Quantitative detection of Helicobacter pylori gene expression in vivo and relationship to gastric pathology.
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.
Peek
R M
RM
Division of Gastroenterology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2279, USA. richard.peek@mcmail.vanderbilt.edu
van Doorn
L J
LJ
Donahue
J P
JP
Tham
K T
KT
Figueiredo
C
C
Blaser
M J
MJ
Miller
G G
GG
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004381:Duodenal Ulcer / Q000382:microbiology / Q000473:pathology; D005753:Gastric Mucosa / Q000382:microbiology / Q000473:pathology*; D015870:Gene Expression; D016481:Helicobacter Infections / Q000382:microbiology* / Q000473:pathology; D016480:Helicobacter pylori / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014774:Virulence
D001426:Bacterial Proteins; C408644:IceA2 protein, Helicobacter pylori; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; C405087:iceA1 protein, Helicobacter pylori