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Inactivated Expressing the Receptor-Binding Domain Of Inactivated expressing the receptor-binding domain of bacterial adhesins elicit antibodies inhibiting hemagglutination Nani van Gerven, Henri de Greve, Jean-Pierre Hernalsteens To cite this version: Nani van Gerven, Henri de Greve, Jean-Pierre Hernalsteens. Inactivated expressing the receptor- binding domain of bacterial adhesins elicit antibodies inhibiting hemagglutination. Veterinary Micro- biology, Elsevier, 2008, 131 (3-4), pp.369. 10.1016/j.vetmic.2008.04.001. hal-00532410 HAL Id: hal-00532410 https://hal.archives-ouvertes.fr/hal-00532410 Submitted on 4 Nov 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Accepted Manuscript Title: Inactivated Salmonella expressing the receptor-binding domain of bacterial adhesins elicit antibodies inhibiting hemagglutination Authors: Nani Van Gerven, Henri De Greve, Jean-Pierre Hernalsteens PII: S0378-1135(08)00134-X DOI: doi:10.1016/j.vetmic.2008.04.001 Reference: VETMIC 3997 To appear in: VETMIC Received date: 13-11-2007 Revised date: 25-3-2008 Accepted date: 10-4-2008 Please cite this article as: Van Gerven, N., De Greve, H., Hernalsteens, J.- P., Inactivated Salmonella expressing the receptor-binding domain of bacterial adhesins elicit antibodies inhibiting hemagglutination, VeterinaryMicrobiology (2007), doi:10.1016/j.vetmic.2008.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. * Manuscript 1 Inactivated Salmonella expressing the receptor-binding domain of 2 bacterial adhesins elicit antibodies inhibiting hemagglutination 3 4 Nani Van Gerven1*, Henri De Greve2,3, Jean-Pierre Hernalsteens1 5 6 1Onderzoeksgroep Genetische Virologie, Vrije Universiteit Brussel, B-1050 Brussels, Belgium 7 2Department Molecular and Cellular Interactions, VIB, B-1050 Brussels, Belgium 8 3Onderzoeksgroep Ultrastructuur, Vrije Universiteit Brussel, B-1050 Brussels, Belgium 9 10 *Corresponding author. Mailing address: Onderzoeksgroep Genetische Virologie, Faculteit van 11 de Wetenschappen, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. Phone: 12 32 2 6291857. Fax: 32 2 6291912. E-mail: [email protected] 13 14 Abstract 15 We examined the potential of inactivated Salmonella strains to induce protective 16 antibodies against two adhesins of pathogenic E. coli. The receptor-binding domains of the 17 F17a-G adhesin of F17a fimbriae and of the FimH adhesin of type 1 fimbriae were fused to the 18 translocator domain of the autotransporter AIDA-I. An IgG response against F17a-G or FimH 19 was induced afterAccepted immunization of mice with acetone-inactivated ManuscriptSalmonella displaying the 20 corresponding fimbrial receptor-binding domain. These sera inhibit in vitro agglutination of 21 erythrocytes by E. coli carrying these fimbriae. Our results demonstrate that induced and 22 subsequently acetone-inactivated Salmonella are useful delivery vehicles for the stimulation of 23 an IgG antibody response against heterologous antigens. 1 Page 1 of 17 24 Keywords: AIDA-I/autodisplay/F17 fimbriae/Salmonella/vaccine/Type 1 fimbriae 25 26 1. Introduction 27 Initial attachment, mediated through different fimbriae, allows bacteria to colonize 28 specific host tissues (Klemm and Schembri, 2000). Although present on most 29 Enterobacteriaceae, type 1 fimbriae are virulence factors of avian pathogenic Escherichia coli 30 (APEC) (Pourbakhsh et al., 1997) and uropathogenic E. coli (UPEC) (Wu et al., 1996). F17a 31 fimbriae, on the other hand, can be found on particular bovine enterotoxigenic E. coli (ETEC) 32 strains (Mainil et al., 2000). The adhesion of type 1 and F17a fimbriae to their receptors is 33 mediated by the adhesin subunit, respectively FimH or F17a-G, which is located at the tip of 34 the fimbriae. Because attachment is essential for infection, anti-adhesin antibodies can play an 35 important role in protective immunity (Langermann et al., 1997). Immunization with complete 36 fimbriae or inactivated bacteria carrying these may however mostly generate antibodies against 37 the repeated major subunits, and not against the adhesins. 38 Adhesins are unstable when expressed on their own, but expressing only the N-terminal 39 receptor-binding domain (RBD) overcomes this problem (Buts et al., 2003; Hung et al., 2002). 40 Purifying large quantities of adhesins is nonetheless not cost-effective; therefore the use of an 41 antigen delivery system can be advantageous. We examined the potential of killed Salmonella 42 cells, expressingAccepted the F17a-G or FimH RBD fusedManuscript to the translocator domain of the 43 autotransporter AIDA-I (Benz and Schmidt, 1989), to elicit an antibody response to these 44 adhesins. Vaccination using inactivated bacteria avoids the safety issues related to the use of 45 live-attenuated bacteria and allows the immunization with bacteria expressing the antigens at 46 high levels, which in live-attenuated bacteria can interfere with the viability of the bacteria. 2 Page 2 of 17 47 2. Materials and methods 48 2.1. Bacterial strains and plasmids 49 Bacterial strains and plasmids used in this study are listed in Table I. Bacteria were grown 50 at 37°C on Luria-Bertani (LB) agar plates or in LB broth, supplemented with carbenicillin (100 51 mg/l) or kanamycin (50 mg/l) when required. 52 2.2. Cloning 53 Fusion of the RBD of the F17a-G or FimH adhesins to the AIDA-I translocator domain 54 was carried out using the MultiSite Gateway® Three-Fragment Vector Construction Kit 55 (Invitrogen). The primers used are shown in Table II. 56 The plasmids pGV5141 or pGV5153 were obtained after site-specific LR recombination 57 between the destination vector pDESTR4-R3 and the plasmids pGV5159 and pGV5153, 58 together with pGV5125 or pGV5292. The resulting plasmids pGV5141 and pGV5153 contain 59 a translational gene fusion coding for the F17a-G or the FimH receptor-binding domain, fused 60 to the translocator domain of AIDA-I, under the control of the PBAD promoter. 61 2.3. Expression of the RBD fused to AIDA-I 62 Recombinant gene expression was analyzed in E. coli K514 and Salmonella (Van Gerven 63 et al., 2008). The presence of the fusion protein in bacterial whole-cell lysates was examined by 64 SDS-PAGE and Western blotting, using a rabbit antiserum raised against the F17a-G or FimH 65 RBD. Outer membraneAccepted (OM)-enriched fractions ofManuscript bacteria were prepared using the sarcosyl 66 method (Rizos et al., 2003). 67 2.4. Preparation of the purified RBDs 3 Page 3 of 17 68 The RBD of F17a-G and FimH was purified using gel filtration and affinity 69 chromatography (Bouckaert et al., 2005; Buts et al., 2003). The purity of the resulting proteins 70 was confirmed by SDS-PAGE, followed by Coomassie Blue staining. 71 2.5. Immunization of mice 72 Female BALB/c mice were immunized intraperitoneally (i.p.) or subcutaneously (s.c.) 73 with 100 µl of a 1 mg/ml suspension of powder of induced and subsequently acetone- 74 inactivated bacterial cultures, prepared as described (Harlow and Lane, 1988), in sterile 75 phosphate buffered saline (PBS). Blood samples were collected from the tail vein of each 76 mouse before the first immunization and after two, four, six and eight weeks. The immune 77 response to the F17a-G or FimH RBD or to LPS of S. Typhimurium (Sigma) was tested by 78 ELISA, both on individual samples as on pools, as described (Van Gerven et al., 2008). 79 Experiments were repeated two independent times. Animals were handled following all 80 relevant national guidelines and institutional policies. 81 2.6. Agglutination assays 82 Hemagglutination assays were performed with bovine or rabbit erythrocytes in 96-well, 83 round-bottom microtiter plates and on glass slides as described (Brunder et al., 2001). Yeast 84 agglutination assays were performed on glass slides, using a 1% suspension of live commercial 85 baker’s yeast in PBS. Mice sera were used starting from a dilution of 1:20. 86 3. Results andAccepted discussion Manuscript 87 3.1. Construction and expression of the F17a-G-AIDA-I and FimH-AIDA-I fusion 88 proteins 89 Since, in the case of live-attenuated vaccines, surface expression enhances the immune 90 response to heterologous antigens (Haddad et al., 1995; Liljeqvist and Stahl, 1999), the RBD of 4 Page 4 of 17 91 the FimH and F17a-G adhesins were targeted to the surface of the bacteria by fusing them to 92 the translocator domain of the autotransporter protein AIDA-I. Expression of the fusion 93 proteins was confirmed by SDS-PAGE of whole cell lysates of L-arabinose-induced bacteria 94 and subsequent Western blotting, using a rabbit antiserum raised against the RBD of F17a-G 95 (Fig. 1A) or FimH (Fig. 1B). 96 To determine the cellular localization of the F17a-G or FimH RBD, OM-enriched 97 fractions were prepared of non-induced and L-arabinose-induced E. coli K514 and S. 98 Typhimurium DV6181 transformed with plasmid pGV5141 or pGV5153. In both strains, and 99 for both plasmids, the fusion protein was mainly recovered in the sarcosyl-insoluble fraction, 100 containing the bacterial OM (data not shown). 101 3.2.
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