Bioinformatics analysis of small RNAs in Helicobacter pylori and the role of NAT‑67 under tinidazole treatment

Abstract

Helicobacter pylori (Hp) infection is a major cause of gastrointestinal disease. However, the pathogenesis of gastric mucosa injury by Hp has remained elusive. Small non‑coding RNA (sRNA) is a type of widespread RNA in prokaryotic organisms and regulates bacterial growth, reproduction and virulence. In the present study, Hp sRNA profiles were generated to reveal the sequences and possible functions of sRNA by bioinformatics analysis. The role of sRNA in tinidazole (TNZ) treatment was also explored. Total sRNAs of HP26695 were sequenced using an Illumina HiSeq2000. Detected Tags were then compared with a known sRNA database to build an sRNA profile. Reverse transcription‑quantitative (RT‑q)PCR products were sequenced directly and agarose gel electrophoresis was used to identify NAT‑67 and 5'ureB‑sRNA in HP. Furthermore, HP was treated with TNZ for 6, 12 and 24 h. The bacterial concentration was measured, the expression of NAT‑67, 5'ureB‑sRNA and ceuE was determined by RT‑qPCR and superoxide dismutase (SOD) activity and reactive oxygen species (ROS) production were detected. A total of 163 sRNA tags were predicted in Hp through bioinformatics analysis. Among them, 35 tags were evolutionarily aconserved in different Hp strains. By target prediction, it was indicated that certain candidate sRNAs were associated with bacterial oxidative stress, virulence and chemotaxis. It was also observed that NAT‑67 and 5'ureB‑sRNA were downregulated in TNZ‑treated HP. TNZ treatment inhibited the growth of Hp, which was accompanied by downregulation of ceuE and SOD activity, as well as upregulation of ROS. RNA sequencing and bioinformatics are valuable in predicting the expression profile and function of sRNA in HP. sRNA‑targeted genes may be associated with virulence, oxidative stress and chemokines. Downregulation of NAT‑67 by TNZ may be involved in Hp oxidative stress regulation, which may comprise one of the mechanisms of the antibacterial effects of TNZ.

Keywords: Helicobacter pylori; small rna; naT-67; tinidazole; oxidative stress.

Figure 1.

(A) Venn diagram presenting the Hp candidate sRNAs length distribution and the annotation of candidate sRNAs. Gray, total candidate sRNAs; blue, sequence conservative sRNAs; green, secondary structure evolutionarily conserved sRNAs; light blue, both conservative. Annotation and secondary structure of Hp candidate sRNAs. Gene locations of representative sRNAs were shown in (B) sRNA008, (C) sRNA014, (F) sRNA099 and (G) sRNA078. Their respective secondary structures are presented below. (D) Secondary structure of sRNA008. (E) Secondary structure of sRNA014. (H) Secondary structure of sRNA099. (I) Secondary structure of sRNA078. Hp, Helicobacter pylori; sRNA, small non-coding RNA.

Figure 2.

Target prediction of candidate sRNAs. Targets associated with (A) virulence factors, (B) chemotaxis and (C) oxidative stress are provided. Blue, type IV secretion system-associated genes; yellow, cag-pathogenicity island protein; red, vacuolating cytotoxin-associated genes; purple, chemotaxis protein-encoding genes; pale yellow, methyl-accepting chemotaxis protein-associated genes; green, ceuE; orange, peroxidase. Hp, Helicobacter pylori; sRNA, small non-coding RNA.

Figure 3.

Verification of five known sRNA of Hp including Nat-39, Nat-67, IG-443, IG-524 and 5′ureB-sRNA. (A) Reverse transcription-quantitative PCR was performed, and the amplified products of five sRNAs were electrophoresed in a 2% agarose gel. 5′ureB-sRNA and Nat-67 can be detected clearly and Nat-39, IG-443 and IG-524 were weakly detected. (B) 5′ureB-sRNA PCR products were sequenced. (C) Nat-67 PCR products were sequenced. sRNA, small non-coding RNA; Hp, Helicobacter pylori.

Figure 4.

Effect of TNZ on the expression of sRNA and oxidative stress levels in HP. (A) Growth curve of Hp SS1. (B) Cell viability in the presence of TNZ was determined with an ultraviolet spectrophotometer. (C and D) The expression of (C) 5′ureB-sRNA and (D) NAT-67 was determined by RT-qPCR. (E) ROS levels. (F) The expression of ceuE was detected by RT-qPCR. (G) SOD activity. Values are expressed as the mean ± standard error of the mean (n=5). *P<0.05, **P<0.01 vs. Control. CFU, colony-forming units; RT-qPCR, reverse transcription-quantitative PCR; ROS, reactive oxygen species; SOD, superoxide dismutase; Hp, Helicobacter pylori; sRNA, small non-coding RNA; TNZ, tinidazole.

Figure 4.

Effect of TNZ on the expression of sRNA and oxidative stress levels in HP. (A) Growth curve of Hp SS1. (B) Cell viability in the presence of TNZ was determined with an ultraviolet spectrophotometer. (C and D) The expression of (C) 5′ureB-sRNA and (D) NAT-67 was determined by RT-qPCR. (E) ROS levels. (F) The expression of ceuE was detected by RT-qPCR. (G) SOD activity. Values are expressed as the mean ± standard error of the mean (n=5). *P<0.05, **P<0.01 vs. Control. CFU, colony-forming units; RT-qPCR, reverse transcription-quantitative PCR; ROS, reactive oxygen species; SOD, superoxide dismutase; Hp, Helicobacter pylori; sRNA, small non-coding RNA; TNZ, tinidazole.