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

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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 References This article cites 42 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/177/1/430.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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, Malmo¨University 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 vi- tronectin were analyzed by flow cytometry. The wild-type strains and the ␮ 54–608 Rabbits were immunized i.m.
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