Transcriptional Profiling of Type II Toxin–Antitoxin Genes of Helicobacter pylori under Different Environmental Conditions: Identification of HP0967–HP0968 System

Abstract

Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and is responsible for causing peptic ulcers and gastric carcinoma. The expression of virulence factors allows the persistence of H. pylori in the stomach, which results in a chronic, sometimes uncontrolled inflammatory response. Type II toxin–antitoxin (TA) systems have emerged as important virulence factors in many pathogenic bacteria. Three type II TA systems have previously been identified in the genome of H. pylori 26695: HP0315–HP0316, HP0892–HP0893, and HP0894–HP0895. Here we characterized a heretofore undescribed type II TA system in H. pylori, HP0967–HP0968, which is encoded by the bicistronic operon hp0968–hp0967 and belongs to the Vap family. The predicted HP0967 protein is a toxin with ribonuclease activity whereas HP0968 is an antitoxin that binds to its own regulatory region. We found that all type II TA systems were expressed in H. pylori during early stationary growth phase, and differentially expressed in the presence of urea, nickel, and iron, although, the hp0968–hp0967 pair was the most affected under these environmental conditions. Transcription of hp0968–hp0967 was strongly induced in a mature H. pylori biofilm and when the bacteria interacted with AGS epithelial cells. Kanamycin and chloramphenicol considerably boosted transcription levels of all the four type II TA systems. The hp0968–hp0967 TA system was the most frequent among 317 H. pylori strains isolated from all over the world. This study is the first report on the transcription of type II TA genes in H. pylori under different environmental conditions. Our data show that the HP0967 and HP0968 proteins constitute a bona fide type II TA system in H. pylori, whose expression is regulated by environmental cues, which are relevant in the context of infection of the human gastric mucosa.

Gene Expression Profiling of Transcription Factors of Helicobacter pylori under Different Environmental Conditions

“…Our group recently reported that antibiotics affect the expression of virulence factors in H. pylori (Cardenas-Mondragon et al, 2016). Whereas the environmental conditions tested here mostly up regulated expression of the transcription regulators analyzed, exposure to different antibiotics resulted predominantly in gene repression ( Figures 3, 4 ).…”

“…Recently, we reported the transcriptional profiling of type II toxin-antitoxin genes under different environmental conditions (Cardenas-Mondragon et al, 2016). The type II antitoxins function as transcriptional repressors of their own expressions (Yamaguchi and Inouye, 2011) and also regulate the expression of other genes related with cellular functions such as biofilm formation, persistence, and the general stress response (Wang and Wood, 2011; Hu et al, 2012).…”

“…A bacterial suspension was prepared in Brucella broth (BB), and adjusted to an optical density of 0.1 at 600 nm (2 × 10 6 CFU/ml). H. pylori was then grown at 37°C for 24 h (logarithmic growth phase) or 48 h (stationary growth phase) in BB supplemented with 10% decomplemented fetal bovine serum (BB + FBS) under the following conditions: adjusted to pH 5.5, or containing either urea [5 mM CO(NH 2 ) 2 ], nickel [250 mM NiCl 2 ], or iron [150 mM (NH 4 ) 2 Fe(SO 4 ) 2 ⋅6H 2 O] as previously described (Contreras et al, 2003; Wen et al, 2003; Vannini et al, 2014; Cardenas-Mondragon et al, 2016). Fold-changes in transcription were determined by calculating the relative expression of transcription regulator genes under different environmental conditions as compared to expression in BB + FBS.…”

“…Biofilm formation on abiotic surface (polystyrene) was analyzed using 6-well polystyrene plates, inoculated with 3 ml of a bacterial suspension (in BB + FBS, at a final concentration of OD 600nm = 0.1) in each well. The plates were incubated during 3 days at 37°C under microaerophilic conditions as previously reported (Cardenas-Mondragon et al, 2016). Supernatant (planktonic) and adhered (sessile) bacteria were recovered for RNA extraction.…”

“…The antibiotics kanamycin (Km, 50 μg/mL), chloramphenicol (Cm, 30 μg/mL) or tetracycline (Tc, 10 μg/mL) were added and the cultures were incubated for 1 h as previously described (Christensen-Dalsgaard et al, 2010; Cardenas-Mondragon et al, 2016). Antibiotics were used at the minimal inhibitory concentrations that have been reported for E. coli and S. enterica (Christensen-Dalsgaard et al, 2010; Maisonneuve et al, 2011; Silva-Herzog et al, 2015; Li et al, 2016).…”