Beta-defensins and analogs in Helicobacter pylori infections: mRNA expression levels, DNA methylation, and antibacterial activity

Abstract

Antimicrobial peptides can protect the gastric mucosa from bacteria, but Helicobacter pylori (H. pylori) can equally colonize the gastric apparatus. To understand beta-defensin function in H. pylori-associated chronic gastritis, we investigated susceptibility, human beta-defensin mRNA expression, and DNA methylation changes to promoters in the gastric mucosa with or without H. pylori infection. We studied the expression of HBD2 (gene name DEFB4A), HBD3 (DEFB103A), and HBD4 (DEFB104) using real-time PCR in 15 control and 10 H. pylori infection patient gastric specimens. This study demonstrates that H. pylori infection is related to gastric enhancement of inducible HBD2, but inducible HBD3 and HBD4 expression levels remained unchanged. HBD2 gene methylation levels were overall higher in H. pylori-negative samples than in H. pylori-positive samples. We also assessed antimicrobial susceptibility using growth on blood agar. The H. pylori strain Tox+ was susceptible to all defensins tested and their analogs (3N, 3NI). These results show that HBD2 is involved in gastritis development driven by H. pylori, which facilitates the creation of an epigenetic field during H. pylori-associated gastric tumorigenesis.

Fig 1. HBD2 gene expression analysis and DNA methylation assay in gastric mucosal tissues.

mRNA and genomic DNA was extracted from 15 H. pylori-negative patients (N) and -positive (G) patients. (A) A qPCR assay measured HBD2 mRNA expression. (B) Schematic representation of HBD2 sequence: TTS (+1), NFKB sites and primers positions used for methylation analysis are indicated. The white lollipop represents CpG dinucleotide positions.

Fig 2. HBD3 gene expression analysis and DNA methylation assay in gastric mucosal tissues.

mRNA and genomic DNA was extracted from 15 H. pylori-negative patients (N) and -positive (G) patients. (A) qPCR measured HBD3 mRNA expression. (B) Schematic representation of HBD3 sequence: TTS (+1), STAT1, NFKB sites, and primer positions used for methylation analysis are indicated. The white lollipop represents CpG dinucleotide positions.

Fig 3. HBD4 gene expression analysis and DNA methylation assay in gastric mucosal tissues.

mRNA and genomic DNA was extracted from 15 H. pylori-negative patients (N) and positive (G) patients. (A) qPCR measured HBD4 mRNA expression. (B) Schematic representation of HBD4 sequence: TTS (+1), AP-2, NFKB sites, and primer positions used for methylation analysis are indicated. The white lollipop represents CpG dinucleotide positions.

Fig 4. The 3NI analog has increased antibacterial activity against H. pylori in contrast to wild-type peptides.

The antimicrobial activities of wild-type HBD2, HBD3, and HBD4 and the analogs 3N and 3NI were evaluated at two concentrations (2.5 and 12.5 μM) against H. pylori with 0, 50, 100, and 200 mM NaCl. Error bars show standard deviations (SDs) from 3 independent experiments. Statistical analysis was performed concerning salt point 0 at both two concentrations.