Inactivated expressing the receptor-binding domain of bacterial adhesins elicit 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￿

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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 response against heterologous .

1 Page 1 of 17 24 Keywords: AIDA-I/autodisplay/F17 fimbriae/Salmonella//Type 1 fimbriae

25

26 1. Introduction

27 Initial attachment, mediated through different fimbriae, allows to colonize

28 specific host tissues (Klemm and Schembri, 2000). Although present on most

29 Enterobacteriaceae, type 1 fimbriae are factors of avian pathogenic

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 , 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 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 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

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 , 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. Induction of anti-adhesin antibodies using acetone-inactivated bacteria

102 In order to test the immunogenicity of the proteins expressed by the Salmonella strains,

103 mice were immunized i.p. or s.c. with a suspension of L-arabinose-induced and subsequently

104 acetone-inactivated cultures of DV6181 containing plasmid pGV5141. Acetone treatment was

105 chosen as inactivation method, since immunization with acetone-inactivated S. enterica serovar

106 Typhi protected against challenge in mice (Esposito et al., 1969) and humans (Pittman and

107 Bohner, 1966). After two weeks, a strong IgG antibody response was detected in the

108 immunized mice. This response further increased in time. A comparison of s.c. and i.p. 109 immunizationsAccepted showed that, although i.p. immunization Manuscript was superior in evoking an anti-LPS 110 response (data not shown), both were equally effective in eliciting an antibody response to

111 F17a-G (Fig. 2A).

112 To determine the requirements for efficient immunization, the ability to elicit anti-F17a-

113 G responses of E. coli MG1655 containing plasmid pGV5153 and of S. Typhimurium

5 Page 5 of 17 114 DV6181, carrying the plasmid pPLHD673, and therefore expressing the F17a-G RBD in the

115 periplasm, was tested. Mice were immunized i.p. with L-arabinose-induced and subsequently

116 acetone-inactivated bacteria and, as negative controls, with acetone-inactivated MG1655 and

117 DV6181 bacteria. The lack of IgG antibody response against the F17a-G RBD after these

118 immunizations, as shown in Fig. 2B, suggested that both the fusion to AIDA-I and the use of a

119 Salmonella strain as carrier were necessary to efficiently raise antibodies against the RBD. A

120 comparison with acetone-inactivated cells of E. coli 25KHO9st, a clinical isolate carrying

121 complete F17a fimbriae, was also done. Again, no noticeable antibody response against F17a-

122 G was detected in ELISA (Fig. 2B). This can be due to an immunodominance of the repeated

123 major fimbrial subunit F17a-A. It is also possible that fusion to AIDA-I is more efficient

124 because more copies of the F17a-G are present.

125 To test the general applicability of the immunization with acetone-inactivated bacteria,

126 the experiment was repeated with S. Typhimurium DV6181 containing the FimH-AIDA-I

127 fusion. Again, a clear IgG antibody response was detected after immunization (Fig. 2C).

128 3.3. Inhibition of in vitro bacterial attachment by immune sera

129 Inhibition of binding of F17a-G to GlcNAc-containing receptors was tested via

130 hemagglutination of E. coli 25KHO9st with bovine erythrocytes. A 1:40 dilution of pooled sera

131 of five mice immunized with acetone-inactivated S. Typhimurium DV6181 expressing the 132 F17a-G-AIDA-IAccepted fusion completely inhibited agglutination, Manuscript and a 1:80 dilution of these sera 133 largely inhibited agglutination. Sera of naive mice or sera of mice immunized with acetone-

134 inactivated S. Typhimurium DV6181 did not inhibit agglutination, when used at the same

135 dilutions (data not shown). To evaluate the ability of the immune sera to inhibit binding of

136 FimH to mannose-containing receptors, we performed both agglutination assays with yeast and

6 Page 6 of 17 137 hemagglutination assays with rabbit erythrocytes. E. coli K514 cells were unable to agglutinate

138 yeast cells or rabbit erythrocytes in the presence of a 1:80 dilution of pooled sera of five mice

139 immunized with acetone-inactivated S. Typhimurium DV6181 expressing the FimH-AIDA-I

140 fusion. Sera of naive mice or sera of mice immunized with the controls did not inhibit

141 agglutination, when used at the same dilution (data not shown). Since the sequence

142 of FimH is conserved among E. coli of different origin (Vandemaele et al., 2004), we tested the

143 inhibitory effect of the sera on agglutination of clinical E. coli isolates. As expected, the sera

144 obtained after immunization with acetone-inactivated S. Typhimurium DV6181 expressing the

145 FimH-AIDA-I fusion could also inhibit agglutination of yeast cells and rabbit erythrocytes by

146 the human UPEC strain UTI89 and by the APEC strains APEC 5 and APEC 60 (data not

147 shown).

148 While there has been a considerable interest in using live-attenuated enteric organisms as

149 vaccine vectors, little attention has been focused on non-living vectors. Although i.p.

150 immunization with acetone-inactivated Salmonella bacteria, displaying the RBD of the FimH

151 and F17a-G adhesins did not generate the mucosal IgA response that can in principle be

152 obtained with live-attenuated Salmonella bacteria (data not shown), a fast and efficient IgG

153 response against the heterologous antigens was obtained. Since mucosal vaccination with

154 inactivated whole cells has already shown promise against toxigenic enteric diseases caused by 155 ETEC and VibrioAccepted cholerae (Osorio et al., 2007), Manuscript other ways of administration than i.p. 156 immunization might elicit a mucosal immune response. In addition, a systemic immune

157 response can protect against UPEC (Langermann et al., 1997; Langermann et al.,

158 2000).

7 Page 7 of 17 159 In conclusion, this study proves that in vitro induced and subsequently acetone-

160 inactivated Salmonella bacteria could be used as delivery vehicles for foreign antigens, and

161 surface expression accounts for a better stimulation of an IgG antibody response against these

162 heterologous antigens.

163 Acknowledgements

164 The research is funded by a Ph.D. grant of the Institute for the Promotion of Innovation

165 through Science and Technology in Flanders (IWT-Vlaanderen). The authors thank the Fonds

166 voor Wetenschappelijk Onderzoek – Vlaanderen for the DNA sequencing equipment (grant

167 FWOAL215).

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191 Berzins, K., 1995. Surface display compared to periplasmic expression of a malarial antigen

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194 Harlow, E., Lane, D., 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor

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222 Osorio, M., Bray, M.D., Walker, R.I., 2007. Vaccine potential for inactivated shigellae. 223 Vaccine 25,Accepted 1581-1592. Manuscript 224 Pittman, M., Bohner, H.J., 1966. Laboratory assays of different types of field trial typhoid

225 vaccines and relationship to efficacy in man. J. Bacteriol. 91, 1713-1723.

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227 J.M., 1997. Localization of the in vivo expression of P and F1 fimbriae in chickens

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230 efficacious surface exposure of antigenic UreA fragments from in

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233 receptor-binding domain of the bacterial adhesin F17a-G on bacteriophage M13. Antonie

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235 Vandemaele, F., Vandekerchove, D., Vereecken, M., Derijcke, J., Dho-Moulin, M., Goddeeris,

236 B.M., 2003. Sequence analysis demonstrates the conservation of fimH and variability of

237 fimA throughout avian pathogenic Escherichia coli (APEC). Vet. Res. 34, 153-163.

238 Vandemaele, F.J., Hensen, S.M., Goddeeris, B.M., 2004. Conservation of deduced amino acid

239 sequence of FimH among Escherichia coli of bovine, porcine and avian disease origin. Vet.

240 Microbiol. 101, 147-152.

241 Wu, X.R., Sun, T.T., Medina, J.J., 1996. In vitro binding of type 1-fimbriated Escherichia coli

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243 9630-9635. 244 Figure legendsAccepted Manuscript 245 Fig. 1. Expression of the F17a-G or FimH receptor-binding domain (RBD), fused to the

246 translocator domain of AIDA-I. (A) S. Typhimurium DV6181 (pGV5141) (B) DV6181

247 (pGV5153). Bacterial cultures were induced by the addition of 0.2% L-arabinose (Ara). Lanes

11 Page 11 of 17 248 1 and 2: whole cell lysates, lanes 3 and 4: acetone-inactivated bacteria. Subsequent Western

249 blotting was carried out, using an antibody raised against the RBD of F17a-G or FimH.

250

251 Fig. 2. Antibody response against the F17a-G or FimH RBD in mice, immunized with acetone-

252 inactivated bacteria. Serum IgG responses were determined by ELISA, using the F17a-G or

253 FimH RBD as coated antigen. Two-fold dilutions starting from 1:100 are shown. Symbols

254 represent a pool of five mice. (A) Response to F17a-G four weeks after immunization with S.

255 Typhimurium DV6181 (pGV5141), expressing the F17a-G-AIDA-I fusion. Pre-immune serum

256 (□), serum after subcutaneous (s.c.) (▲) or intraperitoneal (i.p.) (■) immunization with

257 DV6181 (pGV5141) or after i.p. () immunization with the negative control, Salmonella

258 DV6181. (B) Response to F17a-G six weeks after i.p. immunization with S. Typhimurium

259 DV6181 or E. coli MG1655 harboring different plasmids. Pre-immune serum (♦),

260 S. Typhimurium DV6181 negative control (■), DV6181(pGV5141), expressing the F17a-G-

261 AIDA-I fusion (●), DV6181 (pPLHD673), expressing F17a-G in the periplasm (▲), E. coli

262 MG1655 negative control (□), MG1655 (pGV5141), expressing the F17a-G-AIDA-I fusion

263 (○), MG1655 (pPLHD673), expressing F17a-G in the periplasm (Δ) and E. coli 25KHO9st,

264 expressing complete F17a fimbriae (▼). (■), (▲), (□), (Δ) and (▼) all fall into the same OD-

265 range. (C) Response to FimH after i.p. immunization. Pre-immune sera before immunization 266 with the S. TyphimuriumAccepted DV6181 negative controlManuscript (Δ) or with DV6181 (pGV5153), 267 expressing the FimH-AIDA-I fusion (▲), sera four weeks after immunization with DV6181

268 (pGV5153) (♦), six weeks after immunization with DV6181 (○) or DV6181 (pGV5153) (●).

269

12 Page 12 of 17 Figure 1

Accepted Manuscript

Page 13 of 17 Figure 2

Accepted Manuscript

Page 14 of 17 Table 1

Table I. Bacterial strains and plasmids used in this study

Plasmid or strain Description Source or reference

S. Typhimurium Attenuated guaB fliCfljBA derivative of S. Typhimurium (Adriaensen, 2006)

DV6181 1491S96, a pathogenic isolate from a chicken.

E. coli K514 hsdR derivative of E. coli C600 (Colson et al., 1965)

E. coli E2787 Clinical O126:H27 isolate expressing the AIDA-I Dr. H. Imberechts, CODA

autotransporter

E. coli MG1655 Wild type E. coli K12 strain E. coli Genetic Stock

Center

E. coli 25KHO9st Spontaneous non-toxigenic derivative of a bovine ETEC strain (Lintermans et al., 1988)

producing F17a fimbriae

APEC 5 Clinical APEC isolated from a broiler chick (Vandemaele et al., 2003)

APEC 60 Clinical APEC isolated from a laying hen (Vandemaele et al., 2003)

E. coli UTI89 Clinical UPEC isolated from a human cystitis patient (Mulvey et al., 2001)

pGV5159 The araBAD promoter (PBAD) and the araC gene in (Van Gerven et al., 2008)

pDONRP4-P1

pPLHD673 Contains the F17a-G adhesin (amplified with primers F17-30 This study

and F17-32 and cloned in the pBAD/Myc-His A vector

between the filled-in XbaI site and the EcoRI site) expressed in

the periplasm under the control of the PBAD promoter

pDONR221 Donor vector used to clone the RBD of the bacterial adhesins Invitrogen pDONRP2R-P3 AcceptedDonor vector used to clone the AIDA-I Manuscript translocator domain Invitrogen pDESTR4-R3 Destination vector Invitrogen

pGV5125 F17a-G RBD in pDONR221 (Van Gerven et al., 2008)

pGV5292 FimH RBD in pDONR221 (position 305060 to 305608 of This study

accession number AE016771), amplified from E. coli

K514 using primers Fim3 and FimH6

Page 15 of 17 pGV5291 AIDA-I translocator domain in pDONRP2R-P3 (position This study

2827 to 3909 of accession number X65022), amplified

from E. coli E2787, using primers AIDA10 and AIDA5 pGV5141 Contains F17a-G-AIDA-I under the control of PBAD This study pGV5153 Contains FimH -AIDA-I under the control of PBAD This study

Accepted Manuscript

Page 16 of 17 Table 2

1 Table II. Primers used for the construction of the fusion proteins

Primer Sequence (5’ → 3’) (attB sites are underlined, EcoRI site is double underlined)

AIDA10 GGGGACAGCTTTCTTGTACAAAGTGGGCGTTGCCGGAGCTTATGATTACACA

AIDA5 GGGGACAACTTTGTATAATAAAGTTGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC

ATTATCAGAAGCTGTATTTTATCCC

F17-30 GCAGTTTCATTTATTGGCAGT

F17-32 CCCGAATTC AACACTGCCTGCAGATGAACCGTTATA

GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAGAAGGAGATATATCATGAAACGAGTTA FimH3 TTACCCTGTTTGCTGT

GGGGACCACTTTGTACAAGAAAGCTGGGTTGCCGCCCTGAAAATAAAGATTCTCGCCAGT FimH6 AGGCACCACCACATCATTATTG

2

Accepted Manuscript

1 Page 17 of 17