Identification of Immunogenic Antigens of Helicobacter Pylori Via The

Home , Escherichia coli, Helicobacter pylori

FEMS Microbiology Letters 186 (2000) 251^256 www.fems-microbiology.org

Identi¢cation of immunogenic antigens of Helicobacter pylori via the Escherichia coli hemolysin secretion system1

Simone Spreng a, Ivaylo Gentschev a;*, Werner Goebel a, Hans-Joachim Mollenkopf a;2,

Mathias Eck b, Hans Konrad Mu«ller-Hermelink b, Bernd Schmausser b;3 Downloaded from https://academic.oup.com/femsle/article/186/2/251/689303 by guest on 27 September 2021

a Theodor-Boveri-Institut, Lehrstuhl fu«r Mikrobiologie, D-97074 Wu«rzburg, Germany b Institut fur Pathologie, Universitat Wu«rzburg, D-97080 Wu«rzburg, Germany

Received 7 March 2000; received in revised form 27 March 2000; accepted 28 March 2000

Abstract

We describe a new procedure allowing the generation and detection of immunogenic antigens from Helicobacter pylori via the hemolysin secretion apparatus of Escherichia coli. The gene (or gene fragment) encoding the H. pylori protein (or protein domain) is inserted in-frame into a residual portion of the hemolysin gene (hlyA), encoding the HlyA secretion signal (HlyAs). These fusion proteins are secreted efficiently by E. coli. This new approach allows the identification of immunodominant antigens by using sera derived from H. pylori-infected patients suffering from different gastroduodenal pathologies. Three immunodominant antigens bearing the ureB (urease B-subunit), flaA

(flagellin A-subunit), and an unknown ORF (HP0888) encoding an E. coli FecE analogous protein fused to hlyAs were identified and characterized. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Escherichia coli hemolysin; Secretion of hlyA fusion protein; Helicobacter pylori antigen

1. Introduction secretion apparatus recognizes a signal sequence of about

50^60 amino acids in length (HlyAs), leading to direct The Escherichia coli K-hemolysin secretion system is the secretion of the entire protein into the extracellular me- prototype of type I secretion systems and consists of two dium without the formation of periplasmic intermediates inner membrane proteins, HlyB and HlyD, and the outer [19]. The HlyAs can also be fused to the C-terminus of membrane protein, TolC [25,26]. HlyB and HlyD are spe- heterologous proteins, leading to the e¤cient secretion of ci¢c determinants of the transport apparatus of the K-he- such proteins by many Gram-negative bacteria, like E. coli, molysin, the third component, TolC, is a multifunctional Salmonella spp., Vibrio cholerae and Yersinia spp. [10,24]. protein in the outer membrane of E. coli and is part of at In this study, we used the hemolysin secretion system to least four di¡erent export systems [26]. The hemolysin create several H. pylori hemolysin fusion proteins and tested their immunogenicity with sera of patients with H. pylori-associated diseases. H. pylori infection, which is becoming a major health problem worldwide, causes chronic gastritis and gastric or duodenal ulcer and is associated with severe diseases, like gastric cancer and MALT-type lymphoma [8,27]. Antibi- otics combined with a proton pump inhibitor are the standard therapy for patients with H. pylori gastritis. * Corresponding author. Tel.: +49 (931) 888-4408; However, in recent times, an increasing number of Fax: +49 (931) 888-4402; E-mail: [email protected] H. pylori strains are described, which are resistant to ther- apeutic antibiotics. The intention of our approach was to 1 The sequence data from Helicobacter pylori were produced by TIGR develop a new method for the easy detection of immuno- and can be obtained from http://www.tigr.org/ genic proteins, which could be used for the development of 2 Present address: Max-Planck Institut fu«r Infektionsbiologie, D-10117 Berlin, Germany. novel diagnostic tools and/or new vaccines against H. py- 3 Also corresponding author. lori.

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2. Materials and methods nodes and when the established histological (cytological appearance, growth pattern, lymphoepithelial lesions) 2.1. Bacterial strains and nucleic acids and immunohistological (CD19, CD20 and CD22 ; CD53, CD103 and CD233) criteria were ful¢lled [12]. All bacterial strains and nucleic acids used in this study Monoclonality of the tumors was shown either by mono- are presented in Table 1. clonal light-chain expression (U or V) or in doubtful cases additionally by polymerase chain reaction (PCR) using 2.2. Isolation and cultivation of H. pylori from patients and consensus primers of the J and V regions for the immu- DNA isolation noglobulin receptor heavy chain rearrangement. Patients with non-H. pylori-associated gastroduodenal Helicobacter pylori strain 018/3 (Institute of Pathology, diseases determined by histopathology were used as con- University of Wu«rzburg, Germany) was freshly isolated trols (n = 3). from antrum-biopsy specimens of a patient with chronic Downloaded from https://academic.oup.com/femsle/article/186/2/251/689303 by guest on 27 September 2021 active gastritis undergoing routine endoscopy. After ho- 2.4. Construction of recombinant plasmid pMOS1 mogenization, the biopsy was plated on selective agar (Biomerieux, Frankfurt, Germany) and H. pylori was A vector system for direct cloning of heterologous genes grown under microaerobic conditions for 96 h. Single col- upstream of the last 183 bp of the E. coli hemolysin gene onies were then multiplied microaerobically on Colombia (hlyAs) was developed. This 3P part of hlyAs encodes the blood agar (Oxoid, Basingstoke, UK) containing 10% in- C-terminal 61-amino acid secretion signal of E. coli hemo- activated de¢brinated horse blood (30 min at 56³C) for lysin (HlyAs) which is recognized by the HlyB/HlyD/TolC 96 h. For DNA isolation, bacteria were harvested from secretion machinery required for HlyA protein export. 10 agar plates and washed in 10 mmol Tris-bu¡ered saline Into the single NsiI site of plasmid pMOhly1 [9], a SrfI (TBS), pH 7.4. linker consisting of the oligonucleotide SrfI(3n+1) Chromosomal H. pylori DNA was isolated with a DNA 5P-tgcccgggcatgc-3P (underlined sequence indicates the isolation kit (Nucleobond AXG 20, Macherey-Nagel, Du«- SrfI recognition site) was inserted. The resulting hlyAs ren, Germany) according to the manufacturer's instruc- cassette of the new plasmid pMOS1 is therefore out-of- tions. frame for the translation of HlyAs, leading to a translation stop of the signal. The insertion of an ORF (gene/gene 2.3. Patients' sera fragment) which can reconstitute the original open reading frame results in hlyAs fusion proteins. This prevents the Sera were obtained from patients with H. pylori-associ- generation of false-positive clones that do not express and ated diseases, such as chronic gastritis (n = 5), gastric car- secrete any fusion protein in the subsequent screening for cinoma (n = 5) and gastric MALT-type lymphoma (n = 3). HlyAs fusion proteins. Diagnosis was performed by histopathology. An eradica- tion therapy was excluded in all patients on the basis of 2.5. Cloning of H. pylori gene fragments and identi¢cation clinical data. Chronic gastritis was classi¢ed based on the of secreted fusion proteins Sydney system [21]. Gastric carcinoma was diagnosed according to the classi¢cation of Lauren [18]. Gastric Random DNA fragments were derived from chromoso- MALT-type lymphoma was diagnosed when the tumor mal H. pylori DNA. The fragments were generated by was restricted to the stomach and its contiguous lymph ultrasonication of high molecular mass genomic DNA.

Table 1 Bacterial strains and nucleic acids Relevant characteristics Source/position/reference Strains E. coli DH10b FP, mcrA v-(mrr-hsdRMS.mcrBC), P80dlacZvM15,v lacX174, recA1, Gibco Para D139v(ara, leu), galU, galK, strR H. pylori 018/3 Institute of Pathology, University Wu«rzburg Plasmids pMOhly1 ApR, hlyC, hlyB, hlyD, hlyAs (encoding the last C-terminal 61 aa of HlyA) [7] pMOS1 derivative of pMOhly1 this work Oligonucleotides SrfI (3n+1) 5P-tgcccgggcatgc-3P this work M1: chly 5P-atcactggcattaccgga-3P 4332^4349a [13] M2:hly 5P-tatgggttatacgccctggagatt-3P 1288^1311a[13] aPosition.

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The fragment size was analyzed on agarose gel and fragments with an average size of 500 bp were used. Since the shearing of chromosomal DNA does not lead exclusively to blunt-ended fragments, the ends were blunt-ended with the Klenow fragment of DNA polymerase I and 5P- phosphorylated by T4 polynucleotide kinase. Ligation into the plasmid pMOS1 was carried out using an alter- native blunt-end ligation protocol as described by Mollen- kopf et al. [22]. The procedure uses linearized vector DNA, T4-DNA ligase, ATP and restriction enzyme in one step. Ligation mixtures were electroporated into E. coli DH10b (Table 1). The transformed cells were

plated on cellulose nitrate overlaying selective media. Col- Downloaded from https://academic.oup.com/femsle/article/186/2/251/689303 by guest on 27 September 2021 ony immunostaining using an anti-serum against the C- terminal secretion signal of HlyA (anti-HlyAs antiserum, Jarchau et al. [16]) was performed as described by Spreng and Gentschev [23].

2.6. Sequencing of DNA Fig. 1. Plasmid map of pMOS1. Phly, promoter of the K-hemolysin de- The H. pylori inserts were sequenced with primers terminant; hlyAs, fragment encoding the C-terminal signal of K-hemolysin; hlyC, hlyB, hlyD, genes of the E. coli hemolysin operon; bla, gene M1 and M2 by use of the Pharmacia T7-sequencing kit encoding L-lactamase. The gene sizes are drawn out of scale. The SrfI (Pharmacia Biotech, Uppsala, Sweden). Sequences of the restriction site is in italics and underlined. H. pylori a¡ected genes were identi¢ed by sequence com- parison using TIGR/H. pylori genome database [1]. pressing a hemolysin fusion protein were tested for their

ability to secrete HlyAs fusion products by isoltion of 3. Results the cultures' supernatant proteins. Immunoblot analysis with polyclonal antibodies directed to the hemolysin secre-

3.1. Construction of pMOS1 tion signal showed that most of the HlyAs fusion proteins were secreted into the supernatant ( s 1 Wgml31). Several reports were published in recent years dealing Their molecular mass varied from 8 to 30 kDa (data not with the expression and secretion of HlyA fusion proteins shown). [3,10]. Most of the described fusion proteins were trans- located by various C-terminal parts of HlyA (the last 50^ 3.3. Detection of immunodominant antigens of H. pylori 60 aa) used as secretion signal [10,14,16]. For the easy detection of immunogenic antigens from H. pylori,we All seropositive clones were characterized by immuno- constructed a new vector, pMOS1 (Fig. 1, see Section 2), blotting with di¡erent sera obtained from patients with which allows generation, expression and secretion of het- H. pylori-associated diseases such as chronic gastritis erologous antigens as HlyAs-fusion proteins with an e¤- (n = 5), gastric carcinoma (n = 5) and gastric MALT-type ciency comparable to the natural substrate HlyA. lymphoma (n = 3), which were diagnosed by histopathology [12]. Patients with non-H. pylori-associated gastroduo- 3.2. Isolation and characterization of antigens from denal diseases determined by histopathology were used as H. pylori secreted as HlyAs fusion proteins controls (n = 3) (see Table 2). Three colonies (Hp 1^3), secreting immunodominant DNA fragments were generated through random shear- proteins showing a positive reaction with more than 50% ing of high molecular mass chromosomal DNA from H. of the tested sera (Fig. 2A) were analyzed by DNA se- pylori by ultrasonication, made blunt and phosphorylated quencing. The inserted H. pylori sequences were ¢nally and were subsequently cloned into the SrfI site of pMOS1. identi¢ed by computer homology search using the The ligation mixture was electroporated into E. coli TIGR/H. pylori genome database (Fig. 2B). DH10b and plated directly on nitrocellulose ¢lters over- Analysis of the colony HP 1 showed that the inserted laying 2UYT agar plates supplemented with 100 Wgml31 fragment encoded a part of the urease B-subunit (HP0072) ampicillin. A total of 100 ¢lters (about 27 000 colonies) from aa 207^342 (Fig. 2B). The urease B-subunit fusion were screened by colony immunoblot analysis [23] and protein was recognized by all sera from patients with about 80 secreting random clones were identi¢ed with H. pylori-associated diseases (¢ve patients with chronic anti-HlyAs antibodies (data not shown). All colonies ex- gastritis, ¢ve patients with gastric carcinoma and three

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in iron transport, for example the FecE protein of E. coli (46% amino acid similarity identi¢ed by the BLAST search program, Internet service). The FecE homologous protein was recognized by 10/13 sera from patients with H. pylori- associated diseases (four patients with chronic gastritis, ¢ve patients with gastric carcinoma and one patient with gastric MALT-type lymphoma) and by one control serum (Table 2). To test a possible cross reaction of the sera with the signal part of hemolysin, we used as negative control supernatants from strain E. coli DH10b/pMOhly1, which secretes only the hemolysin secretion signal. The serum of

only one patient with chronic gastritis reacted with the Downloaded from https://academic.oup.com/femsle/article/186/2/251/689303 by guest on 27 September 2021 hemolysin secretion signal, probably due to the infection of this patient with hemolytic E. coli (Table 2).

4. Discussion

In this study, we present a new method for the easy detection of immunogenic antigens (or protein domains) from H. pylori using the E. coli hemolysin secretion system. The successful cloning of random heterologous chromo- Fig. 2. (A) Western blot analysis of H. pylori fusion proteins prepared somal fragments and the direct generation of hemolysin from 2 ml culture supernatant of the producing strains with anti-HlyAs antiserum diluted 1:1000. (B) HlyAs fusion proteins (Hp 1^3). The posi- fusion proteins was already demonstrated with Salmonella tion of the H. pylori-speci¢c gene fragments is indicated by arrows. aa, typhimurium [22] and Listeria monocytogenes [11]. By using amino acids; N-HlyAP, the ¢rst 34 aa of K-hemolysin, marked by a Salmonella DNA, we found that up to 1.5% of all colonies black box; HlyAs, C-terminal signal, indicated by a hatched box. The showed secretion of HlyAs fusion proteins [22]. In the case proteins sizes are drawn out of scale. of H. pylori, however, only 0.3% of all tested clones were positive for secretion of fusion proteins. Speculatively, this patients with gastric MALT-type lymphoma) and by one di¡erence may be due to the fact that H. pylori utilizes a control serum (Table 2). di¡erent codon usage [1] compared to Salmonella and E. In the case of colony Hp2, we detected a part of the £aA coli and possibly di¡erent transcription/termination con- gene encoding aa 62^209 of the £agellin A subunit (Fig. trol mechanisms, or an appropriate in-frame insertion oc- 2B) (HP0601). The £agellin A-subunit fusion protein was curs allowing for an epitope to be expressed. recognized by 12/13 sera from patients with H. pylori-as- We identi¢ed three immunogenic H. pylori antigen frag- sociated diseases (four patients with chronic gastritis, ¢ve ments, including the urease B-subunit, the £agellin A pro- patients with gastric carcinoma and three patients with tein, the major £agellin, and an unknown protein, which gastric MALT-type lymphoma). Flagellin A was recog- shows a homology to the FecE protein (46% aa similarity) nized by the serum of one control patient (Table 2). of E. coli. The ¢rst two proteins are immunogenic [17] and Colony Hp3 harbors an insertion of an unknown ORF associated with the ability of H. pylori to colonize the (HP0888), aa 154^202 (Fig. 2B). Interestingly, this ORF gastric mucosa [20]. Interestingly, the third immunogenic shows a homology to some ATP-binding proteins involved protein we found (FecE analogous protein) seems to be a

Table 2 Detection of immunogenic proteins from H. pylori with di¡erent sera obtained from patients su¡ering from chronic gastritis (n = 5), gastric carcinoma (n = 5), MALT-lymphoma (n = 3) and from three H. pylori-negative individuals Chronic gastritis Carcinoma Lymphoma Control Patient: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Urease B part + + + + + + + + + + + + + 33+ Flagellin A part 3 +++++++++++++33 FecE part + + 3 +++++++33+ 3 + 3 HlyAs control 333+ 333333333333 For detection of immunoreactivity against various patient sera, 20 Wg supernatant proteins of each colony were used. Part: H. pylori part of the fusion protein. +, positive reaction; 3, negative reaction.

FEMSLE 9390 27-4-00 S. Spreng et al. / FEMS Microbiology Letters 186 (2000) 251^256 255 part of an iron-uptake system (Fec-Exb system) in desministerium fu«r Bildung, Wissenschaft, Forschung und H. pylori allowing the bacteria to overcome iron-limitation Technologie (BMBF 01 KI9210 and 01 KI9608) and the [4,20]. The analogous protein in E. coli is an ATPase, Fond der Chemischen Industrie. which is responsible for the energy supply of this system. Systems for the uptake of iron are important for bacterial survival in the iron-depleted environment, a situation, References which occurs during H. pylori infection in the human stomach. The fact that most of the patients' sera reacted [1] Alm, R.A., Ling, L.S., Moir, D.T., King, B.L., Brown, E.D., Doig, with this protein shows the antigenic nature of this FecE P.C., Smith, D.R., Noonan, B., Guild, B.C., deJonge, B.L., Carmel, analogous protein. G., Tummino, P.J., Caruso, A., Uria-Nickelsen, M., Mills, D.M., However, other important H. pylori proteins, for exam- Ives, C., Gibson, R., Merberg, D., Mills, S.D., Jiang, Q., Taylor, ple proteins of the cag pathogenicity island (PAI) [5] were D.E., Vovis, G.F. and Trust, T.J. (1999) Genomic-sequence compar- ison of two unrelated isolates of the human gastric pathogen Heli- not found to be secreted. Based on the presence of a 40-kb Downloaded from https://academic.oup.com/femsle/article/186/2/251/689303 by guest on 27 September 2021 cobacter pylori. Nature 397, 176^180. PAI, H. pylori strains are subdivided into two types. Type [2] Blaser, M.L., Perez-Perez, G.L., Kleanthous, H., Cover, T.L., Peek, I strains, containing the cag PAI, exhibit increased viru- R.M., Chyou, P.H., Stemmermann, G.N. and Nomura, A. (1995) lence and are associated with severe complications of the Infection with Helicobacter pylori strains possessing CagA is associ- H. pylori infection such as gastric ulcer, carcinoma and ated with an increased risk of developing adenocarcinoma of the MALT-type lymphoma [2,6,7]. Type II strains lacking stomach. Cancer Res. 55, 2111^2115. [3] Blight, M.A. and Holland, J.B. (1994) Heterologous protein secretion the cag PAI are more frequently isolated from asympto- and the versatile Escherichia coli haemolysin translocator. Trends matic carriers. Biotechnol. 12, 450^455. In our study, we used a clinical strain, which was freshly [4] Braun, V. and Killmann, H. (1999) Bacterial solutions to the iron- isolated from biopsy specimens. This strain bears the cag supply problem. Trends Biochem. Sci. 24, 104^109. PAI as determined by PCR analysis for cagA, a genomic [5] Censini, S., Lange, C., Xiang, Z., Crabtree, J.E., Ghiara, P., Boro- dovsky, M., Rappuoli, R. and Covacci, A. 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(1997) MALT-type lymphoma of the stomach is may be that the N-terminal secretion signal of these PAI associated with Helicobacter pylori strains expressing the CagA pro- proteins, which are secreted by type II or type III secretion tein. Gastroenterology 112, 1482^1486. [8] Foreman, D. (1993) Eurogast Study Group. An international associ- systems (for review see 15), interferes in the fusion product ation between Helicobacter pylori infection and gastric cancer. Lancet with the HlyA speci¢c signal during the translocation 341, 359^362. (Gentschev et al., unpublished data). [9] Gentschev, I., Mollenkopf, H.-J., Sokolovic, Z., Ludwig, A., Tengel, On the other side, with the hemolysin secretion system, C., Hess, J., Demuth, A. and Goebel, W. (1994) Synthesis and secre- we were able to secrete immunogenic antigens of H. pylori tion of bacterial antigens by attenuated Salmonella via the Escherichia coli hemolysin secretion system. Behring Inst. Mitt. 95, 57^66. as hemolysin fusion proteins. Furthermore, our secretion [10] Gentschev, I., Mollenkopf, H.-J., Sokolovic, Z., Hess, J., Kaufmann, system can be used as an e¤cient delivery system for se- S.H.E. and Goebel, W. (1996) Development of antigen-delivery sys- creted antigens in di¡erent Gram-negative vaccine carriers, tems, based on the Escherichia coli hemolysin secretion pathway. e.g. Salmonella, Shigella, Vibrio and EHEC [24]. This of- Gene 179, 133^140. fers the opportunity to use these carriers for the presenta- [11] Gentschev, I., Dietrich, G., Mollenkopf, H.-J., Sokolovic, Z., Hess, J., Kaufmann, S.H.E. and Goebel, W. (1997) The Escherichia coli tion of immunogenic H. pylori antigens in order to design hemolysin secretion apparatus ^ a versatile antigen delivery system new subunit vaccines against H. pylori on the basis of in attenuated Salmonella. Behring Inst. Mitt. 98, 103^113. recombinant live bacteria. The therapeutical consequences [12] Greiner, A. and Mu«ller-Hermelink, H.K. (1996) Recent advances in of these studies are currently under investigation. gastric extranodal B-cell lymphoma. Curr. Diagn. Pathol. 3, 91^98. [13] Hess, J., Wels, W., Vogel, M. and Goebel, W. (1986) Nucleotide sequence of a plasmid-encoded hemolysin determinant and its com- parison with a corresponding chromosomal hemolysin sequence. Acknowledgements FEMS Lett. 34, 1^11. [14] Hess, J., Gentschev, I., Goebel, W. and Jarchau, T. (1990) Analysis We would like to thank J. Daniels, D. Beier, G. Dietrich of the haemolysin secretion system by PhoA-HlyA fusion proteins. and M. Dietrich for critically reading the manuscript and Mol. Gen. Genet. 224, 201^208. [15] Hueck, C.J. (1998) Type III protein secretion systems in bacterial C. Hu«ttinger for a lot of excellent technical advice. This pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62, work was supported by the Deutsche Forschungsgemein- 379^433. schaft Grant GRK 73/3-97 to B.S., grants from the Bun- [16] Jarchau, T., Chakraborty, T., Garcia, F. and Goebel, W. (1994)

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