Structure of cytotoxic associated antigen A protein of Helicobacter pylori from Bali and Lombok isolates of Indonesia

Abstract

Background and aim: Helicobacter pylori is a well-known zoonotic agent with worldwide distribution. In Indonesia, only one report regarding the variation within the cytotoxic associated antigen A (CagA) protein of H. pylori has been described in the literature, which was conducted in Manado, South Sulawesi. There remains no report concerning the structure of this protein, particularly for the Bali and Lombok isolates. The objective of this study was to investigate the diversity of H. pylori CagA amino acid sequences of Bali and Lombok isolates, to predict their molecular structures and conduct toxicity examination of CagA on gastric cells.

Materials and methods: A total of 36 samples were used in equal proportions for each pathologic condition. DNA extraction was performed to subculture H. pylori Bali isolates. The amplification of the CagA 3' variable region was carried out using the primers P1 (5'-GATAACAGGCAAGCTTTTTGAGG-3') and P2 (5'-CTGCAAAAGATTGTTTGGCAG-3'). The W2, W9, and W35 fragments were selected as a representation of H. pylori Bali isolates, which were modeled through the threading modeling approach using I-TASSER.

Results: According to the 12 CagA sequences obtained and phylogenetic analyses, the H. pylori strain originating from Bali can be grouped within the East Asian genotypes and is identical to the Lombok strain. In addition, the Bali isolates are phylogenetically more closely related to Southeast Asian strains, particularly the Filipino strain. The relationship between degree of inflammation induced and CagA-positive infection was not statistically significant.

Conclusion: The structure of the H. pylori Bali isolate is identical to that of Lombok isolate, which belongs to the same group of East Asian genotypes, and bacterial virulence is not related to structure.

Keywords: Bali; Helicobacter pylori; Lombok; cytotoxic associated antigen A; structure; virulence.

Figure-1

Comparison of cytotoxic associated antigen A testing results based on sex; p=0.003, with Fisher’s exact test = 0.003, and an alpha value of 0.05 and probability of 90.0%.

Figure-2

Comparison of cytotoxic associated antigen A testing results based on age group; p=0.136, with Fisher’s exact test = 0.142, and an alpha value of 0.05, and probability of 90.0%.

Figure-3

Comparison of cytotoxic associated antigen A testing results based on demographic background; p=0.937, with fisher exact test = 0.951 and alpha value of 0.05 and probability of 90.0%.

Figure-4

Comparison of cytotoxic associated antigen A testing results based on the results of endoscopic study; p=0.663, with Fisher’s exact test result = 0.672 and alpha value of 0.05 and probability of 90.0%.

Figure-5

Comparison of cytotoxic associated antigen A testing results based on the degree of inflammation; p=0.769, with Fisher’s exact test result of 0.769 and alpha value of 0.05 and probability of 90.0%.

Figure-6

Phylogenetic tree built using maximum likelihood (ML) algorithm from cytotoxic associated antigen A genes of Helicobacter pylori Bali isolates (W1 to W12), H. pylori Lombok isolates (LOM34, LOM35, and LOM36), as well as H. pylori from several Asian countries. The numbers on the branches represent the percentage of bootstrap used.

Figure-7

Prediction of protein structure of the C-terminal cytotoxic associated antigen A fragment of Helicobacter pylori Bali isolates. (a) W2 fragment with the EPIYA-ABD (East Asian) motif in patient with gastric cancer; (b) W35 fragment with EPIYA-ABD (East Asian) motif in gastritis patients; and (c) W9 fragment with EPIYA ABC (Western) motif.

Figure-8

In silico analysis for the interaction between C-terminus of the cytotoxic associated antigen A protein fragment and SHP2. The numbers in the bracket represent free energy binding. The more negative the free energy binding, the stronger the binding between a ligand and its receptor.