Comparison ofHelicobacter pyloriVirulence Gene Expression In Vitro and in the Rhesus Macaque

Abstract

We used a quantitative real-time reverse transcriptase PCR assay to measure the transcript abundance of 46 known and putative Helicobacter pylori virulence genes, including 24 genes on the Cag pathogenicity island. The expression profile of H. pylori cells grown in vitro was also compared to expression in vivo after experimental infection of rhesus macaques. Transcript abundance in vitro (mid-log phase) ranged from about 0.004 (feoB and hpaA) to 20 (ureAB, napA, and cag25) copies/cell. Expression of most genes was repressed during the transition from logarithmic-to stationary-phase growth, but several well-characterized H. pylori virulence genes (katA, napA, vacA, and cagA) were induced. Comparison of results in the rhesus macaque with similar data from humans showed a strong correlation (r ‫؍‬ 0.89). The relative in vivo expression in the rhesus monkey was highly correlated with in vitro expression during mid-log (r ‫؍‬ 0.89)-and stationary (r ‫؍‬ 0.88)-phase growth. Transcript abundance was on average three-to fourfold reduced in vivo compared to in vitro during mid-log phase. However, when compared to stationary phase, increased expression in vivo was observed for 6 of 7 genes on a contiguous portion of the pathogenicity island, several of which are thought to encode the H. pylori type IV structural pilus and its accessory proteins. These results suggest the possibility that some genes encoding the H. pylori type IV structural pilus and accessory proteins may form an operon that is induced during growth in vivo.

A mutation burst during the acute phase of Helicobacter pylori infection in humans and rhesus macaques

“…2). Biopsies were taken under anaesthesia after an overnight fast 1 week and 1, 2 and 6 months postorogastric H. pylori infection 16 . We sequenced the genome of strain J166 (1,650,561 bp) on a Roche 454-Titanium FLX sequencer, and the genomes of 12 J166 macaque output (J166 output ) strains, three individual bacterial strains cultivated from each of the four biopsies on an Illumina MiSeq to an average coverage of  200.…”

“…in this re-infection study and to 7.5 Â 10 8 c.f.u. in the macaque infection 16 , a cag PAI-negative strain and infection of an immunologically naive host 32 .…”

“…In a previous study, a 4-year-old female rhesus macaque that was confirmed to be free of H. pylori and 'Candidatus H. heilmannii' was inoculated orogastrically with 7.5 Â 10 8 c.f.u. of an overnight liquid culture of H. pylori J166 that was resuspended in 1.5 ml fresh brucella broth 16 . The monkey was biopsied at 1 week and 1, 2 and 6 months post inoculation under anaesthesia after an overnight fast.…”

Helicobacter pylori infection and disease: from humans to animal models

“…These data indicated that cagA was expressed at high levels during the entire time course of infection. Interestingly, some cag genes, such as cagY, were more highly expressed at 1 week post-infection compared with later time points, whereas expression of others, such as cagC, increased between 2 and 3 months and then fell by 4-6 months post-challenge (Boonjakuakul et al, 2005). Thus, data obtained from these independent animal model systems indicate an important role for CagA and other products of the cag pathogenicity island in the development of H. pylori-induced disease, particularly gastric cancer.…”

“…One study used H. pylori-infected Rhesus monkeys to examine expression of genes within the cag island by quantitative real-time reverse transcriptase PCR (Boonjakuakul et al, 2005). These data indicated that cagA was expressed at high levels during the entire time course of infection.…”