Assessment of Helicobacter pylori gene expression within mouse and human gastric mucosae by real-time reverse transcriptase PCR

Abstract

Despite increasing knowledge on the biology of Helicobacter pylori, little is known about the expression pattern of its genome during infection. While mouse models of infection have been widely used for the screening of protective antigens, the reliability of the mouse model for gene expression analysis has not been assessed. In an attempt to address this question, we have developed a quantitative reverse transcriptase PCR (RT-PCR) that allowed the detection of minute amounts of mRNA within the gastric mucosa. The expression of four genes, 16S rRNA, ureA (encoding urease A subunit), katA (catalase), and alpA (an adhesin), was monitored during the course of a 6-month infection of mice and in biopsy samples from of 15 infected humans. We found that the selected genes were all expressed within both mouse and human infected mucosae. Moreover, the relative abundance of transcripts was the same (16S rRNA > ureA > katA > alpA), in the two models. Finally, results obtained with the mouse model suggest a negative effect of bacterial burden on the number of transcripts of each gene expressed per CFU (P < 0.05 for 16S rRNA, alpA, and katA). Overall, this study demonstrates that real-time RT-PCR is a powerful tool for the detection and quantification of H. pylori gene expression within the gastric mucosa and strongly indicates that mice experimentally infected with H. pylori provide a valuable model for the analysis of bacterial gene expression during infection.

FIG. 1

Quantitative real-time PCR of alpA expression. Six reactions, each initiated with a different number of template molecules (3 × 10⁶ to 30 copies/sample), were incubated with primers specific for alpA in the amplifications reactions. Standard 10-fold dilution series are indicated (curves numbered from 1 to 6). One infected mouse stomach sample with an unknown amount of alpA transcripts is shown (curve I). Two negative controls obtained by introducing water instead of DNA (curve II) or by omitting the reverse transcription step on DNaseI-treated total RNA corresponding to sample I (curve III) are also indicated (A). The standard curve is the plot of the crossing point, intersection of the best-fit line through the log-linear region and the noise band, versus the log copy numbers. The parameters of the linear regression (error and regression coefficient r) are indicated (B).

FIG. 2

Levels of H. pylori gene mRNA during a 6-month course of infection in mice. The numbers of cDNA copies corresponding to 16S rRNA (A), ureA (B), katA (C), and alpA (D) genes were determined in stomachs from Swiss mice over time. Fifty mice that had been inoculated with a suspension of H. pylori X47-2AN were divided in 10 groups (each containing five mice) that were sacrificed at different times (H, hour; W, week[s]; M, months). The numbers of cDNA copies per half stomach normalized to the expression of mouse G3PDH were determined. Horizontal bars represent the geometric means for mice (n = 5) sacrificed at each time point.

FIG. 3

Levels of H. pylori gene expression in biopsy samples from asymptomatic patients and in mice stomachs. The numbers of cDNA copies corresponding to 16S rRNA, ureA, katA, and alpA genes were determined in two adjacent biopsy samples from the stomach of each of the 15 infected patients and 5 noninfected controls (A). To allow a comparison, the results from Fig. 2 corresponding to 15 mice (data for three groups) [3 weeks, 1 month, and 2 months postinfection]) were pooled (B). The numbers of cDNA copies per two biopsy samples normalized to the expression of human or mouse G3PDH were determined as described in the text. Geometric means and standard deviations are indicated. After DNase I treatment of each RNA preparation, no target gene expression was detected on the cDNA corresponding to the five negative human and five mouse controls (<10 copies/70 ng of total RNA).

FIG. 4

Influence of bacterial burden on expression levels of H. pylori genes during infection in mice. (A) Quantitative H. pylori culture of gastric tissue on half of the antrum of each mouse was performed on five mouse stomachs at 1 h and 1 week postinfection. (B) The amounts of 16sRNA, ureA, alpA, and katA transcripts were determined for the corresponding mouse stomachs at 1 h and 1 week postinfection. The results were normalized by the number of CFU contained in each preparation used for RT-PCR (cDNA copies/CFU). Horizontal bars represent the geometric means for mice (n = 5) sacrificed at each point.