Objective:To investigate potential mechanism of Helicobacter pylori metabolites antagonizing host innate immunity.Methods:RNA sequencing and pathway enrichment analysis were used to analyze only LPS-stimulated gastric mucosal cells GES-1,GES-1 cells co-treated with LPS and Helicobacter pylori culture supernatant,and untreated GES-1 cells.The culture supernatant of He-licobacter pylori was filtered by a 3KD ultrafiltration tube,and the filtered filtrate(metabolite part)and the retained solution(protein part)were treated with LPS-stimulated GES-1 cells to detect activity of NF-κB pathway,phosphorylation level of NF-κB,secretion levels of NF-κB pathway effectors TNF-α,IL-6 and IL-8.Identification of key metabolites by untargeted metabolic mass spectrometry.Results:Compared with GES-1 cells stimulated only by LPS,after co-treated with LPS and Helicobacter pylori culture supernatant,expression levels of various genes were regulated and tended to the level of GES-1 in untreated gastric mucosal cells,mainly in the NF-κB pathway.After co-treatment with LPS and culture supernatant of Helicobacter pylori,activity of NF-κB pathway was inhibited(P<0.05).Helicobacter pylori metabolites could inhibit the activity of NF-κB pathway,inhibit phosphorylation of NF-κB,and inhibit the secretion of NF-κB pathway effectors TNF-α,IL-6 and IL-8(P<0.05).1,5 and 25 μmol/L of Helicobacter pylori metabolite 2-D-Glu-copyranose(2DG)treatment inhibited activity of NF-κB pathway and phosphorylation of NF-κB in GES-1 cells,and secretion of NF-κB pathway effectors TNF-α,IL-6 and IL-8 were inhibited(P<0.05).After 2DG treatment,activity of NF-κB in GES-1 cells with TLR3,TLR4,TLR5,TLR6,TLR7,TLR8,TLR9 and TLR10 knockout were significantly decreased(P<0.05);while there was no significant changes in activity of NF-κB in TLR1 and TLR2 knockout GES-1 cells.Both TLR1 and TLR2 interactions were attenuated in GES-1 cells after 2DG treatment.Molecular docking showed that 2DG could bind to TLR2 amino acid disabled R321,K347 and F349,the binding energy was-12 kcal/mol.TLR2 wild-type and mutant plasmids(R321K,K347R,F349A)were constructed,and TLR2-knockout GES-1 cells were respectively transfected.It was found that 2DG treatment did not reduce NF-κB activity in GES-1 cells transfected with TLR2 mutant.Conclusion:Helicobacter pylori metabolite 2DG can interact with TLR2,reduce the formation of het-erodimers between TLR2 and TLR1,and inhibit the activity of innate immune NF-κB pathway.

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.
Animals ; Base Sequence ; China ; DNA, Ribosomal/chemistry/genetics ; Molecular Sequence Data ; Phylogeny ; RNA, Ribosomal, 28S/genetics ; Ruminants ; Sequence Alignment ; Sequence Analysis, DNA/veterinary ; Theileria/*classification/genetics/isolation & purification ; Theileriasis/*parasitology