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. 0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0378-1097(00)00157-9 FEMSLE 9390 27-4-00 252 S. Spreng et al. / FEMS Microbiology Letters 186 (2000) 251^256 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 ac- cording 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. FEMSLE 9390 27-4-00 S. Spreng et al. / FEMS Microbiology Letters 186 (2000) 251^256 253 The fragment size was analyzed on agarose gel and frag- ments 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-hemoly- sin; 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.