Control of gene expression in Helicobacter pylori using the Tet repressor

Abstract

The lack of a versatile system to control gene expression in Helicobacter pylori has hampered efforts to study H. pylori physiology and pathogenesis. To overcome these limitations, we evaluated the utility of an inducible system based on the well-characterized Tet repressor (TetR) and Tet operator (tetO). As validation of this system, we introduced three copies of tetO into the promoter region upstream of the cagUT operon (encoding two virulence factors required for function of the H. pylori Cag type IV secretion system) and expressed tetR by introducing a codon-optimized gene into the chromosomal ureA locus. Introduction of the tetO copies upstream of cagUT did not disrupt promoter activity, as determined by immunoblotting for CagT. The subsequent introduction of tetR, however, did repress CagT synthesis. Production of CagT was restored when strains were cultured in the presence of the inducer, anhydrotetracycline. To demonstrate one potential application of this new tool, we analyzed the function of the Cag type IV secretion system. When the modified H. pylori strains were co-cultured with AGS cells, activity of the Cag type IV secretion system was dependent on the presence of anhydrotetracycline as evidenced by inducer-dependent induction of IL-8 secretion, CagA translocation, and appearance of type IV secretion system pili at the bacteria-host interface. These studies demonstrate the effectiveness of the tetR-tetO system to control gene expression in H. pylori and provide an improved system for studying H. pylori physiology and pathogenesis.

Genes Required for Assembly of Pili Associated with the Helicobacter pylori cag Type IV Secretion System

“…When cocultured in vitro with gastric epithelial cells, H. pylori forms structures termed "pili" at the bacterium-host cell interface (29)(30)(31)(32)(33)(34)(35)(36)(37). Previous studies reported that a cag PAI mutant strain does not produce pili (29,(31)(32)(33)(34)(35)(36), but thus far, there has been very little effort to identify specific genes that are required for pilus production.…”

“…Rabbit anti-CagT antiserum has been described previously (32). To generate CagC antiserum, three peptides representing residues 31 to 46, 60 to 80, and 106 to 115 of CagC were synthesized (39, 40) (Covance, Princeton, NJ) (Fig.…”

“…When H. pylori is cocultured with gastric epithelial cells, filamentous structures can be detected at the bacterium-host cell interface (29)(30)(31)(32)(33)(34)(35)(36)(37). The assembly of these structures is dependent on the presence of the cag PAI (31,32,(34)(35)(36), and CagA has been visualized at the tips of the structures (29,31,37).…”

“…The assembly of these structures is dependent on the presence of the cag PAI (31,32,(34)(35)(36), and CagA has been visualized at the tips of the structures (29,31,37). Based on these observations, it has been suggested that the structures are components of the cag T4SS utilized for translocation of CagA into host cells, and the structures have been termed "pili," analogous to pilus-like structures in the A. tumefaciens T4SS (38).…”

“…More recently, one study reported that cagI and cagL are required for pilus production (35). Another recent study used a tetR-tetO system to regulate H. pylori gene expression and reported that the cagU-cagT operon is required for pilus production, but experiments were not done to determine which of these two genes is essential (32).…”