Gut Commensal Parabacteroides goldsteinii MTS01 Alters Gut Microbiota Composition and Reduces Cholesterol to Mitigate Helicobacter pylori-Induced Pathogenesis

Abstract

Helicobacter pylori infection is closely associated with various gastrointestinal diseases and poses a serious threat to human health owing to its increasing antimicrobial resistance. H. pylori possesses two major virulence factors, vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), which are involved in its pathogenesis. Probiotics have recently been used to eradicate H. pylori infection and reduce the adverse effects of antibiotic-based therapies. Parabacteroides goldsteinii MTS01 is a novel next-generation probiotic (NGP) with activities that can alleviate specific diseases by altering the gut microbiota. However, the mechanism by which P. goldsteinii MTS01 exerts its probiotic effects against H. pylori infection remains unclear. Our results showed that administration of P. goldsteinii MTS01 to H. pylori-infected model mice altered the composition of the gut microbiota and significantly reduced serum cholesterol levels, which mitigated H. pylori-induced gastric inflammation. In addition, the pathogenic effects of H. pylori VacA and CagA on gastric epithelial cells were markedly abrogated by treatment with P. goldsteinii MTS01. These results indicate that P. goldsteinii MTS01 can modulate gut microbiota composition and has anti-virulence factor functions, and thus could be developed as a novel functional probiotic for reducing H. pylori-induced pathogenesis.

Keywords: Helicobacter pylori; Parabacteroides goldsteinii; cholesterol; gut microbiota; pathogenesis.

Figure 1

Murine model for experimental studies. Mice were divided into four groups for the treatments with vehicle-control (PBS, n = 9), P. goldsteinii MTS01 (PG, n = 10), H. pylori (HP, n = 8), and P. goldsteinii MTS01 + H. pylori (n = 10). Mice were intragastrically administered P. goldsteinii MTS01 (2×10⁸ CFU/100 μl) once daily for a total of nine weeks and continuously inoculated with P. goldsteinii MTS01 and H. pylori (1×10⁹ CFU/100 μl) once daily for additional two weeks. Mice were euthanized and the samples of blood, stomach, and stool were prepared for further analysis.

Figure 2

P. goldsteinii MTS01 and H pylori alter the gut microbiota communities. Diversity index of (A) ACE, (B) Fisher, (C) Shannon, and (D) Simpson were performed to analyze microbiota diversity in murine intestinal stool. *P < 0.05 was assessed using Student’s t-test. (E) Analysis of the gut microbiota diversity. PCoA was conducted to analyze the gut microbiota composition in mice administered with vehicle-control (black), P. goldsteinii MTS01 (blue), H. pylori (red), and P. goldsteinii MTS01 + H. pylori (green).

Figure 3

Relative abundance of the gut microbiota in phyla. The bacteria taxonomic profiles at phylum in the gut microbiota in P. goldsteinii MTS01-treated and H. pylori-infected mice. (A) Top seven most abundant phyla in the four murine groups were analyzed. Relative abundance of (B) Firmicutes, (C) Bacteroidetes, (D) Verrucomicrobia, (E) Actinobacteria, (F) Proteobacteria, (G) Tenericutes, and (H) Patescibacteria in the gut microbiota community were assessed. *P < 0.05.

Figure 4

Relative abundance of the gut microbiota in genera. The bacteria taxonomic profiles at genus in the gut microbiota in mice administrated with P. goldsteinii MTS01 and H. pylori. (A) Top twenty most abundant genera among four treated groups were shown. Relative abundance of (B) Akkermansia, (C) Bifidobacterium, (D) Butyricicoccus, (E) Lachnospiraceae_UCG-004, (F) Candidatus_Saccharimonas, (G) Clostridium_sensu_stricto_1, (H) Lachnospiraceae_UCG-010, (I) Dehalobacterium in the gut microbiota were analyzed. *P < 0.05.

Figure 5

Differences in the gut microbiota composition in P. goldsteinii MTS01-treated and H pylori-infected mice. LEfSe analysis exhibited the abundance bacterial species compared with (A) control and H. pylori; (B) H. pylori and P. goldsteinii MTS01+H. pylori.

Figure 6

Circular taxonomic and phylogenetic tree of the gut microbiota diversity. Compared the effect of P. goldsteinii MTS01 and H. pylori altered microbiota composition in each group. Cladogram showed enriched taxa of gut microbiome in mice treated with (A) control and H. pylori; (B) H. pylori and P. goldsteinii MTS01+H. pylori.

Figure 7

Putative functions of microbiota community. The microbial functionality profiles altered by P. goldsteinii MTS01 and H. pylori were analyzed using PICRUSt2 to generate KEGG pathway. Functional features showed the comparison between (A) control and H. pylori, and (B) H. pylori and P. goldsteinii MTS01+H. pylori.

Figure 8

P. goldsteinii MTS01 lowers serum triglyceride/cholesterol. Sera from mice were collected and the levels of (A) triglyceride, (B) total cholesterol, (C) LDL/VLDL, and (D) HDL were analyzed. *P < 0.05.

Figure 9

P. goldsteinii MTS01 mitigates H. pylori-induced gastric inflammation. Mouse gastric tissues were subjected to IHC staining with specific antibodies against COX-2, IL-1β, and TNF-α (original magnification: 200×). The magnified images are displayed below each cropped area. Scale in each panel, 1000 μm, and in each magnified image, 100 μm. The intensity of COX-2, IL-1β, and TNF-α expression for IHC staining in gastric tissues are shown in the right panels. "*" indicated P < 0.05.

Figure 10

P. goldsteinii MTS01 ameliorates H. pylori-induced pathogenesis. (A) AGS cells were pretreated with P. goldsteinii MTS01 for 30 min and then infected with H. pylori at an MOI of 100 for 6 h. Cell vacuolation and elongation were observed by using a phase-contrast microscope. (B) The proportion of elongated cells were counted. (C) CagA phosphorylation were analyzed by western blot assay. Relative protein expression levels were normalized to β-actin and indicated under each lane. (D) The level of NF-κB activation was assessed using luciferase assay. *P < 0.05.