Influence and Mechanism of Bacteroides fragilis Type VI Secretory System on the Intestinal Barrier

doi:10.13560/j.cnki.biotech.bull.1985.2023-0840

Abstract

Abstract:

【Objective】 To investigate the effect and mechanism of Bacteroides fragilis type VI protein secretion system（T6SS）on the intestinal barrier. 【Method】 Suicide vector was used to construct B. fragilis T6SS mutant strain. The dextran sulfate sodium（DSS）salt-induced colitis mouse model was constructed, and supplemented mice with PBS, B. fragilis WT, and B. fragilis ΔT6SS, respectively. The disease characters and intestinal barrier integrity were compared among the three groups. Quantitative PCR and immunohistochemistry were used to detect the expressions of tight junction proteins. Untargeted metabolomics was used to compare gut differential metabolites in each group of mice.【Result】 The deletion of T6SS did not affect bio-viability of B. fragilis. B. fragilis WT showed the improvements in body weight loss and colon length compared to PBS controls, demonstrating the protection to DSS-induced colitis; while this protection was lost with the absence of T6SS. The fluorescein isothiocyanate dextran concentration in the mouse serum and histological examination in intestinal pathological sections indicated that B. fragilis T6SS improved the integrity of the intestinal barrier in mice. T6SS mutation affected intestinal tight junction protein expression, confirmed by quantitative PCR and immunohistochemistry. Untargeted metabolomics analysis revealed 96 up-regulated differentially expressed metabolites in the B. fragilis WT group, compared to the B. fragilis ΔT6SS group, with multiple metabolites enriched in cholinergic synaptic metabolism and glycerophospholipid metabolism-related pathways. 【Conclusion】 B. fragilis T6SS alters intestinal metabolome and increases the expression of tight junction protein in intestinal cells, thus improving the permeability of intestinal barrier, being involved in the protection of B. fragilis to the intestinal barrier.

Key words: microbiota, T6SS, intestinal barrier, intestinal inflammation, metabolites

LEI Qi-yi, XU Yang, LI Peng-fei. Influence and Mechanism of Bacteroides fragilis Type VI Secretory System on the Intestinal Barrier[J]. Biotechnology Bulletin, 2024, 40(3): 286-295.

Figures/Tables 8

References 33

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引物名称Primer name	引物序列Sequence	引物用途Primer purpose
pLGB-13 vector	F: TCAAGTTGTGCGTGATGTG	pLGB-13载体引物
	R: GGATTCATACAAGCGGTC	pLGB-13 vector primer
T6SS 5' flanking	F: CGCGGATCC CCGCATACGCCGGAAGT	T6SS基因上游同源臂扩增引物
	R: CAGTTTTAATCAGGGAACGAACA	5' flanking amplification of T6SS genes
T6SS 3' flanking	F: cgtgttcgttccctgattaaaactgTA CCTGAGCCGATACGACAAA	T6SS基因下游同源臂扩增引物
	R: tgcctgcagTGCTACGGGCATAAACCCT	3' flanking amplification of T6SS genes
T6SS mutation	F: TCCCAGCAGGGTAGCGTC	T6SS缺陷株筛选引物
	R: AGTGCATTCTGAATGTCCGTATT	Mutant selection
Claudin1	F: GGGGACAACATCGTGACCG	Claudin1荧光定量引物
	R: AGGAGTCGAAGACTTTGCACT	qPCR primer for Claudin1 amplification
Claudin2	F: CAACTGGTGGGCTACATCCTA	Claudin2荧光定量引物
	R: CCCTTGGAAAAGCCAACCG	qPCR primer for Claudin2 amplification
Claudin3	F: ACCAACTGCGTACAAGACGAG	Claudin3荧光定量引物
	R: CAGAGCCGCCAACAGGAAA	qPCR primer for Claudin3 amplification
Claudin4	F: TGGAGGACGAGACCGTCAA	Claudin4荧光定量引物
	R: CACGGGCACCATAATCAGCA	qPCR primer for Claudin4 amplification
Occludin	F: TTGAAAGTCCACCTCCTTACAGA	Occludin荧光定量引物
	R: CCGGATAAAAAGAGTACGCTGG	qPCR primer for Occludin amplification
ZO-1	F: GCTTTAGCGAACAGAAGGAGC	ZO-1荧光定量引物
	R: TTCATTTTTCCGAGACTTCACCA	qPCR primer for ZO-1 amplification
Jam	F: TCACGTTCAGTTCTGTGACCC	Jam荧光定量引物
	R: TGACCTCCCCGTAGTTCTGG	qPCR primer for Jam amplification
Muc1	F: GGCATTCGGGCTCCTTTCTT	Muc1荧光定量引物
	R: TGGAGTGGTAGTCGATGCTAAG	qPCR primer for Muc1 amplification
Gapdh	F: GTCTCCTCTGACTTCAACAGCG	Gapdh荧光定量引物
	R: ACCACCCTGTTGCTGTAGCCAA	qPCR primer for Gapdh amplification

引物名称Primer name	引物序列Sequence	引物用途Primer purpose
pLGB-13 vector	F: TCAAGTTGTGCGTGATGTG	pLGB-13载体引物
	R: GGATTCATACAAGCGGTC	pLGB-13 vector primer
T6SS 5' flanking	F: CGCGGATCC CCGCATACGCCGGAAGT	T6SS基因上游同源臂扩增引物
	R: CAGTTTTAATCAGGGAACGAACA	5' flanking amplification of T6SS genes
T6SS 3' flanking	F: cgtgttcgttccctgattaaaactgTA CCTGAGCCGATACGACAAA	T6SS基因下游同源臂扩增引物
	R: tgcctgcagTGCTACGGGCATAAACCCT	3' flanking amplification of T6SS genes
T6SS mutation	F: TCCCAGCAGGGTAGCGTC	T6SS缺陷株筛选引物
	R: AGTGCATTCTGAATGTCCGTATT	Mutant selection
Claudin1	F: GGGGACAACATCGTGACCG	Claudin1荧光定量引物
	R: AGGAGTCGAAGACTTTGCACT	qPCR primer for Claudin1 amplification
Claudin2	F: CAACTGGTGGGCTACATCCTA	Claudin2荧光定量引物
	R: CCCTTGGAAAAGCCAACCG	qPCR primer for Claudin2 amplification
Claudin3	F: ACCAACTGCGTACAAGACGAG	Claudin3荧光定量引物
	R: CAGAGCCGCCAACAGGAAA	qPCR primer for Claudin3 amplification
Claudin4	F: TGGAGGACGAGACCGTCAA	Claudin4荧光定量引物
	R: CACGGGCACCATAATCAGCA	qPCR primer for Claudin4 amplification
Occludin	F: TTGAAAGTCCACCTCCTTACAGA	Occludin荧光定量引物
	R: CCGGATAAAAAGAGTACGCTGG	qPCR primer for Occludin amplification
ZO-1	F: GCTTTAGCGAACAGAAGGAGC	ZO-1荧光定量引物
	R: TTCATTTTTCCGAGACTTCACCA	qPCR primer for ZO-1 amplification
Jam	F: TCACGTTCAGTTCTGTGACCC	Jam荧光定量引物
	R: TGACCTCCCCGTAGTTCTGG	qPCR primer for Jam amplification
Muc1	F: GGCATTCGGGCTCCTTTCTT	Muc1荧光定量引物
	R: TGGAGTGGTAGTCGATGCTAAG	qPCR primer for Muc1 amplification
Gapdh	F: GTCTCCTCTGACTTCAACAGCG	Gapdh荧光定量引物
	R: ACCACCCTGTTGCTGTAGCCAA	qPCR primer for Gapdh amplification