Comparative genomics of two Vietnamese Helicobacter pylori strains, CHC155 from a non-cardia gastric cancer patient and VN1291 from a duodenal ulcer patient

Abstract

Helicobacter pylori is involved in the etiology and severity of several gastroduodenal diseases; however, plasticity of the H. pylori genome makes complete genome assembly difficult. We report here the full genomes of H. pylori strains CHC155 and VN1291 isolated from a non-cardia gastric cancer patient and a duodenal ulcer patient, respectively, and their virulence demonstrated by in vitro infection. Whole-genome sequences were obtained by combining long- and short-reads with a hybrid-assembly approach. Both CHC155 and VN1291 genome possessed four kinds of genomic island: a cag pathogenicity island (cagPAI), two type 4 secretion system islands within an integrative and conjugative element (tfs ICE), and prophage. CHC155 and VN1291 carried East Asian-type cagA and vacA s1m1, and outer membrane protein genes, including two copies of oipA. Corresponded to genetic determinants of antibiotic resistance, chromosomal mutations were identified in CHC155 (rdxA, gyrA, and 23S rRNA) and VN1291 (rdxA, 23S rRNA, and pbp1A). In vitro infection of AGS cells by both strains induced the cell scattering phenotype, tyrosine phosphorylation of CagA, and promoted high levels of IL8 secretion, indicating fully intact phenotypes of the cagPAI. Virulence genes in CHC155 and VN1291 genomes are crucial for H. pylori pathogenesis and are risk factors in the development of gastric cancer and duodenal ulcer. Our in vitro studies indicate that the strains CHC155 and VN1291 carry the pathogenic potential.

Figure 1

Overview of the genomes of H. pylori strain CHC155 (A) and VN1291 (B). The plot displays the genomes of CHC155 and VN1291 as circular chromosomes of 1,696,601 bp and 1,702,481 bp. The black outermost and innermost rings indicate the genes, which were predicted and annotated using PROKKA v.1.14.6 (https://github.com/tseemann/prokka/). GC skew and GC content are indicated. The genome map of strain CHC155 and VN1291 were visualized from Genbank format by Proksee (https://proksee.ca/).

Figure 2

Overview of the genomic islands (GIs) in strain CHC155 and VN1291 genomes. Locations of four genomic islands in both genomes are visualized by color scheme using SnapGene v.6.2.2. The genetic schemes of GIs in two strains (A–H) were plotted by genoplotR v.0.8.11. (A–D) GIs in strain CHC155; (A) cagPAI, (B) KHP30-like prophage, (C) tfs3 ICE_1, and (D) tfs3 ICE_2. (E–H) GIs of strain VN1291; (E) cagPAI, (F) KHP40-like prophage, (G) hybrid tfs3_4 ICE_1, and (H) tfs3 ICE_2. The black color indicates the flanking genes of GIs: sel1 (left) and murI (right) in cagPAI, comGF (left) and gene encodes DNA-binding protein (right) in KHP30-like prophage, gene encodes RloF protein (left) and hsdR (right) in tfs3 ICE_1, hsdM (left) and gene encodes RecJ protein (right) in tfs3 ICE_2. The red arrow indicates cagA, an effector protein of cagPAI, and the yellow arrow indicates ctkA (cell-translocating kinase A), an accessory gene of tfs3 ICE. Some genes of interest in KHP30-like prophage and KHP40-like prophage are marked and colored.

Figure 3

Flow diagram for phylogenomic analysis of H. pylori CHC155 and VN1291. Whole-genome sequences of strains CHC155, VN1291, and 35 reference strains were annotated by Prokka. Subsequently, the genome feature (.gff) pangenome was analyzed by Roary and exported as a core gene alignment. Using this alignment, the phylogenetic analysis was performed and exported as a Newick tree, which was visualized in a radical image. The flow diagram was drown in PowerPoint (Office 365, https://www.microsoft.com/vi-vn/microsoft-365).

Figure 4

Phylogenetic tree constructed from core single nucleotide polymorphisms of H. pylori CHC155, VN1291, and 35 reference strains, which were representative of different geographical populations. The tree map was visualized by iTOL v.6.7.3 (https://itol.embl.de/). The Newick format was generated from IQTREE2, H. pylori population was annotated into different colors.

Figure 5

CagA type and VacA type CHC155 and VN1291 strains. (A) The EPIYA motifs (three black boxes) and neighboring regions in the C-terminal region of the CagA protein from four strains: Shi470, F57, CHC155, and VN1291. CagA of either strain CHC155 or VN1291 is an ABD type. (B) Characterization of VacA in strain CHC155. The black boxes in the figure display the alignment of VacA protein sequences of strain CHC155 and VN1291 compared with the signal region, s1 (ACB41503.1)/s2(BAV60359) (upper box), and intermediate region, m1(BAV60412.1)/m2 (BAB87691.1) (bottom box). The VacA alleles are characterized by deletions in the s- and m-regions corresponding to s1 and m1 types.

Figure 6

H. pylori CHC155 and VN1291 induced the hummingbird phenotype and IL8 secretion in human gastric epithelial AGS cells. AGS cells were incubated with or without H. pylori CHC155, VN1291, or 26695 (multiplicity of infection 100) for 6 and 24 h. Bar: 50 μm. (A) Cells 24 h after infection. Red arrows indicate cells with the hummingbird phenotype. (B) Immunoblot analysis of phosphorylated CagA (pY-CagA), CagA, UreB and β-actin. (C) Induction of IL8 during infection (n = 4).