Figure 1
Construction and characterization of the G27 CagA isogenic strains. (a) H. pylori G27 (CagA EPIYA-ABCC) was used to construct isogenic strains that differ primarily in the form of CagA expressed. The C-terminal EPIYA region of WT G27 was replaced with the counter-selectable kan-sacB cassette (gray box) to create the ΔEPIYA strain. The EPIYA-ABT, -ABTC, -ABTCC, -ABTCCC, -ABTCCCC motifs were PCR amplified from clinical isolate K154, while the EPIYA-ABTD was amplified from the clinical isolate K3. Of note, as the EPIYA-B motif of K154 contains a natural alanine to threonine mutation (denoted as BT), the EPIYA-B motif alanine of EPIYA-ABD from K3 was mutated to EPIYT. The ΔEPIYA strain was naturally transformed with vectors carrying the various EPIYA constructs and the kan-sacB cassette was replaced via double homologous recombination. As controls, the entire cagA coding sequence was replaced with a kan-sacB cassette (ΔcagA), a truncated CagA protein was constructed with the addition of a cat cassette (light blue box; cagA::cat), and as a control for genetic manipulation, the ΔEPIYA strain was restored to its WT genotype (ABCC Restorant). Blue-G27 genomic DNA sequence; Green-G27 cagA sequence; White-7.13 flanking sequence; Orange-EPIYA-A motif; Red-EPIYA B motif; Purple-EPIYA-C motifs; Yellow-EPIYA-D motif; (b). The isogenic strains were analyzed for proper deletion, truncation, or expression of CagA by Western blot. For the image on the right, the Western blot image was cropped to show only the region corresponding to CagA (c). AGS cells were infected with each isogenic strain at an MOI of 100 for 8 hrs and whole cell lysates were analyzed by Western blot using anti-CagA and anti-phosphotyrosine antibodies to detect total (CagA) and phosphorylated CagA (pTyr). The Western blot images were cropped to show only the region corresponding to CagA (top) and phosphorylated CagA (bottom). Together, the panel of isogenic strains was able to secrete CagA that could be translocated and phosphorylated in AGS cells.
