3566�pe compared with the isogenic wild

FIGURE 9. Vitronectin in the presence of PE(22–160) and bound to thesurface of NTHi 3655 was still able to inhibit the MAC formation andincrease the survival of NTHi 3655 in NHS. Increasing concentrations ofPE(22–160) (A) or NTHi 3655 (B) preincubated with vitronectin wereadded together with C7, C8, and C9 to C5b-6-coated sheep erythrocytes.After incubation, the hemolysis of the sheep erythrocytes was measuredspectrophotometrically (A414 nm). Factor H was used as a negative control.Hemolysis of sheep erythrocytes in the presence of MAC was set to 100%.C, NTHi 3655 showed a reduced survival upon growth in vitronectin-depleted human serum containing active complement. The NTHi 3655 wasincubated in the presence of 10% NHS or 10% vitronectin-depleted serumboth diluted in DGVB�� and spread on chocolate agar plates to allowdetermination of the number of surviving bacteria. After 10 min, bacteriawere collected and spread on chocolate agar plates to allow determinationof surviving bacteria. The number of bacteria (CFU) at the initiation of theexperiment was defined as 100%. The mean values of three separate ex-periments are shown and error bars correspond to SD. Statistical signifi-cance of differences was calculated using Student’s t test. �, p � 0.05 and���, p � 0.001.

2599The Journal of Immunology

by guest on June 13, 2013http://w

ww

.jimm

unol.org/D

ownloaded from

type when low vitronectin concentrations were used. However, thePE-deficient NTHi bound vitronectin at higher concentrations,suggesting the presence of a second vitronectin binding proteinindependently of PE. Moreover, when human serum was used as asource of vitronectin, similar results were obtained.

In addition to rendering the bacteria more resistant to com-plement-induced lysis, the bacteria-vitronectin interaction alsocontributes to the attachment and invasion into host cells. Thevitronectin binding of Pseudomonas aeruginosa and Neisseriagonorrhoeae is involved in attachment and internalization ofthe bacteria into human epithelial cells (27, 45). Since vitronectinalso is a component of the ECM, binding of H. influenzae to ex-posed ECM components may contribute to bacterial adherence,which is an essential step in the bacterial pathogenesis. These in-teractions appear to contribute to the spread of bacteria throughtissue barriers into secondary sites of infection. Previous studieshave shown that H. influenzae can interact with ECM and recon-stituted basement membranes from cultured human epithelial cells(17). Binding to ECM proteins makes the bacteria able to reachdeeper tissue layers of the mucosa. We demonstrate that the H.influenzae wild-type and PE-expressing E. coli transformants bothbound to immobilized vitronectin. In contrast, when PE was de-leted in H. influenzae, a significantly decreased binding was ob-served. Thus, PE-mediated bacterial attachment to vitronectin maybe an important factor in initial colonization and bacterial spread tonew sites of infection.

Three heparin-binding domains of the vitronectin molecule (res-idues 82–137, 175–219, and 348–376) have been identified (1,28). We found that heparin inhibited the binding between PE-ex-pressing H. influenzae and vitronectin. However, the interactionbetween PE and vitronectin is not dependent on ionic interactionsbetween amino acids. In addition to NTHi PE, Hsf from encap-sulated H. influenzae has been shown to interact with vitronec-tin (13). Hsf also interacts with both soluble and immobilizedvitronectin and this interaction is inhibited by heparin and con-tributes to bacterial serum resistance. Despite the fact that PE isa low molecular mass protein (16 kDa) compared with Hsf (245kDa), it is a potent binder of vitronectin. We compared thevitronectin-binding capacity of PE and Hsf and found that thesetwo outer membrane proteins were approximately equally effi-cient in vitronectin-binding capacity (T. Hallstrom and K. Rie-beck, unpublished observation).

To define the vitronectin-binding domains in PE, peptides span-ning the entire PE molecule were analyzed for vitronectin bindingin ELISA. PE(84–108) displayed the highest affinity for vitronec-tin and showed a dose-dependent binding to vitronectin. A rela-tively large area of PE (aa 41–108) is involved in binding to vi-tronectin. However, the highest signal in the ELISA analysis wasobtained with the peptide comprising PE(84–108), which includesthe adhesive domain mediating attachment to epithelial cells (20,21). PE(84–108) has been shown to have affinity for the lung car-cinoma cell line A549, the conjunctival epithelial cell line Chang,and the pharynx-derived cell line Detroit. Another proof of itsimportance for the interaction with epithelial cells is that PE(84–108) also is able to inhibit the attachment of NTHi to the epithelialcell surface. Furthermore, in a pulmonary clearance model, miceimmunized with PE(84–108) developed Abs, resulting in a signif-icantly faster clearance upon challenge with NTHi. In the light ofthe ELISA results, it is suggested that two binding sites exist in thePE molecule. However, the most significant PE-dependent vitro-nectin-binding domain is located within aa 84–108, since it waspossible to fully block the PE-vitronectin interaction with PE(84–108). Interestingly, PE(84–108) at a low concentration (2 �g/ml)increased binding of vitronectin to whole bacteria. The explanation

for this is at present unclear. However, PE(84–108) at �10 �g/mlsignificantly inhibited the binding between vitronectin and NTHi.The precise binding domain of PE on the vitronectin molecule is atpresent unknown and is an impetus for future studies.

The formation of the lytic pore, i.e., MAC, occurs by transitionof hydrophilic complement components C5b, C6, C7, and C8, re-sulting in polymerization of C9. Vitronectin inhibits both the as-sembly and formation of MAC by inhibiting the C5b-7 complex atits membrane binding site (2). The insertion of the complex intothe cell membrane is thereby inhibited, preventing lysis of themicrobe. Vitronectin bound to recombinant PE(22–160) and to thesurface of the bacteria (NTHi 3655) maintained its inhibitory ac-tivity and inhibited lysis in a hemolytic assay. Consequently, thisinactivation protects the pathogen. In addition, when bacteria wereincubated in serum lacking vitronectin but with the complementsystem preserved, a statistically significant decrease in survivalwas seen with NTHi 3655. Thus, this demonstrated that binding tovitronectin protects NTHi against the attack of the complementsystem.

In recent years, it has been demonstrated experimentally that H.influenzae interferes with the complement system in several ways.NTHi has been shown to interact with C4BP, factor H, and factorH-like protein 1, the soluble inhibitors of the classical/lectin andalternative pathways, and thereby uses these interactions as aprotection against complement-mediated attacks (11, 14). In thepresent study, we have identified a novel interaction betweenPE of NTHi and vitronectin, the regulator of the terminal path-way. PE significantly contributes to NTHi serum resistance byinteracting with vitronectin, and consequently contributes toNTHi virulence. This interaction may also contribute to bacte-rial colonization and spread of NTHi. These observations pro-vide an excellent background for in vivo studies using animalmodels to fully establish the in vivo significance of the vitro-nectin binding in NTHi pathogenesis.

DisclosuresThe authors have no financial conflict of interest.

References1. Schvartz, I., D. Seger, and S. Shaltiel. 1999. Vitronectin. Int. J. Biochem. Cell

Biol. 31: 539–544.2. Preissner, K. T. 1991. Structure and biological role of vitronectin. Annu. Rev. Cell

Biol. 7: 275–310.3. Podack, E. R., K. T. Preissner, and H. J. Muller-Eberhard. 1984. Inhibition of C9

polymerization within the SC5b-9 complex of complement by S-protein. ActaPathol. Microbiol. Immunol. Scand. Suppl. 284: 89–96.

4. Preissner, K. T., E. R. Podack, and H. J. Muller-Eberhard. 1985. The membraneattack complex of complement: relation of C7 to the metastable membrane bind-ing site of the intermediate complex C5b-7. J. Immunol. 135: 445–451.

5. Kilian, M. 2003. Haemophilus. In Manual of Clinical Microbology. P. R. Murray,ed. ASM Press, Washington, DC, pp. 623–635.

6. Broome, C. V. 1987. Epidemiology of Haemophilus influenzae type b infectionsin the United States. Pediatr. Infect. Dis. J. 6: 779–782.

7. St. Geme, J. W. III. 1993. Nontypeable Haemophilus influenzae disease: epide-miology, pathogenesis, and prospects for prevention. Infect. Agents Dis. 2: 1–16.

8. Cripps, A. W., and D. C. Otczyk. 2006. Prospects for a vaccine against otitismedia. Expert. Rev. Vaccine 5: 517–534.

9. Foxwell, A. R., J. M. Kyd, and A. W. Cripps. 1998. Nontypeable Haemophilusinfluenzae: pathogenesis and prevention. Microbiol. Mol. Biol. Rev. 62: 294–308.

10. Kraiczy, P., and R. Wurzner. 2006. Complement escape of human pathogenicbacteria by acquisition of complement regulators. Mol. Immunol. 43: 31–44.

11. Hallstrom, T., H. Jarva, K. Riesbeck, and A. M. Blom. 2007. Interaction withC4b-binding protein contributes to nontypeable Haemophilus influenzae serumresistance. J. Immunol. 178: 6359–6366.

12. Eberhard, T., and M. Ullberg. 2002. Interaction of vitronectin with Haemophilusinfluenzae. FEMS Immunol. Med. Microbiol. 34: 215–219.

13. Hallstrom, T., E. Trajkovska, A. Forsgren, and K. Riesbeck. 2006. Haemophilusinfluenzae surface fibrils contribute to serum resistance by interacting with vi-tronectin. J. Immunol. 177: 430–436.

14. Hallstrom, T., P. F. Zipfel, A. M. Blom, N. Lauer, A. Forsgren, and K. Riesbeck.2008. Haemophilus influenzae interacts with the human complement inhibitorfactor H. J. Immunol. 181: 537–545.

2600 PE INTERACTS WITH VITRONECTIN

by guest on June 13, 2013http://w

ww

.jimm

unol.org/D

ownloaded from

15. Murphy, T. F., J. M. Bernstein, D. M. Dryja, A. A. Campagnari, andM. A. Apicella. 1987. Outer membrane protein and lipooligosaccharide analysisof paired nasopharyngeal and middle ear isolates in otitis media due to non-typable Haemophilus influenzae: pathogenetic and epidemiological observations.J. Infect. Dis. 156: 723–731.

16. Hultgren, S. J., S. Abraham, M. Caparon, P. Falk, J. W. St Geme, 3rd, andS. Normark. 1993. Pilus and nonpilus bacterial adhesins: assembly and functionin cell recognition. Cell 73: 887–901.

17. Virkola, R., M. Brummer, H. Rauvala, L. van Alphen, and T. K. Korhonen. 2000.Interaction of fimbriae of Haemophilus influenzae type b with heparin-bindingextracellular matrix proteins. Infect. Immun. 68: 5696–5701.

18. Fink, D. L., B. A. Green, and J. W. St Geme, 3rd. 2002. The Haemophilusinfluenzae Hap autotransporter binds to fibronectin, laminin, and collagen IV.Infect. Immun. 70: 4902–4907.

19. St. Geme, J. W. III, D. Cutter, and S. J. Barenkamp. 1996. Characterization of thegenetic locus encoding Haemophilus influenzae type b surface fibrils.J. Bacteriol. 178: 6281–6287.

20. Ronander, E., M. Brant, E. Eriksson, M. Morgelin, O. Hallgren,G. Westergren-Thorsson, A. Forsgren, and K. Riesbeck. 2009. The non-typeableHaemophilus influenzae adhesin protein E: characterization and biological activ-ity. J. Infect. Dis. 199: 522–531.

21. Ronander, E., M. Brant, H. Janson, J. Sheldon, A. Forsgren, and K. Riesbeck.2007. Identification of a novel Haemophilus influenzae protein important foradhesion to epithelial cells. Microbes Infect. 10: 87–96.

22. Melhus, A., A. Hermansson, A. Forsgren, and K. Prellner. 1998. Intra- and in-terstrain differences of virulence among nontypeable Haemophilus influenzaestrains. APMIS 106: 858–868.

23. Forsgren, A., M. Brant, A. Mollenkvist, A. Muyombwe, H. Janson, N. Woin, andK. Riesbeck. 2001. Isolation and characterization of a novel IgD-binding proteinfrom Moraxella catarrhalis. J. Immunol. 167: 2112–2120.

24. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1963. The preparation ofI-131-labelled human growth hormone of high specific radioactivity. Biochem. J.89: 114–123.

25. Nordstrom, T., A. Forsgren, and K. Riesbeck. 2002. The immunoglobulin D-binding part of the outer membrane protein MID from Moraxella catarrhaliscomprises 238 amino acids and a tetrameric structure. J. Biol. Chem. 277:34692–34699.

26. Mollenkvist, A., T. Nordstrom, C. Hallden, J. J. Christensen, A. Forsgren, andK. Riesbeck. 2003. The Moraxella catarrhalis immunoglobulin D-binding pro-tein MID has conserved sequences and is regulated by a mechanism correspond-ing to phase variation. J. Bacteriol. 185: 2285–2295.

27. Leroy-Dudal, J., H. Gagniere, E. Cossard, F. Carreiras, and P. Di Martino. 2004.Role of �v�5 integrins and vitronectin in Pseudomonas aeruginosa PAK inter-action with A549 respiratory cells. Microbes Infect. 6: 875–881.

28. Liang, O. D., S. Rosenblatt, G. S. Chhatwal, and K. T. Preissner. 1997. Identi-fication of novel heparin-binding domains of vitronectin. FEBS Lett. 407:169–172.

29. Murphy, T. 2001. Haemophilus infections. In Harrison’s Principles of InternalMedicine. F. Braunwald, D. Kaspar, S. Hauser, D. Longo, and L. Jameson, eds.McGraw-Hill, New York, pp. 939–942.

30. Bandi, V., M. A. Apicella, E. Mason, T. F. Murphy, A. Siddiqi, R. L. Atmar, andS. B. Greenberg. 2001. Nontypeable Haemophilus influenzae in the lower respi-ratory tract of patients with chronic bronchitis. Am. J. Resp. Crit. Care Med. 164:2114–2119.

31. Bandi, V., M. Jakubowycz, C. Kinyon, E. O. Mason, R. L. Atmar,S. B. Greenberg, and T. F. Murphy. 2003. Infectious exacerbations of chronicobstructive pulmonary disease associated with respiratory viruses and non-type-able Haemophilus influenzae. FEMS Immunol. Med. Microbiol. 37: 69–75.

32. Groenewegen, K. H., and E. F. Wouters. 2003. Bacterial infections in patientsrequiring admission for an acute exacerbation of COPD; a 1-year prospectivestudy. Respir. Med. 97: 770–777.

33. Patel, I. S., T. A. Seemungal, M. Wilks, S. J. Lloyd-Owen, G. C. Donaldson, andJ. A. Wedzicha. 2002. Relationship between bacterial colonisation and the fre-quency, character, and severity of COPD exacerbations. Thorax 57: 759–764.

34. Rosell, A., E. Monso, N. Soler, F. Torres, J. Angrill, G. Riise, R. Zalacain,J. Morera, and A. Torres. 2005. Microbiologic determinants of exacerbation inchronic obstructive pulmonary disease. Arch. Intern. Med. 165: 891–897.

35. King, P. T., J. Ngui, D. Gunawardena, P. W. Holmes, M. W. Farmer, andS. R. Holdsworth. 2008. Systemic humoral immunity to non-typeable Haemophi-lus influenzae. Clin. Exp. Immunol. 153: 376–384.

36. Greiff, L., M. Andersson, J. S. Erjefalt, C. G. Persson, and P. Wollmer. 2003.Airway microvascular extravasation and luminal entry of plasma. Clin. Physiol.Funct. Imaging 23: 301–306.

37. Greiff, L., I. Erjefalt, C. Svensson, P. Wollmer, U. Alkner, M. Andersson, andC. G. Persson. 1993. Plasma exudation and solute absorption across the airwaymucosa. Clin. Physiol. 13: 219–233.

38. Persson, C. G., I. Erjefalt, U. Alkner, C. Baumgarten, L. Greiff, B. Gustafsson,A. Luts, U. Pipkorn, F. Sundler, C. Svensson, et al. 1991. Plasma exudation as afirst line respiratory mucosal defence. Clin. Exp. Allergy 21: 17–24.

39. Narkio-Makela, M., J. Jero, and S. Meri. 1999. Complement activation and ex-pression of membrane regulators in the middle ear mucosa in otitis media witheffusion. Clin. Exp. Immunol. 116: 401–409.

40. Narkio-Makela, M., J. Hellwage, O. Tahkokallio, and S. Meri. 2001. Comple-ment-regulator factor H and related proteins in otitis media with effusion. Clin.Immunol. 100: 118–126.

41. Blom, A. M. 2004. Strategies developed by bacteria and virus for protection fromthe human complement system. Scand. J. Clin. Lab. Invest. 64: 479–496.

42. Zipfel, P. F., R. Wurzner, and C. Skerka. 2007. Complement evasion of patho-gens: common strategies are shared by diverse organisms. Mol. Immunol. 44:3850–3857.

43. Attia, A. S., S. Ram, P. A. Rice, and E. J. Hansen. 2006. Binding of vitronectinby the Moraxella catarrhalis UspA2 protein interferes with late stages of thecomplement cascade. Infect. Immun. 74: 1597–1611.

44. Hood, D. W., K. Makepeace, M. E. Deadman, R. F. Rest, P. Thibault, A. Martin,J. C. Richards, and E. R. Moxon. 1999. Sialic acid in the lipopolysaccharide ofHaemophilus influenzae: strain distribution, influence on serum resistance andstructural characterization. Mol. Microbiol. 33: 679–692.

45. Dehio, M., O. G. Gomez-Duarte, C. Dehio, and T. F. Meyer. 1998. Vitronectin-dependent invasion of epithelial cells by Neisseria gonorrhoeae involves �v in-tegrin receptors. FEBS Lett. 424: 84–88.

2601The Journal of Immunology

by guest on June 13, 2013http://w

ww

.jimm

unol.org/D

ownloaded from