Haemophilus Influenzae Surface Fibrils Contribute to Serum Resistance by Interacting with Vitronectin

Haemophilus influenzae Surface Fibrils Contribute to Serum Resistance by Interacting with Vitronectin

This information is current as Teresia Hallström, Elena Trajkovska, Arne Forsgren and of October 2, 2021. Kristian Riesbeck J Immunol 2006; 177:430-436; ; doi: 10.4049/jimmunol.177.1.430 http://www.jimmunol.org/content/177/1/430 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Haemophilus inﬂuenzae Surface Fibrils Contribute to Serum Resistance by Interacting with Vitronectin1

Teresia Hallstro¨m, Elena Trajkovska, Arne Forsgren, and Kristian Riesbeck2

Vitronectin inhibits the membrane attack complex of the complement system and is found both in plasma and the extracellular matrix. In this study, we have identified the outer membrane protein Haemophilus surface fibrils (Hsf) as the major vitronectin- binding protein in encapsulated H. influenzae type b. A H. influenzae mutant devoid of Hsf showed a significantly decreased binding to both soluble and immobilized vitronectin as compared with the wild-type counterpart. Moreover, Escherichia coli- expressing Hsf at the surface strongly adhered to immobilized vitronectin. Importantly, the H. influenzae Hsf mutant had a markedly reduced survival as compared with the wild-type bacterium when incubated with normal human serum. A series of truncated Hsf fragments were recombinantly manufactured in E. coli. The vitronectin binding regions were located within two separate binding domains. In conclusion, Hsf interacts with vitronectin and thereby inhibits the complement-mediated bactericidal activity, and thus is a major H. influenzae virulence factor. The Journal of Immunology, 2006, 177: 430–436. Downloaded from

aemophilus influenzae is a Gram-negative human patho- Both pilus and nonpilus adhesins of H. influenzae have dis- gen responsible for a variety of diseases. Encapsulated played adherence to ECM proteins. H. influenzae pili, which hem- H H. influenzae strains belong to one of six serotypes (a–f), agglutinate human erythrocytes and adhere to human oropharyn- of which type b is the most virulent serotype (1, 2). The most geal epithelial cells (10–16), exhibit adherence to fibronectin and serious and sometimes life-threatening conditions are invasive dis- heparin-binding ECM proteins. The nonpilus adhesin Haemophi- http://www.jimmunol.org/ eases (e.g., septicemia, epiglottitis, and meningitis) caused by en- lus adhesion and penetration protein was reported as a binder of 3 capsulated H. influenzae serotype b (Hib) (3). In contrast, non- fibronectin, laminin, and collagen IV (8). typable H. influenzae accounts for the majority of local disease and The major nonpilus adhesin in Hib is Haemophilus surface upper and lower respiratory tract infections (e.g., bronchitis, si- fibrils (Hsf) (17). The hsf gene is highly conserved among encap- nusitis, and acute otitis media) and is after pneumococci the second sulated H. influenzae strains and encodes a 2414-aa-long protein most common pathogen isolated from children with acute otitis consisting of three repetitive domains with high sequence similar- medium (1, 2). ity. Hsf is found as short, thin surface fibrils at the bacterial surface A crucial factor in the pathogenesis of both encapsulated and

and is associated with adherence to epithelial cells (16). In 25% of by guest on October 2, 2021 nonencapsulated H. influenzae involves the initial adherence to the all unencapsulated strains, a homologue to the Hsf protein, H. in- mucosa in the respiratory tract (4). If the bacteria manage to over- fluenzae adhesin (Hia), can be found (17–19). The hia gene, which come the mucociliary escalator, they may colonize and cause dam- is shorter than the hsf gene, encodes for a protein with a size of age to the epithelial cells and breakdown of tight junctions (5, 6). 1098 aa and harbors only one domain that corresponds to the three Consequently, the bacteria reach the basement membrane and the repetitive domains in Hsf. However, Southern blot analysis has extracellular matrix (ECM), and may penetrate into deeper tissue layers and consequently into the circulation. Studies on the inter- revealed that hsf and hia are alleles of the same locus with 81% action of H. influenzae and tissue samples from the human respi- similarity and 72% identity (17). ratory tract show that H. influenzae has been associated with dis- The complement system is the first line of innate defense against rupted epithelial cells and exposed ECM proteins such as pathogenic microorganisms, and activation of this system leads to fibronectin, collagen, vitronectin, and laminin (7–9). a cascade of protein deposition on the bacterial surface, resulting in formation of the membrane attack complex (MAC) and opso- nization of the pathogen, followed by phagocytosis. A regulatory component of MAC is the multifunctional glycoprotein vitronectin Medical Microbiology, Department of Laboratory Medicine, Lund University, Malmo¨ that is found both in plasma and in the ECM (20). It exists as a University Hospital, Malmo¨, Sweden 75-kDa protein in the ECM and is found in plasma as two trun- Received for publication November 1, 2005. Accepted for publication April 6, 2006. cated forms: 75 and 65 kDa. The costs of publication of this article were defrayed in part by the payment of page Both Hib and nontypable H. influenzae bind surface-associated charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. vitronectin equally well, and it has been suggested that adhesins 1 This work was supported by grants from the Alfred O¨ sterlund Foundation, the Anna are involved in the binding because both fimbriated and nonfim- and Edwin Berger Foundation, the Crafoord Foundation, the Greta and Johan Kock briated strains adhere to a similar degree (9). In this study, we Foundation, the Swedish Medical Research Council, the Swedish Society of Medi- demonstrate that Hsf is the major vitronectin-binding protein in cine, and the Cancer Foundation at the University Hospital in Malmo¨. Hib. A H. influenzae mutant devoid of Hsf displayed a decreased 2 Address correspondence and reprint requests to Dr. Kristian Riesbeck, Medical Mi- crobiology, Department of Laboratory Medicine, MalmoÜniversity Hospital, Lund binding to both soluble and immobilized vitronectin. Furthermore, University, SE-205 02 Malmo¨, Sweden. E-mail address: [email protected] Hsf-dependent interaction with vitronectin was inhibited by hep- 3 Abbreviations used in this paper: Hib, H. influenzae serotype b; ECM, extracellular arin. Interestingly, H. influenzae wild type survived a significantly matrix; Hia, H. influenzae adhesin; Hsf, H. influenzae surface fibril; MAC, membrane attack complex; NHS, normal human serum; pAb, polyclonal Ab; OMP, outer mem- longer time as compared with the Hsf mutant counterpart when brane protein. exposed to normal human serum (NHS). Finally, we show that two

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 431 separate binding domains of Hsf are involved in the vitronectin SDS-PAGE and Western blots binding. Recombinant proteins were subjected to SDS-PAGE (10%) (25) and stained with Coomassie brilliant blue R-250 (Bio-Rad). Electrophoretical Materials and Methods transfer of protein bands from the gel to an Immobilon-P membrane (Mil- lipore) was done at 35 V overnight to transfer the high m.w. complexes. Bacterial strains and culture conditions After transfer, the Immobilon-P membrane was blocked in PBS with 0.1% The type b strain H. influenzae Eagan and the clinical capsule-deficient H. Tween 20 (PBS-Tween) containing 5% milk powder. After several wash- influenzae isolate RM804 have been described in detail (21, 22). Bacteria, ings in PBS-Tween, the membrane was incubated with rabbit anti-Hsf an- wild type, and mutants were routinely cultured in brain-heart-infusion liq- tiserum diluted 1/100 in PBS-Tween, including 2% milk powder, for 1 h at uid broth supplemented with NAD and hemin (both at 10 ␮g/ml) or on room temperature. HRP-conjugated goat anti-rabbit antiserum diluted 1/1000 was added after washings in PBS-Tween. After incubation for 40 chocolate agar plates at 37°C in a humid atmosphere containing 5% CO2. The Hsf-deficient mutant was cultured in the presence of 15 ␮g/ml kana- min at room temperature and additional washings in PBS-Tween, devel- mycin (Merck). The Streptococcus pyogenes was a clinical isolate from our opment was performed with ECL Western blotting detection reagents (Am- department and was grown in brain-heart infusion liquid broth. Escherichia ersham Biosciences). To analyze the purity of vitronectin obtained from coli BL21 (DE3) and DH5␣ were grown in Luria Bertani liquid broth, human plasma (Sigma-Aldrich), 2 ␮g was subjected to SDS-PAGE and whereas Hsf transformants were cultured with 50 ␮g/ml ampicillin Coomassie stained. (Sigma-Aldrich). Flow cytometry analysis Antibodies The Hsf protein expression and the capacity for H. influenzae to bind vitronectin were analyzed by flow cytometry. The wild-type strains and the ␮ 54–608 Rabbits were immunized i.m. with 200 g of rHsf emulsified in CFA Hsf-deficient mutants were grown in broth overnight and washed once in (Difco and BD Biosciences), and boosted on days 18 and 36 with the same PBS containing 2% BSA (PBS-BSA). Bacteria (108) were incubated with dose of protein in IFA. Blood was drawn 3 wk later. To increase the rabbit anti-Hsf pAb. Bacteria were washed and incubated for 30 min on ice Downloaded from specificity, the anti-Hsf antiserum was affinity purified with Sepharose- with FITC-conjugated goat anti-rabbit pAb (Dakopatts), diluted according conjugated rHsf. To ensure that the polyclonal Ab (pAb) reacted with rHsf, to the manufacturers’ instructions. After three additional washes, the bac- the pAb was analyzed in ELISA. Hsf (100 ng/well) were immobilized in teria were analyzed in a flow cytometer (EPICS, XL-MCL; Corixa). To microtiter plates and incubated at increasing concentrations of the anti- analyze H. influenzae binding to vitronectin, bacteria were incubated with serum, followed by HRP-conjugated goat anti-rabbit pAb diluted 1/1000 0.1–2.5 ␮g of vitronectin for1hin37°C. After washings, bacteria were (Dakopatts). The FITC-conjugated goat anti-human vitronectin and donkey incubated with goat anti-human vitronectin pAb for 30 min at ice, before anti-goat pAb were purchased from Serotec, and the mouse anti-human

incubation with the FITC-conjugated donkey anti-goat pAb. After addi- http://www.jimmunol.org/ vitronectin and the HRP-conjugated anti-mouse pAb were from Invitrogen tional washes, bacteria were analyzed in the flow cytometer. All incuba- Life Technologies and Dakopatts, respectively. tions were kept in PBS-BSA, and the washings were done with the same buffer. Secondary pAb were added separately as negative controls for each Manufacture of Hsf-deficient H. influenzae strain analyzed. Hsf-expressing E. coli was analyzed for vitronectin binding according to a standard protocol (26). In the competition assay, the H. The 5Ј end of hsf (GenBank accession no. U41852) was amplified as two influenzae wild type was preincubated with increasing concentrations of cassettes using DyNAzyme II DNA Polymerase (Finnzymes) introducing heparin (Heparin Leo, Lo¨vens Kemiske Fabrik, or Sigma-Aldrich) or vi- the restriction enzyme sites BamHI and EcoRI or EcoRI and XhoIinad- tronectin-derived synthetic peptides, followed by 1 ␮g of vitronectin. Pep- dition to specific uptake sequences in the two cassettes (23). Resulting PCR tides spanning the heparin binding domain used in this study were vitro- fragments (825 and 918 bp, respectively) were digested and cloned into 348–361 341–370 ϩ/Ϫ nectin (KKQRFRHRNRKGYR) and vitronectin (APRPSLA

pBluescript SK . A kanamycin resistance gene cassette from pUC4K by guest on October 2, 2021 was amplified by PCR, introducing the restriction enzyme site for EcoRI. KKQRFRHRNRKGYRSQRGHSRGR) (Innovagen). After digestion, the PCR product was ligated into the truncated hsf gene Binding of H. influenzae to immobilized vitronectin fragment. H. influenzae strains Eagan and RM804 were transformed according to the M-IV method of Poje and Redfield (23). Resulting mutants Glass slides were coated with 2 ␮g of human plasma vitronectin, air dried were verified by PCR, and the Hsf expression was analyzed by Western at room temperature, and then washed twice with PBS. The slides were ϭ blot and flow cytometry (Figs. 1 and 2B). incubated with prechilled bacteria at late exponential phase (OD600 0.9) for2hatroom temperature, washed twice with PBS, and followed by DNA cloning and protein expression Gram staining. All truncated Hsf constructs were manufactured using PCR-amplified frag- Serum bactericidal assay hsf H. influ- ments. The open reading frame of the gene (U41852) from NHS was pooled from five healthy volunteers. Inactivated serum was used enzae strain RM804 was used as a template. All Hsf constructs were am- as a control. The H. influenzae wild type and corresponding Hsf mutant plified by PCR using DyNAzyme II DNA Polymerase (Finnzymes) with ϩ were diluted in DGVB2 (2.5 mM veronal buffer (pH 7.3), containing specific primers introducing the restriction enzyme sites BamHI and Hin- 0.1% (w/v) gelatin, 1 mM MgCl , and 0.15 mM CaCl ). Bacteria (104 dIII. The sequence encoding for the signal peptide was excluded. To ex- 2 2 CFU) were incubated in 5% of NHS or heat-inactivated NHS in a final press full-length Hsf, a NcoI restriction enzyme site was introduced. The volume of 100 ␮l at 37°C. At different time points, 10-␮l aliquots were PCR products were cloned into pET26ϩ , except for the full-length Hsf, removed and spread onto chocolate agar plates. After 18 h of incubation at which was cloned into both pET26 and pET16. The resulting plasmids 37°C, CFU were determined. were transformed into the host E. coli DH5␣, followed by transformation into the expressing host E. coli BL21(DE3) (Novagen). All constructs were ELISAs sequenced using the BigDye Terminator Cycle Sequencing v. 3.1 Ready reaction kit (Applied Biosystems). To produce recombinant proteins, bac- Microtiter plates (Nunc-Immuno Module) were coated with 40 ␮M purified rHsf fragments in 0.1 M Tris-HCl (pH 9.0) overnight at 4°C. Plates teria were grown to mid-log phase (OD600 0.5–1.0), followed by 1–3 h of induction with 1 mM isopropyl-1-thio-␤-D-galactoside (Saveen Werner). were washed with PBS-0.05% Tween 20 and blocked for1hatroom Inclusion bodies were purified according to a standard protocol (Novagen). temperature with PBS containing 2% BSA. After washings, the wells were The resulting proteins were examined by SDS-PAGE, Western blots, and incubated for1hatroom temperature with vitronectin (5 ␮g/ml) in 2% ELISA. BSA. Thereafter, the plates were washed and incubated with goat anti- human vitronectin pAb for 1 h. After additional washings, HRP-conjugated Outer membrane protein (OMP) preparations anti-goat pAb was added and incubated at room temperature for 40 min. Plates were developed and measured at an OD of 450 nm. Bacteria grown to stationary phase were washed with 50 mM Tris-HCl To determine the vitronectin concentration in NHS following incubation buffer (pH 8.0). The pellet was resuspended in Tris-HCl buffer containing with wild-type or mutant H. influenzae, microtiter plates were coated with 3% Empigen (Calbiochem) and protease inhibitors (Complete; Roche) goat anti-human vitronectin pAb in 0.1 M Tris-HCl (pH 9.0) at 4°C over- (24). OMPs were extracted by rotating the mixture at 37°C for 2 h. The night. Plates were washed, blocked, and incubated with NHS before (0 bacterial cells, stripped of their outer membranes, were centrifuged, and the min) and after 20-min incubation with RM804 or the corresponding mu- supernatants were collected. Thereafter, the supernatants were analyzed on tant. Thereafter, the plates were washed and incubated with mouse anti- SDS-PAGE and Western blots. human vitronectin mAb, followed by HRP-conjugated anti-mouse pAb. 432 Hsf INTERACTS WITH VITRONECTIN

Protein labeling and competition assays Purified rHsf608–1351 was labeled with 0.05 mol iodine (Amersham Bio- sciences) per molecule of protein, using the chloramine-T method (27). To define saturating conditions of 125I-labeled Hsf608–1351, vitronectin was incubated at increasing concentrations with 125I-labeled Hsf608–1351 in microtiter plates. The competition assays were essentially performed as described elsewhere (26). Briefly, microtiter plates were incubated with 0.065 ␮ g of vitronectin overnight at 4°C in 75 mM NaCO3 (pH 9.6). Thereafter, the wells were washed and blocked, as described above. After four washings, 125I-labeled Hsf608–1351 was added, together with various concentrations of unlabeled proteins diluted in blocking buffer, and followed by an overnight incubation at 4°C. After four washings, the radioactivity was measured in a gamma counter. Results Characterization of a Hsf-deficient H. influenzae mutant Two Hib strains (RM804 and Eagan) were mutated by introduction of a kanamycin resistance gene cassette in the gene encoding for Hsf (Fig. 1A). Resulting mutants were confirmed by PCR, and the absence of Hsf expression was proven by analysis of OMPs in

Western blots using a specific anti-Hsf antiserum (Fig. 1, B and C). Downloaded from The RM804⌬hsf mutant was deficient in a high m.w. complex corresponding to Hsf (Fig. 1C). The H. influenzae Hsf mutant was also analyzed by flow cytometry using anti-Hsf pAb (Fig. 2B). Similar results were obtained with the H. influenzae Eagan wild type and the corresponding mutant (data not shown). http://www.jimmunol.org/ Hsf-deficient H. influenzae shows a significantly decreased binding to vitronectin FIGURE 2. The Hsf-deficient mutant shows a decreased binding to vitronectin. A, Vitronectin from human plasma analyzed for purity by SDS- To determine whether Hsf interacted with vitronectin, soluble vi- PAGE. The fraction contains two truncated forms (75 and 65 kDa, as tronectin (Fig. 2A) at increasing concentrations was incubated with indicated by arrows). B, Flow cytometry profiles of H. influenzae wild type and a Hsf-deficient mutant showed a correlation between Hsf expression and vitronectin binding. The RM804 wild-type isolate and RM804⌬hsf were incubated with a rabbit anti-Hsf antiserum and finally a FITC-conjugated anti-rabbit antiserum. C, The Hsf-deficient mutants showed a sig-

nificantly decreased binding to soluble vitronectin, compared with the by guest on October 2, 2021 wild-type counterpart. RM804 wild type and RM804⌬hsf were incubated with vitronectin, followed by goat anti-vitronectin pAb. Finally, a FITC- conjugated anti-goat antiserum was added. A typical experiment of six is demonstrated. D, H. influenzae Eagan wild type and RM804 wild type bind vitronectin in a dose-dependent manner. The H. influenzae Eagan⌬hsf and RM804⌬hsf mutant displayed a much weaker binding to the different concentrations of vitronectin. Bacteria were incubated with increasing concentrations (0.1–2.5 ␮g) of vitronectin, followed by an anti-vitronectin pAb. FITC-conjugated anti-goat pAb was subsequently added, followed by flow cytometry analysis. The mean values of three experiments are shown. Error bars indicate SD.

H. influenzae and the Hsf mutants, followed by flow cytometry analysis using polyclonal anti-vitronectin Abs (Fig. 2C). The H. influenzae RM804 and Eagan isolates significantly bound vitronectin at a concentration of 0.5–2.5 ␮g. In contrast, a strongly ⌬ FIGURE 1. Construction of a Hsf-deficient H. influenzae mutant. A, decreased vitronectin binding was observed with RM804 hsf and Schematic drawing of Hsf. The numbers above the bars refer to amino acid Eagan⌬hsf as compared with the wild-type counterpart (Fig. 2D). residue positions in the full-length protein. Regions of sequence similarity To further show that Hsf interacts with soluble vitronectin, Hsf- are indicated with Ⅺ. The arrow indicates where the gene was disrupted by expressing E. coli (Fig. 3A) was included in our study. E. coli with a kanamycin casette in the mutant. B, The H. influenzae RM804⌬hsf mu- Hsf at the surface bound vitronectin (1–5 ␮g) in a dose-dependent tant was confirmed by PCR. The H. influenzae RM804 wild type (lane 2) manner, whereas no binding was detected with the control bacteria did not contain a kanamycin cassette, whereas the ⌬hsf mutant did (lane 3). (Fig. 3B). pBluescript containing the hsf casettes and the kanamycin cassette was To investigate the attachment of bacteria to immobilized vitro- used as a positive control (lane 4). C, Western blot analysis of H. influenzae nectin, H. influenzae RM804 and its corresponding Hsf mutant RM804⌬hsf mutant compared with the wild-type counterpart. To extract the OMPs, 3% Empigen was used. Resulting proteins were analyzed by were applied to vitronectin-coated glass slides. The H. influenzae Western blots using a rabbit anti-Hsf antiserum and HRP-conjugated goat RM804 wild type was found to strongly adhere to the vitronectin- anti-rabbit pAb. The RM804⌬hsf mutant lacked the high m.w. complex. A coated glass slides (Fig. 4A). This was in contrast to the H. influ- typical experiment of three is demonstrated. Similar results were obtained enzae RM804⌬hsf mutant that barely bound the immobilized vi- with H. influenzae Eagan and its corresponding mutant. tronectin (Fig. 4B). Similar results were obtained with H. The Journal of Immunology 433

FIGURE 3. Hsf-expressing E. coli binds vitronectin. A, Flow cytometry profiles showing the expression of Hsf on the surface of E. coli. B, Hsf- expressing E. coli bound vitronectin in a dose-dependent manner, whereas E. coli did not. A, Bacteria were incubated with rabbit anti-Hsf, followed by an FITC-conjugated anti-rabbit pAb. B, Bacteria were incubated with increasing concentrations (1–5 ␮g) of vitronectin, followed by an anti- vitronectin pAb. FITC-conjugated anti-goat pAb was subsequently added, followed by flow cytometry analysis. The mean values of three experiments are shown. Error bars indicate SEM. Downloaded from influenzae Eagan and the corresponding Hsf mutant (data not shown). To further prove that Hsf interacts with immobilized vi- FIGURE 5. H. influenzae binds human plasma vitronectin, and the in- tronectin, Hsf-expressing E. coli was tested. In parallel with H. teraction is inhibited by heparin. A, Schematic picture of vitronectin showing the heparin binding domains. B, Inhibition of the Hsf-vitronectin in- influenzae, Hsf-expressing E. coli adhered to the vitronectin- teraction by heparin. The vitronectin binding of H. influenzae (f) and S. coated glass slides (Fig. 4C), whereas only a few bacteria were pyogenes (E) in the absence of heparin was defined as 100%. Vitronectin detected when the control E. coli wild type was analyzed (Fig. 4D). binding of H. influenzae decreased with increasing concentrations of hep- http://www.jimmunol.org/ arin (0.1–500 ␮g/ml), whereas no inhibition could be detected when S. Hsf-vitronectin interaction is inhibited by heparin pyogenes was incubated with heparin. Vitronectin (1 ␮g) binding was mea- The vitronectin molecule harbors different functional groups, sured by flow cytometry, as described in Fig. 2. The mean values of three which are involved in, for example, cell attachment, collagen bind- experiments are shown. Error bars indicate SD. ing, and glycosaminoglycan binding. Three heparin binding domains exist in the N terminus and the C terminus of the vitronectin Ͼ molecule (Fig. 5A) (19, 28). To further investigate the nature of the dependent manner (Fig. 5B). The binding was inhibited 80% ␮ interaction of vitronectin to H. influenzae, a series of blocking when heparin at 10 g/ml was added. BSA did not interfere with experiments with heparin was performed. The H. influenzae wild the binding (data not shown). Another commercial heparin prep- by guest on October 2, 2021 type was incubated with heparin at increasing concentrations, fol- aration showed similar results. To exclude sterical hindrance of the lowed by addition of vitronectin. This commercially available hep- heparin molecule, we also tested the capacity of heparin to block arin inhibited the binding of vitronectin to H. influenzae in a dose- vitronectin binding to group A streptococci (Fig. 5B). A significant vitronectin binding to streptococci was observed in analogy with previously published data (29), whereas any inhibitory effect of heparin on vitronectin binding to group A streptococci was not observed at heparin concentrations up to 500 ␮g/ml. To examine the vitronectin-Hsf interaction in detail, the peptides spanning the heparin binding site, vitronectin348–361 and vitronectin341–370, were preincubated with vitronectin, followed by addition of H.

FIGURE 4. The H. influenzae RM804⌬hsf mutant does not bind immobilized vitronectin. A, The H. influenzae wild type was able to adhere at a high density on vitronectin-coated glass slides, whereas B, H. influenzae RM804⌬hsf mutant adhered poorly. C, E. coli-expressing Hsf at the bac- FIGURE 6. Both H. influenzae ⌬hsf mutants were more serum sensitive terial cell surface strongly adhered to the vitronectin-coated glass slide. In than the H. influenzae wild types. Eagan, RM804, Eagan⌬hsf mutant, and contrast, D, E. coli adhered poorly. Glass slides were coated with vitro- RM804⌬hsf mutant were incubated in the presence of 5% NHS. The nectin and incubated with the bacteria. After several washes, bacteria were RM804 wild type was also incubated with 5% heat-inactivated NHS. Num- Gram stained. A typical experiment of three is presented. Similar results bers of bacteria (CFU) before addition of NHS were defined as 100%. The were obtained with Eagan and its corresponding mutant. mean values of three experiments are shown. Error bars indicate SD. 434 Hsf INTERACTS WITH VITRONECTIN

and incubated with increasing concentrations of vitronectin. Bound vitronectin was detected by an anti-human vitronectin pAb, followed by incubation with an HRP-conjugated anti-goat pAb, as can be seen in Fig. 7. Hsf54–2414 bound soluble vitronectin, and the interaction was dose dependent. To deﬁne the vitronectin binding domain of Hsf, recombinant proteins spanning the entire molecule were manufactured. Vitro- nectin was incubated with immobilized Hsf fragments, and the interaction was quantiﬁed by ELISA. Interestingly, two major binding domains were found, i.e., Hsf608–1351 and Hsf1536–2414 (Fig. 8).

The binding of Hsf608–1351 to vitronectin is dose dependent and specific FIGURE 7. Recombinantly expressed Hsf54–2414 binds vitronectin in a dose-dependent manner. Hsf54–2414 (10 ␮g/ml) was coated on microtiter The interaction between vitronectin and the most efficient binding plates and incubated with increasing concentrations of vitronectin, fol- domain (Hsf608–1351) was further confirmed using a competition lowed by detection with goat anti-human vitronectin pAb and HRP-con- assay after the saturated conditions of vitronectin and 125I-labeled jugated anti-goat pAb. The background binding was subtracted from all the Hsf608–1351 had been defined (Fig. 9A). Vitronectin was incubated samples. Mean values of two experiments are shown, and error bars indi- 125 608–1351 with I-labeled Hsf in the presence of increasing Downloaded from cate SD. Hsf608–1351 concentrations. Unlabeled Hsf608–1351 specifically inhibited the binding between 125I-labeled Hsf608–1351 and vitronectin (Fig. 9B). A total of 95 nM Hsf608–1351 was required to block influenzae. These peptides did not block the vitronectin binding. 125 608–1351 Thus, the N-terminal part of the vitronectin molecule is most likely the vitronectin/ I-labeled Hsf interaction by 50% (IC50). involved in the Hsf-vitronectin interaction. Discussion http://www.jimmunol.org/ Hsf is crucial for H. influenzae survival in human serum In the present work, we demonstrate a novel interaction between Vitronectin plays a major role in the complement cascade by in- the encapsulated respiratory pathogen Hib and the important com- hibiting the MAC of complement (19). To analyze the importance plement inhibitor vitronectin. Complement resistance is crucial for of Hsf in H. influenzae survival when exposed to NHS, the wild- bacterial virulence. Binding of complement inhibitors such as vi- type strains RM804 and Eagan, in addition to the corresponding tronectin, C4BP, or factor H is an efficient strategy used by serum- mutants, were tested in a serum bactericidal assay. The wild-type resistant pathogens (30–32). Several studies have indicated that strains were significantly more resistant to NHS as compared with complement proteins and regulators are present in the human re- the mutants devoid of Hsf (Fig. 6). Both the wild-type strain and spiratory tract (33, 34). Moreover, complement activity can be by guest on October 2, 2021 the mutant were resistant to heat-inactivated NHS. detected in the ECM during inflammation (34, 35). Vitronectin was bound to wild-type bacteria, but not to the mu- Hsf is a major adhesin in Hib (17). It is a large, highly conserved tant after incubation with NHS for 20 min. Quantification of re- autotransporter protein, which extrudes as thin fibrils from the bac- sidual vitronectin in serum was performed with ELISA after bacterial surface. Flow cytometry analysis of the Hsf-deficient mutant teria were spun down. Interestingly, the vitronectin serum revealed that Hsf is the major vitronectin-binding protein in en- concentration decreased with 13.3 Ϯ 2.5% after exposure to H. capsulated H. influenzae. Hsf-expressing H. influenzae and E. coli influenzae, whereas no difference was seen with the Hsf-deficient transformants bound soluble vitronectin at increasing concentra- mutants. Taken together, Hsf significantly contributed to H. influ- tions (Figs. 2 and 3B). This finding is in contrast to what has been enzae serum resistance. shown in a previous study, in which no binding of H. influenzae to soluble vitronectin was found (9). In addition to H. influenzae, The vitronectin binding regions are located within two separate Staphylococcus aureus, E. coli, and ␤-hemolytic streptococci are domains of Hsf efficient binders of soluble vitronectin (29, 36). Furthermore, we To further analyze the interactions of Hsf with vitronectin, Hsf54–2414 demonstrate that the Hsf-expressing H. influenzae and E. coli was recombinantly produced in E. coli, coated on microtiter plates, transformants both bound to immobilized vitronectin (Fig. 4, A and

FIGURE 8. The active vitronectin binding domains of Hsf are located between Hsf608–1351 and Hsf1536–2414. Truncated proteins derived from Hsf are shown. All fragments were tested for binding to vitronectin by ELISA; 40 ␮Mof each fragment was coated on microtiter plates and incubated with 5 ␮g/ml vitronectin. Bound vitronectin was detected with goat anti-human vitronectin pAb, followed by HRP-conjugated anti-goat pAb. Results are mean values of three experiments and error bars indicate SD. The Journal of Immunology 435

complement cascade, and inhibits attachment to the cell membrane and induction of cell lysis of the bacteria (37, 38). It also binds the C5b-9 complex and blocks the tubular polymerization of C9, which is responsible for induction of the cell lysis. These two mechanisms make it impossible for the complement to attach to the surface of the bacteria, resulting in bacterial survival. Interest- ingly, it has been suggested that Yersinia enterocolitica OMP YadA acts in a similar fashion as vitronectin by sterically hinder- ing the formation of the MAC (39). In addition to inhibiting the complement system, vitronectin is involved in attachment and spreading of endothelial cells to the ECM and in wound healing by binding the thrombin-antithrombin III complex (20, 38). Further- more, vitronectin promotes the coagulation cascade by binding and activating the plasminogen activator inhibitor 1. Localization of the vitronectin binding domains of Hsf is an important step in defining the function of Hsf. Recombinantly produced Hsf54–2414 bound vitronectin in a dose-dependent manner (Fig. 7). In addition, Hsf608–1351 and Hsf1536–2414 from the clinical isolate H. influenzae RM804 contained two vitronectin binding domains. Hsf608–1351 displayed the highest affinity for vitronectin Downloaded from and showed both a dose-dependent and specific binding to vitronectin (Fig. 9). These data were confirmed by dot-blot assay, in which nitrocellulose membranes were coated with vitronectin and incubated with 125I-labeled Hsf fragments (data not shown). Dur- ing preparation of this manuscript, St. Geme and coworkers (40) demonstrated that Hsf binds Chang epithelial cells via two acidic http://www.jimmunol.org/ binding domains. These domains comprise aa 537–652 and 1904– 2022. They also identified a third binding pocket (Hsf1214–1338), which did not bind to Chang cells. Our strongest binding domains contain two of these three adhesive binding pockets. Hsf is an- chored in the outer membrane by its C-terminal translocator domain, and one Hsf fiber may thus be able to bind two vitronectin 608–1351 FIGURE 9. The binding between Hsf and vitronectin is specific molecules and stabilizing adherence despite the physical forces in because Hsf608–1351 competes with iodine-labeled Hsf608–1351. A, To define the respiratory tract, which includes the mucociliary escalator, saturating conditions, increasing concentrations of 125I-labeled Hsf608–1351 by guest on October 2, 2021 were incubated with vitronectin. B, 125I-labeled Hsf608–1351 (60 kcpm/well) sneezing, and coughing (41). was added together with increasing concentrations of unlabeled Hsf608–1351 Vitronectin is also a component of the ECM (20). Binding of H. to microtiter plates coated with vitronectin. Binding at the lowest compet- influenzae to exposed ECM components may contribute to bacte- itor (i.e., unlabeled Hsf608–1351) concentration was defined as 100%. The rial adherence, which is an essential step in the bacterial patho- mean values of three experiments are shown. Error bars correspond to SD. genesis. One hypothesis is that these interactions contribute to the spread of bacteria through tissue barriers into secondary infection sites. Previous studies have shown that H. influenzae can interact C). In contrast, when Hsf was deleted in H. influenzae, a signifi- with ECM and reconstituted basement membranes from cultured cantly decreased binding was observed (Fig. 4B). Three heparin human epithelial cells (12). Binding to ECM proteins makes the binding domains of the vitronectin molecule (residues 82–137, bacteria able to reach deeper tissue layers of the mucosa. Hsf- 175–219, and 348–376) have been identified (Fig. 5A) (20, 28). mediated bacterial attachment to vitronectin may be an important Heparin inhibited the binding between Hsf-expressing H. influen- factor in initial colonization and spread of the bacteria to new sites zae and vitronectin. Two different commercial preparations of hep- of infection. The ability to bind vitronectin is of great importance arin were tested with similar results. To further prove the speci- for several bacterial species. Neisseria gonorrhoeae probably uses ficity of the heparin blocking, S. pyogenes was included in our vitronectin as a bridge for attachment and invasion of human cells study. The binding of S. pyogenes to vitronectin was not inhibited (41). Binding of N. gonorrhoeae to specific integrins can trigger by heparin (Fig. 5B). This result suggests that the interaction be- endocytosis of vitronectin and consequently engulfment of the tween heparin and H. influenzae is specific. Peptides encompassing bacteria. The interaction with the integrin receptor occurs by direct the C-terminal heparin binding domain, vitronectin348–361 and vi- binding or through binding of vitronectin. In addition, vitronectin tronectin341–370, were also used in blocking experiments. How- mediates attachment of Candida albicans to endothelial cells and ever, any blocking could not be detected with the two peptides, of Pneumocystis carinii to bronchial epithelial cells in the lower suggesting that the N-terminal heparin binding domains are in- respiratory tract (42, 43). volved in the interaction. In conclusion, we have presented several lines of evidence on H. The H. influenzae devoid of Hsf had a markedly reduced sur- influenzae Hsf binding to vitronectin, a factor that inhibits the vival as compared with the wild type when exposed to NHS (Fig. MAC formation in the complement system, preventing comple- 6). The ability to bind vitronectin suggests that Hsf uses the ca- ment-induced cell lysis. This interaction may also contribute to pacity of vitronectin to inhibit the complement-mediated attack. bacterial colonization and spread of H. influenzae. Hsf is the major An important function of vitronectin is inhibition of the MAC of vitronectin-binding protein and consists of two separate binding the complement cascade that is the first line of defense (20, 37). domains. Hsf binding to vitronectin contributes to H. influenzae Vitronectin binds C5b-7, which is one of the end products in the serum resistance and, consequently, virulence. 436 Hsf INTERACTS WITH VITRONECTIN

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