Deletion of Helicobacter pylori vacuolating cytotoxin gene by introduction of directed mutagenesis

Figure 1 The strategy for disruption of vacA gene by directed mutagenesis. LA and RA were PCR-amplified from H pylori NCTC 11638 genomic DNA, and the kanamycin resistance gene, from pEGFP-N1. PCR products with different restriction sites on both sides were digested with corresponding endonucleases, and then cloned into pBluescript II SK digested with the same enzymes. Because there is an EcoR II site in RA, pLA and pkm^r were firstly joined together, resulting in pLK, which then was joined with pRA, resulting in pLKR. The plasmid pLKR was transformed into H. pyloi NCTC 11638, where the Km^r marked mutation was introduced into the genome by homologous recombination, resulting in the vacA^-Km^r mutant strain.

Figure 2 Nucleotide sequence of cysS gene and the downstream sequence amplified from the vacA^-Km^r mutant H pylori. The 1398 bp cysS ORF and the 795 bp km^r ORF are shown. Primers la1, la2, ra1, ra2, and rs for amplifying LA, RA, and ASm are indicated. -35 signal, -10 signal, and rbs of vacA gene serving km ^r gene in the mutant strain are also shown.

Figure 3 PCR amplification for the determination of homolo-gous recombination in Km^r mutant strain. Genomic DNA of NCTC 11638 wild strain and Km^r mutant strain were respec-tively PCR-amplified for the fragments ASm and ASp using the primers ra1 and rs. A single 3.8 kb product ASm was am-plified from Km^r mutant strain as compared with the 3.0 kb product ASp amplified from the wild strain.

Figure 4 Gastric cancer cells SGC-7901 were co-cultured with the supernatant either from Helicobacter pylori NCTC 11638 or its vacA-knockout mutant strain. 12 h after the incubation, A: cells co-cultured with NCTC 11638 developed vacuoles in the cytosol; B: cells co-cultured with vacA^- mutant strain developed no vacuoles at all.