Amino acid deletions at positions 893 and 894 of cytotoxin-associated gene A protein affect Helicobacter pylori gastric epithelial cell interactions

Abstract

Background: Helicobacter pylori (H. pylori) persistently colonizes the human gastric mucosa in more than 50% of the global population, leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma. Cytotoxin-associated gene A (CagA) protein, an important oncoprotein, has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus, which play crucial roles in pathogenesis. Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer.

Aim: To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function.

Methods: We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894. The cagA gene was amplified and mutated into cagA-NT and cagA-NE (sequence characteristics of strains from nongastric cancer patients), cloned and inserted into pAdtrack-CMV, and then transfected into AGS cells. The expression of cagA and its mutants was examined using real-time polymerase chain reaction and Western blotting, cell elongation via cell counting, F-actin cytoskeleton visualization using fluorescence staining, and interleukin-8 (IL-8) secretion via enzyme-linked immunosorbent assay.

Results: The results revealed that pAdtrack/cagA induced a more pronounced hummingbird phenotype than pAdtrack/cagA-NT and pAdtrack/cagA-NE (40.88 ± 3.10 vs 32.50 ± 3.17, P < 0.001 and 40.88 ± 3.10 vs 32.17 ± 3.00, P < 0.001) at 12 hours after transfection. At 24 hours, pAdtrack/cagA-NE induced significantly fewer hummingbird phenotypes than pAdtrack/cagA and pAdtrack/cagA-NT (46.02 ± 2.12 vs 53.90 ± 2.10, P < 0.001 and 46.02 ± 2.12 vs 51.15 ± 3.74, P < 0.001). The total amount of F-actin caused by pAdtrack/cagA was significantly lower than that caused by pAdtrack/cagA-NT and pAdtrack/cagA-NE (27.54 ± 17.37 vs 41.51 ± 11.90, P < 0.001 and 27.54 ± 17.37 vs 41.39 ± 14.22, P < 0.001) at 12 hours after transfection. Additionally, pAdtrack/cagA induced higher IL-8 secretion than pAdtrack/cagA-NT and pAdtrack/cagA-NE at different times after transfection.

Conclusion: Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity, which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic H. pylori.

Keywords: Cytotoxin-associated gene A; Glu-Pro-Ile-Tyr-Ala; Helicobacter pylori; Hummingbird phenotype; Interleukin-8.

Figure 1

Identification of cytotoxin-associated gene A and its mutated genes. A: The cytotoxin-associated gene A (cagA) gene was amplified by polymerase chain reaction (PCR) from the HZT strain. Lane M: DNA marker; lane 1 cagA gene; lane 2 Control; B: The mutated gene was amplified by splicing by overlap extension (SOE) PCR. Lane M: DNA marker; lane 1 cagA-NT gene; lane 2 cagA-NE gene; lane 3 Control; C: Sanger sequencing results; D: The target genes were amplified by PCR from eukaryotic expression recombinant plasmids. Lane M: DL8000 DNA marker; lane 1 pAdtrack/cagA; lane 2 pAdtrack/cagA-NT; lane 3 pAdtrack/cagA-NE; E: Double enzyme identification results. Lane M: DL8000 DNA marker; lane 1 pAdtrack/cagA; lane 2 pAdtrack/cagA-NT; lane 3 pAdtrack/cagA-NE. cagA: Cytotoxin-associated gene A.

Figure 2

Detection of each plasmid transfection efficiency and expression of each cytotoxin-associated gene A in AGS cells. A: Images under an ordinary light microscope and fluorescence photos after each cytotoxin-associated gene A (cagA) transfection into AGS (100 ×). Scale bar = 100 μm; B: The mRNA expression levels in AGS cells; C: The protein levels in AGS cells. CagA proteins were quantified using by Western blot analysis.

Figure 3

Observation of cell morphology at different times after transfection (100 ×). Representative micrographs of AGS cells transfected with pAdtrack/cytotoxin-associated gene A were shown. Scale bar = 100 μm. cagA: Cytotoxin-associated gene A.

Figure 4

Induction of AGS cells hummingbird phenotype. A: The percentage of hummingbird cells at 12 hours and 24 hours after transfection into AGS. The number of hummingbird cells was quantitated in triplicate in 5 random fields and the results were mean ± SD of three independent experiments. Errors bars represent standard errors of the means. ^aP < 0.05, ^bP < 0.01; B: Representative micrographs of transfected AGS cells as indicated (100 ×). 1 and 2 represented cell morphology at 12 hours and 24 hours after transfection, respectively. Scale bar = 100 μm.

Figure 5

Quantification of the F-actin cytoskeleton with FITC phalloidin stain. A: The average fluorescence intensity of F-actin at 12 hours and 24 hours after transfection. The data were mean ± SD of three independent experiments. Errors bars represented standard errors of the means. ^aP < 0.05, ^bP < 0.01; B: Representative micrographs of F-actin cytoskeleton as indicated (400 ×). AGS cells were labeled for F-actin using FITC phalloidin and the nucleus was stained with DAPI. F-actin was shown in green, and the nucleus was labeled with blue. 1 and 2 represented the F-actin cytoskeleton at 12 hours and 24 hours after transfection, respectively. Scale bar = 25 μm.

Figure 6

The secretion of interleukin-8 after transfection into AGS cells. The data were mean ± SD of three independent experiments. Errors bars represented standard errors of the means. ^aP < 0.05, ^bP < 0.01. The solid line was the statistical result between the two groups, and the dashed line was the statistical result in multiple groups.