Serodiagnosis and Bacterial Genome of Helicobacter pylori Infection

Abstract

The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori.

Keywords: CagA; VacA; antibody; genome.

Figure 1

ROC curve of five strains. Four antigens from clinically isolated strains (#3, #5, #6 and #8) and kit original antigen (SL-Wako) were reacted with H. pylori negative and positive sera. ROC analysis was performed.

Figure 2

Genome rings. A genome ring was created to visualize the characteristics of each strain. From the outside, gray is the number of all ORFs, red is the forward ORF, blue is the reverse ORF, orange is the number of tRNAs, green is the number of rRNAs, and the innermost is the GC content.

Figure 3

Dot plot analysis. Dot plot analysis was performed for #3 and #5 (A), #3 and #6 (B), and #3 and #8 (C). Dot plot analysis is a graph for comparing the amino acid sequence of a protein and the base sequence of a nucleic acid to clarify the homology. The vertical axis is arranged from bottom to top and the horizontal axis is arranged from left to right and is plotted at the matching points.

Figure 4

Comparison around cagA genes of 4 strains. The site that encodes about 30 genes including cagA is called cag pathogenicity island (CagPAI). No major mutation was found in 4 strains of CagPAI. However, it was found that the position of CagPAI started from about 1 million bp in the #8 strain and from about 600,000 bp in the other three strains.

Figure 5

Comparison of vacA genes of 4 strains. The arrows of vacA and its surrounding genes are shown. The vacA genes of #3 and #5 were located at 1 million positions in the forward direction. In contrast, the vacA genes of #6 and #8 were present at 600,000 positions in reverse directions.

Figure 6

Protein productions of CagA (A) and VacA (B). The protein expression levels of CagA and VacA were examined using Western blotting (mean ± SE). The expression levels of CagA were significantly reduced at #6 and #8 compared to #3 and at #8 compared with #5. Although there was no significant difference in VacA expression level, there was, however, a tendency for the expression level to be low in #6 and #8.

Figure 7

Chromosome structure. From the results of the dot plot analysis and the map around the genes of cagPAI and vacA, we considered the possibility that mutations, such as inversion, occurred among the 4 strains in the genome. Most of the sequences are the same for #3 and #5. It was found that the first half of the sequence of #3 was reversed in the second half of the sequence in #6, and the second half of the sequence including vacA in #6 was reversed in the first half of the sequence in #6. The middle sequence containing cagA should be approximately homologous. There are many mutations in #8 compared to #3, #5 and #6. The positions of cagA and vacA are also completely different in #8.