Weizmannia coagulans BCF-01: a novel gastrogenic probiotic for Helicobacter pylori infection control

Figures & data

Table 1. Primers of qPCR.

Species	Gene symbol	Forward primer (5’-3’)	Reverse primer (5’-3’)
Bacteria	CagA	GTTGATAACGCTGTCGCTTCA	GGGTTGTATGATATTTTCCATAA
Bacteria	VacA	CACCCACTTGATTATTCACTCTTAAA	CCACCCAATAACACGCAAA
Bacteria	16S v3-v4	CCTACGGGNGGCWGCAG	GACTACHVGGGTATCTAATCC
Mouse	RORγ	GACCCACACCTCACAAATTGA	AGTAGGCCACATTACACTGCT
Mouse	Foxp3	ACCATTGGTTTACTCGCATGT	TCCACTCGCACAAAGCACTT
Mouse	IL-17α	TTTAACTCCCTTGGCGCAAAA	CTTTCCCTCCGCATTGACAC
Mouse	TGF-β	GCCTGAGTGGCTGTCTTTTG	AGTTGGTATCCAGGGCTCTCC
Mouse	IL-10	CTTACTGACTGGCATGAGGATCA	GCAGCTCTAGGAGCATGTGG
Mouse	Arg-1	TGTCCCTAATGACAGCTCCTT	GCATCCACCCAAATGACACAT
Mouse	IL-1β	GAAGTTGACGGACCCCAAAA	CCACAGCCACAATGAGTGATAC
Mouse	IFN-γ	ATGAACGCTACACACTGCATC	CCATCCTTTTGCCAGTTCCTC
Mouse	IL-4	GGTCTCAACCCCCAGCTAGT	GCCGATGATCTCTCTCAAGTGAT
Mouse	TNF-α	CCTGTAGCCCACGTCGTAG	GGGAGTAGACAAGGTACAACCC
Mouse	IL-12	CAATCACGCTACCTCCTCTTTT	CAGCAGTGCAGGAATAATGTTTC
Mouse	IL-6	AGTCCTTCCTACCCCAATTTCC	GTCTTGGTCCTTAGCCACTCC
Mouse	GAPDH	AGCTTGTCATCAACGGGAAG	TTTGATGTTAGTGGGGTCTCG
Human	IL-6	CACTGGTCTTTTGGAGTTTGAG	GGACTTTTGTACTCATCTGCAC
Human	IL-1α	CTTCTGGGAAACTCACGGCA	AGCACACCCAGTAGTCTTGC
Human	IL-1β	TGAGCTCGCCAGTGAAATGA	AGATTCGTAGCTGGATGCCG
Human	TGF-β	GGCCAGATCCTGTCCAAGC	GTGGGTTTCCACCATTAGCAC
Human	ZO-1	CAACATACAGTGACGCTTCACA	CAACATACAGTGACGCTTCACA
Human	GAPDH	GGGAGCCAAAAGGGTCATCA	TGATGGCATGGACTGTGGTC

Figure 1. Characterization and colonization ability of gastrogenic W. coagulans strains.

A. Survival rate of different W. coagulans strains in simulated intestinal fluid and simulated gastric fluid. B. Representative picture of anti bacteria test. C. Diameter of inhibition zones. D. Adhesion rate of different W. coagulans strains adhering to GES-1 cells using the plate coating method. E. Adhesion rate of Hp adhering to GES-1 cells. F. Representative fluorescent picture of Hp and BCF-01 adhering to GES-1 cells. G. Fluorescent intensity pictures of the in-vivo colonization experiment. *p < .05; **p < .01; ***p < .001.

Figure 2. Safety evaluation of gastrogenic W. coagulans BCF-01 in male mice.

A. Body weight change in different groups of mice. B. Concentration of ALT, AST, and UREA in the blood. C. Representative picture of HE staining of vital organs.

Figure 3. W. coagulans BCF-01 inhibits Hp infection in a mouse model.

A. Grouping and flowchart of the mouse experiment. B. Body weight change in different groups of mice. C. Representative picture of HE staining of gastro. D. Urease test of gastro contents and fecal Hp antigen detection of feces. E. Transcript levels of CagA in different groups of mice. F. Transcript levels of VacA in different groups of mice.

Figure 4. 16S rRNA sequencing and bioinformatic analysis of mouse intestinal content.

A. Alpha diversity of the microbiome. B. Beta diversity of the microbiome. C. Histogram of the community barplot analysis. D. LefSe analysis of the microbiome. *p < .05; **p < .01.

Figure 5. W. coagulans BCF-01 regulates mucosal immunity through the TLR4-NFκB-pyroptosis signaling pathway.

A. Transcript levels of RORγ, Foxp3, IL-17α, and TGF-β in gastro contents B. Transcript levels of IL-10 and Arg-1 in gastro contents C. Transcript levels of IL-1β, IFN-γ, and IL-4 in gastro contents. D. Transcript levels of TNF-α, IL-12, and IL-6 in gastro contents. E. Protein levels of RORγ and Foxp3. F. Protein level of key genes in the TLR4-NFκB-pyroptosis signaling pathway. *p < .05; **p < .01; ***p < .001.

Figure 6. W. coagulans BCF-01 downregulates the TLR4-NFκB-pyroptosis signaling pathway in macrophages.

A. Transcript levels of IL-6, IL-1α, and IL-1β in GES-1 cells. B. Transcript levels of IL-1β and TGF-β in RAW264.7 cells. C. Protein levels of key genes in the TLR4-NFκB- pyroptosis signaling pathway in RAW264.7 cells. D. Transcript levels of IL-1β and TGF-β in BMDM cells. *p < .05; **p < .01; ***p < .001.

Figure 7. Schematic diagram summarizing the working model used in this study.

Data availability statement

The data that support the findings of this study have been deposited in the CNGB Sequence Archive (CNSA) of the China National GeneBank DataBase (CNGBdb) under accession number CNP0005043.