Additional file 1 of Evolution and function of genetic polymorphisms in the Helicobacter pylori type IV secretion pilus protein CagL in gastric carcinogenesis

Supplementary Material 1: Supplementary Figure S1. Cloning strategy and genetic complementation of H. pylori cagL. (A) CagL and flanking genes cagN and cagI from H. pylori strain 7.13 were amplified by PCR and cloned into the pGEM-Teasy vector (Promega Corporation, Madison, Wisconsin, USA) to generate plasmid pGEM-Teasy-7.13cagNLI. (B) The entire cagL gene from its start codon to the cagI stop codon was excised using the indicated primers and replaced by the kanamycin resistance gene (aphA3) to generate plasmid pGEM-Teasy-7.13 ∆cagL::aphA3 for transformation into 7.13. to create the ∆cagL deletion mutant. (C) Afterwards, we inserted a BglII restriction site in plasmid pGEM-Teasy-7.13cagNLI by the QuikChange site-directed mutagenesis (Agilent Technologies, La Jolla, California, USA) immediately after the cagL stop codon using the indicated primers. This site was used to insert the chloramphenicol resistance cassette (cat) with compatible BamHI sites to generate plasmid pGEM-Teasy-7.13 ∆cagLNK wt::cat. (D) pGEM-Teasy-7.13 ∆cagLNK wt::cat was further modified to introduce N58Y and K59E mutations using the indicated primers and QuikChange kit. The resulting complementation plasmid pGEM-Teasy-7.13 ∆cagLYE::cat as well as pGEM-Teasy-7.13 ∆cagLNK wt::cat were transformed into the 7.13 ∆cagL deletion mutant to restore CagL expression by the NK wild-type (wt) or YE alleles, respectively. Blue arrows indicate the position and names of primers. All primers are listed in Supplementary Table S3. All clones were checked by restriction digests and sequencing. Supplementary Figure S2. Cross-structure statistics. The RMSD matrix indicates the pairwise similarity between any two CagL models generated in this study. The lower the value, the more similar the models are. Structures 1–5 all have RMSD values of < 0.8 Å relative to each other. Model 6 with the YE motif shows RMSD values > 1.1 Å relative to all other models. The largest structural difference (1.44 Å) is present between the models with the NK and the YE motif. Supplementary Figure S3. Western blot analysis of protein expression in isogenic H. pylori cagL mutant strains. All indicated bacteria were grown for two days on GC agar plates and protein lysates were prepared. Four individual clones each of our complemented CagL NK (wild-type, wt) and YE strains were compared to parental 7.13 wt and ∆cagL deletion mutant. No differences were seen among the strains regarding the expression of T4SS proteins CagL, CagA and CagY with urease B (UreB) as loading control, except for the lack of the CagL band in the ∆cagL deletion variant as expected. Supplementary Figure S4. Binding assays confirm similar attachment capabilities of H. pylori strains to AGS cells and E-cadherin-expressing AGS cells. (A) Cultured AGS gastric epithelial cells and (B) AGS cells that were stably transduced with wt human E-cadherin were cultured to about 80% confluency, followed by infection with the indicated H. pylori strains for 6 hours at an MOI of 50. Both cell lines revealed similar H. pylori binding capacities upon infection. About 40 CFU of bound bacteria per cell were detected, demonstrating similar cell binding of H. pylori to both cell types. Each point represents data from an individual experiment. All graphs display mean values ± SD derived from three independent biological replicates. No significant (ns) statistical differences were observed among the strains. Supplementary Figure S5. Representative PFGE gel from the experiments shown in Fig. 8D. Supplementary Figure S6. All uncropped Western blot files of Fig. 4, Fig. 7 and Supplementary Figure S3.