Elsevier

Gene

Volume 246, Issues 1–2, 4 April 2000, Pages 59-68
Gene

Genetic organization and heterogeneity of the iceA locus of Helicobacter pylori

https://doi.org/10.1016/S0378-1119(00)00054-8Get rights and content

Abstract

The genetic organization and sequence heterogeneity of the iceA locus of Helicobacter pylori was studied, and the existence of two distinct gene families, iceA1 and iceA2, at this locus was confirmed. iceA1 has significant sequence homology to nlaIIIR, encoding an endonuclease in Neisseria lactamica, but the similarity at the protein level is limited, due to frameshift mutations of iceA1 in most H. pylori strains. In only five of the 19 iceA1 strains studied, a full-length open reading frame (ORF), capable of encoding a 228 aa protein, with 52% homology to NlaIII was observed. The region upstream of iceA2 is highly variable in length, containing up to 15 copies of 8 bp tandem repeats. iceA2 can encode proteins of 24, 59, 94, or 129 amino acids, consisting of 14 and 10 aa domains, conserved in all iceA2 strains, flanking 0, 1, 2, or 3 copies of a 35 aa cassette. This 35 aa cassette consists of domains of 13, 16 and 6 aa, respectively. The 13 aa and 6 aa domains are highly conserved, but the 16 aa domain exists in two variants. In total, five distinct iceA2 subtypes were defined. Database searches did not reveal any homologous sequences. Recombinant IceA1 and IceA2 proteins were expressed in Escherichia coli, confirming the predicted ORFs. Genotype-specific PCR primers permitted iceA genotyping in 318 (99.1%) of a worldwide collection of 321 H. pylori strains. The conserved sizes of the amplification products confirmed the worldwide distribution of discrete variants of iceA1 and iceA2.

Introduction

Helicobacter pylori persistently colonizes the human gastric mucosa. Carriage of H. pylori increases the risk of developing duodenal and gastric ulcers, and malignancies including adenocarcinoma of the distal stomach and gastric maltomas (Dunn et al., 1997).

The genetic variability of H. pylori is high (Go et al., 1996) and DNA fingerprinting has revealed substantial heterogeneity (Marshall et al., 1996). Several genes have been identified that are markers for enhanced pathogenicity of H. pylori (Atherton, 1997, Blaser, 1997). cagA (cytotoxin associated gene) is a marker for the presence of a genomic pathogenicity (cag) island of about 37 kbp (Censini et al., 1996, Covacci et al., 1993). Therefore, cagA-positive strains are more pathogenic than cagA-negative strains, which lack the pathogenicity island. Another virulence factor is a cytotoxin, that injures epithelial cells by formation of vacuoles (Cover, 1996, Leunk, 1995, Schmitt and Haas, 1994, Telford et al., 1994), and this protein is encoded by vacA. vacA is present in every H. pylori strain, but the level of cytotoxin production in vitro and risk of disease are each related to the strain's particular vacA genotype (Atherton et al., 1995, Atherton et al., 1997, van Doorn et al., 1998).

Recently, a novel gene was discovered by comparing mRNA transcripts from an ulcer-derived and a gastritis-derived strain of H. pylori in organisms that had adhered to human gastric cells versus non-adherent bacteria. The expression of this gene is upregulated upon contact of the ulcer-derived H. pylori strain with epithelial cells, and it was designated iceA (induced by contact with epithelium) (Peek et al., 1998). Sequence analysis revealed the existence of two distinct variants of the gene, designated iceA1 and iceA2, and only iceA1 was induced following contact with gastric epithelium. Carriage of iceA1 strains is associated with the presence of peptic ulcers (Peek et al., 1998, van Doorn et al., 1998), whereas iceA2 strains are more prevalent among patients with non-ulcer dyspepsia. The present study describes the genetic organization and sequence heterogeneity of the iceA locus of H. pylori strains from various geographic origins. Data from this study may aid our understanding of the genetic organization and variability of H. pylori, as well as its associated pathogenicity.

Section snippets

H. pylori isolates

As part of a worldwide study of H. pylori genotypes, DNA was obtained from 735 H. pylori cultures from 24 different countries, as previously described (van Doorn et al., 1999). Briefly, strains were harvested from plates, and treated with proteinase K. These crude lysates were diluted 1/100 and used directly for PCR. To examine the iceA diversity around the world, 321 H. pylori cultures were randomly selected from this collection, representing strains from Australia (AU, n=19), Brazil (BR, n

Amplification and sequencing of the iceA locus

The sequence variability and genetic organization of iceA were investigated in H. pylori isolates from different geographic origins. iceA is flanked upstream by cysE (a serine acetyltransferase homolog) and downstream by hpyIM (a DNA adenine methylase) (Peek et al., 1998, Xu et al., 1997). Different combinations of forward and reverse primers, located in these flanking genes (Table 1), were used, and amplimers of variable length (approx. 800–1000 bp) from 36 isolates from diverse geographic

Discussion

Although carriage of H. pylori is associated with serious diseases, such as peptic ulcers and gastric malignancies, there is a clear discrepancy between the number of asymptomatic carriers and affected patients. Different H. pylori genotypes, such as vacA and cagA (Atherton, 1997) represent markers for differential clinical outcome. Recently, a novel gene, designated iceA, was discovered, which exists in two different variants, iceA1 and iceA2.

Since hpyIM, encoding a DNA methylase (Xu et al.,

Acknowledgements

C. Figueiredo is supported by the PRAXIS XXI program of the Portuguese Foundation for Science and Technology.

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