Quantitative detection of Helicobacter pylori gene expression in vivo and relationship to gastric pathology

Abstract

The iceA locus of Helicobacter pylori includes one of two mutually exclusive gene families, iceA1 and iceA2. Colonization with iceA1 strains is associated with enhanced acute mucosal inflammation, and adherence to gastric epithelial cells in vitro induces expression of iceA1 but not iceA2 mRNA; however, both transcripts can be detected in vivo. The aim of this study was to determine whether differing levels of iceA transcription in vivo may contribute to disease pathogenesis. RNA from 41 H. pylori-positive gastric biopsy specimens was reverse transcribed to cDNA. Quantitative PCR was performed using biotinylated iceA1, iceA2, and 16S rRNA primers, and binding of biotinylated products to streptavidin-coated plates was detected by hybridization with a fluorescein-labeled probe. iceA genotypes were determined by PCR and sequence analysis. All 41 samples contained detectable H. pylori 16S rRNA, with similar levels in iceA1- (n = 10) and iceA2 (n = 31)-colonized patients (P = 0.34). Biopsy specimens from four (40%) and 19 (61%) persons colonized with iceA1 or iceA2 strains, respectively, had detectable iceA RNA. Acute inflammatory scores were significantly higher in iceA1 RNA-positive patients than in iceA1 RNA-negative, iceA2 RNA-positive, or iceA2 RNA-negative subjects (P </= 0.05 for each). Within the iceA2 RNA-positive group, H. pylori strains with a single 35-amino-acid cassette were associated with significantly higher mucosal iceA2 transcript levels (P = 0.014 versus strains with two cassettes). These results indicate that the levels of transcription of H. pylori iceA1 and iceA2 and of 16S rRNA are independent and that particular iceA2 gene structures are associated with enhanced transcription. The finding that iceA1 transcription levels are significantly associated with the intensity of neutrophilic infiltration suggests that heterogeneity in inflammatory scores among persons colonized with H. pylori iceA1 strains reflects levels of iceA1 transcription in vivo.

FIG. 1

Schematic representation of the genetic organization of iceA1 (A) and iceA2 (B) and the flanking genes, cysE and hpyIM. (A) The hatched regions represent continuous ORFs homologous to nlaIIIR. The top schematic represents cysE, prototype iceA1, and hpyIM from H. pylori strain 60190, and the bottom schematic is a variant from strain CH4 containing an ORF homologous to the complete N. lactamica nlaIIIR ORF. Two potential ATG start sites are shown, as is the major transcriptional start site P₁ (large arrows) recently identified in strain 60190 (13). The presence of minor transcripts originating in the intergenic region between the cysE and iceA1 ORFs is indicated by the small arrows with single asterisks. Read-through transcription from cysE into iceA1 is indicated by the small arrows with two asterisks. The size of the intergenic region between the end of the cysE ORF and the first iceA1 ATG codon is 84 nucleotides for strain 60190 and 25 nucleotides for strain CH4. The positions of PCR primers are indicated by arrows below the 60190 schematic. (B) The top schematic represents cysE, prototype iceA2, and hpyIM from H. pylori strain J178, encoding a protein of 59 aa. The diagrams below represent subsequently identified iceA2 variants (16). Each of the five iceA2 peptide motifs, of 14, 13, 16, 6, and 10 aa, respectively, is represented by a box. The existence of two distinct 16-aa domains is indicated by different patterns. The positions of PCR primers are indicated by arrows. The total number of amino acids in each iceA2 ORF is shown for each variant.

FIG. 2

Sensitivity (A) and specificity (B) of quantitative iceA1 and iceA2 PCR on DNA. (A) Dilutions of plasmid containing either 60190 iceA1 or J178 iceA2 DNA were used as templates for PCR with iceA1- or iceA2-specific oligonucleotide primers (Table 1). The results shown (mean ± SD) are for dilutions corresponding to plasmid DNA from 10⁻¹⁹ to 10⁻²³ mol from a representative standard curve. A line representing mean relative light unit values for negative controls with no template DNA which were run concomitantly is shown for reference. The limit of detection for iceA1 and iceA2 by this assay was 10⁻²² mol of plasmid DNA. (B) Genomic DNA (10 ng) from each of the 41 H. pylori strains was used as a template for quantitative PCR with biotinylated iceA1- and iceA2-specific primers (Table 1). Streptavidin-bound PCR products were hybridized with an allele-specific internal fluorescein-conjugated probe (Table 1), and chemiluminescence was quantitated using a luminometer. Mean relative light units + SD for iceA PCRs are shown for iceA1 and iceA2 strains and for negative controls (solid columns) run concomitantly and consisting of reactions with no template DNA.

FIG. 3

Relationship of H. pylori 16S rRNA levels and detectable expression of iceA1 or iceA2 RNA in gastric tissue from 41 H. pylori⁺ persons. Mean values (⧫) ± SD are shown adjacent to the data points (▴). Among persons carrying iceA2 strains, 16S cDNA levels were significantly higher in iceA2 RNA-positive than in RNA-negative persons (5.4 × 10⁻¹⁹ versus 3.7 × 10⁻¹⁹ mol.; P = 0.008); however, 16S cDNA levels were not significantly different among persons carrying iceA1 strains stratified on the basis of iceA1 expression in vivo (P = 0.1). P values were determined by the Mann-Whitney U test.

FIG. 4

Relationship between acute-inflammatory scores and expression of iceA1 or iceA2 RNA detected in gastric mucosa from 41 H. pylori⁺ persons. Acute inflammation was scored from 0 to 3 by a single pathologist blinded to mucosal levels of iceA RNA. Mean values (⧫) ± SD are shown adjacent to the data points (▴). Each data point reflects averaged scores from two antral biopsy specimens (one data point per patient). Biopsies from four (40%) of 10 iceA1- and 19 (61%) of 31 iceA2-colonized persons had detectable mucosal iceA RNA. Acute inflammatory scores were significantly higher in iceA1 RNA-positive patients compared to all other groups. Statistical significance was determined by the Mann-Whitney U test.

FIG. 5

Relationship of iceA2 gene structure and levels of iceA2 expression in vivo. (A) Among patients harboring iceA2 strains, 19 had detectable levels of iceA2 transcripts. iceA2 from corresponding H. pylori genomic DNA was sequenced using an ALF-Express automated sequencer. Mean values (⧫) ± SD are shown adjacent to the data points (▴). (B) The iceA2/16S rRNA ratio was determined by dividing iceA2 transcript levels by the corresponding 16S rRNA levels. Statistical significance was determined by the Mann-Whitney U test.