Culture Conditions
Strains were cultured from frozen stocks on blood agar plates with antibiotic supplements as previously described.1 Mutant strains were grown on blood agar plates additionally supplemented with kanamycin (20mg/mL), chloramphenicol (20mg/mL), or both antibiotics as required. Liquid cultures were performed in brain heart infusion (BD Difco, Heidelberg, Germany) with yeast extract (2.5 g/L), 10% heat-inactivated horse serum, and a mix of antibiotics (vancomycin (10 mg/L), polymyxin B (3.2 mg/L), amphoter- icin B (4 mg/L), and trimethoprim (5 mg/L). Cultivation was performed at 175 rpm and 37C in microaerobic atmosphere using air-tight jars (Oxoid, Wesel, Germany) and Anaerocult C gas-generating bags (Merck, Darmstadt, Germany).
Escherichia coli strains MC1061 and ER2683
(Supplementary Table 1) were grown under aerobic con-
ditions at 37C on LB agar plates or in LB broth (Lennox L Broth; Thermo Fisher Scientific, Darmstadt, Germany) supplemented with ampicillin (200 mg/mL), kanamycin (20 mg/mL), and/or chloramphenicol (20 mg/mL), as required.
DNA Preparation
Bacterial chromosomal DNA was extracted with the QIAamp DNA Minikit (QIAGEN, Hilden, Germany). Prepa- ration of plasmid DNA was performed using QIAprep Spin Miniprep Kit (QIAGEN).
Sanger Sequencing
PCRs were performed according to standard protocols. Primers were designed using Primer3.2 Amplification products were purified using the QIAquick PCR purification kit (QIAGEN). PCR amplicons were sequenced bidirection- ally using the BigDye terminator v1.1 cycle sequencing kit and the 3130xl genetic analyzer (Thermo Fisher Scientific). Sequence data were analyzed using BioNumerics v6.01 (Applied Maths, Sint-Martens-Latem, Belgium).
Quantitative PCR
The genomic copy number of cagA and efp was deter- mined by quantitative PCR using the standard curve method. Quantitative PCR was performed in the CFX96 Real-Time PCR Detection System (Bio-Rad, Munich, Ger- many) with the QuantiTect SYBR Green PCR Kit (QIAGEN) according to manufacturer’s information. Primer sequences are shown inSupplementary Table 2.
Neutral Red Uptake Assay
Induction of vacuolization in MKN-28 cells by H pylori was analyzed by neutral red uptake assay.3Briefly, 1 x 104 cells/well were seeded into 96-well plates in RPMI 1640 with 10% fetal calf serum (FCS) (Biochrom, Berlin, Ger- many). After 24 hours, cells were infected with H pylori strains at MOI 50 in RPMI 1640 with 1% FCS and incubated
for 24 hours. After removal of medium, cells were incubated with 100mL/well neutral red medium (40 mg/mL) for at least 2 hours at 37C. After washing, the incorporated dye was solubilized by adding 150mL/well neutral red destain solution (50% ethanol [96%], 49% deionized water, 1% glacial acetic acid). Absorbance at 540/690 nm was measured using a microplate reader.
IL8 Induction
Briefly, 1.5 x 105 AGS cells/well were seeded into 24-well plates in RPMI 1640 with 10% FCS (Biochrom). After 24 hours, cells were infected with H pylori strains at MOI 50 in RPMI 1640 with 10% FCS and incubated for 4 hours. The IL8 concentration in the cell culture superna- tants was determined with the BD OptEIA Human IL8 Enzyme-Linked Immunosorbent Assay Set (BD Bio- sciences, San Jose, CA).
BabA Expression
H pylori was harvested from 24-hour-old blood agar plates in phosphate-buffered saline (PBS) and centrifuged (5000g, 4C, 10 minutes). Pellets were homogenized in Tris buffer pH 7.4 by sonication. Equal amounts of protein were separated by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (10%), and analyzed by Western blotting. BabA antibody was kindly provided by Thomas Borén. Peroxidase-labeled AffiniPure goat anti-rabbit IgG (HþL) (Jackson ImmunoResearch Labora- tories, West Grove, PA) was used as secondary antibody. Western blots were developed with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA) and chemiluminescence was detected using the LAS-3000 imaging system (Fujifilm Life Science, Tokyo, Japan).
Le(b) Binding
Le(b) binding of H pylori was determined by an enzyme- linked immunosorbent assay, as previously described.4 Briefly, H pylori was harvested from 24-hour-old blood agar plates in PBS by centrifugation (2795g, 5 minutes, 4C); 2 x 108bacteria were biotinylated with 125mg NHS- LC-biotin for 1 hour. A 96-well Universal Covalent micro- titer plate (Corning Costar, Corning, NY) was coated with 250 ng bovine serum albumin (BSA), or 250 ng Le(b)-BSA. Afterward, the plate was exposed to UV light for 30 seconds in a Stratalinker (Stratagene, Penzberg, Germany). After blocking of the plate with 5% BSA in PBS, 1 x 107 biotinylated bacteria/well were coincubated for 1 hour. Adherent bacteria were fixed with 100 mL para- formaldehyde (2% in 100 mM potassium phosphate, pH 7). After washing with 0.05% Tween 20 in PBS, blocking with 10% FCS in PBS and repeated washing, the plate was incubated with neutravidin-horseradish peroxidase-conju- gate in PBS plus 10% FCS. Afterfinal washing, the plate was incubated with 100 mL/well 3,3’,5,5’-tetramethylbenzidine (BD Biosciences). The reaction was stopped by addition of 1 M H3PO4(50
mL/well), and the extinction at 450 nm was measured using a microplate reader.
Insertion Mutagenesis in H pylori
Mutants in putative R-M genes were generated by nat- ural transformation-mediated allelic exchange5 of the cor- responding target genes. Oligonucleotides used for mutagenesis are provided inSupplementary Table 2. Mutant alleles were constructed by overlap PCR. Briefly, 2 frag- ments of approximately 500 bp omitting a region within the central part of each of the target genes and the aphA-3 and the CAT cassette were amplified via PCR. The resulting overlap fragments were ligated into the plasmid pUC19 via PstI and BamHI restriction sites and propagated in E coli MC1061. The constructed plasmids (Supplementary
Table 3) were used for natural transformation of H pylori
isolates BCM-300 and HE143/09. PCR amplification of the relevant loci confirmed the successful chromosomal replacement of the target gene with the respective mutant allele.
Overexpression of Hpy300XI in E coli
The Type IIG R-M system Hpy300XI (BCM300_01297) of BCM-300 was amplified via PCR and ligated to pRRS (a pUC19 derivative). Oligonucleotides are listed in
Supplementary Table 2. Frameshift correction and stabili-
zation by site-specific mutagenesis was performed as described previously.6 Briefly, in both allele variants, the first repeat region was modified to a nonrepeat sequence of 11 nucleotides in length (CCT CCA CCG CC) to allow stable expression of Hpy300XI in ON status. Additionally, the second repeat in variant 1 was modified to stably comprise 14 nucleotides (CCG CCA CCT CCA CC), which allowed stable expression of the full-length protein containing the naturally occurring C-terminal additional target recognition domain. In variant 2, the second repeat region was modified to a nonrepeat sequence of 12 nucleotides (CCG CCA CCT CCA), resulting in a premature stop codon to prevent full-length translation. All sequence alterations were designed to pre- serve amino acid sequence. Sequence accuracy was confirmed by Sanger sequencing. The resulting constructs
(Supplementary Table 3) were expressed in E coli ER2683
References
1. Moccia C, Krebes J, Kulick S, et al. The nucleotide excision repair (NER) system of Helicobacter pylori: role in mutation prevention and chromosomal import pat- terns after natural transformation. BMC Microbiol 2012; 12:67.
2. Untergasser A, Cutcutache I, Koressaar T, et al. Primer3—new capabilities and interfaces. Nucleic Acids Res 2012;40:e115.
3. Schneider S, Carra G, Sahin U, et al. Complex cellular responses of Helicobacter pylori-colonized gastric adenocarcinoma cells. Infect Immun 2011;79:2362–2371. 4. Nell S, Kennemann L, Schwarz S, et al. Dynamics of Lewis b binding and sequence variation of the babA adhesin gene during chronic Helicobacter pylori infection in humans. MBio 2014;5:e02281–14.
5. Haas R, Meyer TF, van Putten JP. Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis. Mol Microbiol 1993;8:753–760.
6. Krebes J, Morgan RD, Bunk B, et al. The complex methylome of the human gastric pathogen Helicobacter pylori. Nucleic Acids Res 2014;42:2415–2432.
(damþ dcmþ) under the regulation of the Plac promoter
present on pRRS. Genomic DNA of the E coli host ER2683 expressing the modified Hpy300XI alleles was subjected to SMRT sequencing to analyze the resulting methylation profile.
Restriction Analysis
MTase activity for BCM-300 mutant strains disrupted in BCM300_01060 (Hpy300III, M.EcoRI homolog) and BCM300_01362-01364 (Hpy300VI, BccI homolog) was assayed by incubation of 300 ng genomic DNA with EcoRI and BccI, respectively, in 1x CutSmart Buffer (New England Biolabs, Ipswich, MA) for 1 hour at 37C. H pylori 26695, which is naturally deficient for both MTase activities, and wild-type BCM-300 were included as controls. All DNA samples were also incubated without the addition of the respective REase as a negative control. Reactions were analyzed by agarose gel electrophoresis (1% wt/vol).