脆弱拟杆菌六型分泌系统对肠道屏障的影响及机制

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

摘要/Abstract

摘要：

【目的】 探讨脆弱拟杆菌（Bacteroides fragilis）中六型蛋白分泌系统（T6SS）对肠道屏障的影响及作用机制。【方法】 采用自杀载体构建B. fragilis T6SS缺陷株。建立葡聚糖硫酸钠（DSS）诱导的肠炎小鼠模型，并分别补充PBS，B. fragilis WT和B. fragilis ΔT6SS，随后比较三组小鼠疾病特征、肠道屏障完整性的差异。利用荧光定量PCR和免疫组化检测小鼠紧密连接蛋白的表达。采用非靶向代谢组学比较各组小鼠肠道差异代谢物。【结果】 T6SS缺失不影响B. fragilis的生物活性。与PBS对照组相比，B. fragilis WT明显改善小鼠的体重丢失、结肠长度等疾病指标，表现出对DSS诱导的肠炎的保护性，而B. fragilis ΔT6SS随着T6SS的敲除丧失了对DSS诱导的肠炎的保护性。小鼠血清中异硫氰酸荧光素葡聚糖含量和肠道病理切片均表明B. fragilis T6SS能改善小鼠肠道屏障的完整性。荧光定量PCR和免疫组化均表明B. fragilis T6SS影响肠道紧密连接蛋白的表达。非靶向代谢组学分析表明，与B. fragilis ΔT6SS组相比，B. fragilis WT组显著上调96个肠道差异代谢产物，其中多个代谢产物富集到胆碱能突触代谢和甘油磷脂代谢相关通路。【结论】 拟杆菌T6SS改变肠道代谢组并提高肠道细胞紧密连接蛋白的表达，改善肠道屏障的通透性，参与到拟杆菌对肠道屏障的保护性。

关键词: 肠道菌群, 六型分泌系统, 肠道屏障, 肠道炎症, 代谢产物

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

雷棋怡, 徐杨, 李鹏飞. 脆弱拟杆菌六型分泌系统对肠道屏障的影响及机制[J]. 生物技术通报, 2024, 40(3): 286-295.

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.

图/表 8

表1 引物列表

Table 1 Primer sequence

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

图1 基于拟杆菌属细菌tssH基因构建的N-J同源进化树

Fig. 1 Neighbor-Joining homogeneous phylogenetic tree based on tssH gene in Bacteroides genus

图2 B. fragilis T6SS缺陷株的构建 A：全基因组测序证实缺陷株T6SS tssB, tssC, tssH基因序列的缺失。B：B. fragilis野生株和缺陷株的生长曲线。C-D：B. fragilis野生株和缺陷株的细菌转录组和代谢组分析

Fig. 2 Construction of B. fragilis ΔT6SS strain A: Whole genome sequencing showing deletion of tssB, tssC and tssH. B: Growth curve of WT and mutated B. fragilis strains. C-D: Bacterial transcriptomic and metabolism analysis between B. fragilis WT and T6SS mutant

图3 B. fragilis T6SS参与到对DSS诱导肠道炎症的保护性 A：体重下降；B：结肠组织长度代表性图片；C：结肠长度

Fig. 3 B. fragilis T6SS involved in protection on DSS-induced colitis A: Body weight loss; B: representative pictures of colonic tissue length; C: colon length. * P < 0. 05

图4 B. fragilis T6SS有助于DSS诱导肠道炎症中肠道屏障的维持 A：小鼠结肠组织HE染色；B：小鼠血清中荧光素葡聚糖浓度

Fig. 4 B. fragilis T6SS contributes to the maintenance of the intestinal barrier in the DSS-induced colitis A: HE staining of colonic tissues in mice. B: Concentration of fluorescein isothiocyanate-dextran in mouse serum. * P < 0. 05

图5 B. fragilis T6SS有助于上调肠道细胞紧密连接蛋白的表达 A：定量PCR；B：免疫组化

Fig. 5 B. fragilis T6SS upregulates the expressions of intestinal tight junction proteins A: RT-qPCR. B: Immunohistochemistry. * P<0. 05, ** P < 0.01

图6 T6SS对肠道代谢组学的影响 A：PCA图；B：差异代谢物火山图；C：差异代谢物热图

Fig. 6 Effect of T6SS on intestinal metabolome A: PCA plot. B: Volcano blot. C: Heatmap of differentially expressed metabolites

图7 差异代谢物的KEGG富集结果

Fig. 7 KEGG enrichment of differentially expressed metabolites

参考文献 33

[1]	Lee SH. Intestinal permeability regulation by tight junction: implication on inflammatory bowel diseases[J]. Intest Res, 2015, 13(1): 11-18. doi: 10.5217/ir.2015.13.1.11 pmid: 25691839
[2]	Haussner F, Chakraborty S, Halbgebauer R, et al. Challenge to the intestinal mucosa during sepsis[J]. Front Immunol, 2019, 10: 891. doi: 10.3389/fimmu.2019.00891 pmid: 31114571
[3]	Wang YF, Li JW, Wang DP, et al. Anti-hyperglycemic agents in the adjuvant treatment of sepsis: improving intestinal barrier function[J]. Drug Des Devel Ther, 2022, 16: 1697-1711. doi: 10.2147/DDDT.S360348 URL
[4]	Naymagon S, Naymagon L, Wong SY, et al. Acute graft-versus-host disease of the gut: considerations for the gastroenterologist[J]. Nat Rev Gastroenterol Hepatol, 2017, 14(12): 711-726. doi: 10.1038/nrgastro.2017.126 pmid: 28951581
[5]	Mathewson ND, Jenq R, Mathew AV, et al. Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease[J]. Nat Immunol, 2016, 17(5): 505-513. doi: 10.1038/ni.3400 pmid: 26998764
[6]	Rajilić-Stojanović M, de Vos WM. The first 1000 cultured species of the human gastrointestinal microbiota[J]. FEMS Microbiol Rev, 2014, 38(5): 996-1047. doi: 10.1111/1574-6976.12075 pmid: 24861948
[7]	Kim S, Covington A, Pamer EG. The intestinal microbiota: antibiotics, colonization resistance, and enteric pathogens[J]. Immunol Rev, 2017, 279(1): 90-105. doi: 10.1111/imr.12563 pmid: 28856737
[8]	Zafar H, Jr Saier MH. Gut Bacteroides species in health and disease[J]. Gut Microbes, 2021, 13(1): 1-20.
[9]	Coyne MJ, Roelofs KG, Comstock LE. Type VI secretion systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements[J]. BMC Genomics, 2016, 17: 58. doi: 10.1186/s12864-016-2377-z pmid: 26768901
[10]	Wang J, Brodmann M, Basler M. Assembly and subcellular localization of bacterial type VI secretion systems[J]. Annu Rev Microbiol, 2019, 73: 621-638. doi: 10.1146/annurev-micro-020518-115420 pmid: 31226022
[11]	García-Bayona L, Comstock LE. Bacterial antagonism in host-associated microbial communities[J]. Science, 2018, 361(6408): eaat2456. doi: 10.1126/science.aat2456 URL
[12]	Kostow N, Welch MD. Plasma membrane protrusions mediate host cell-cell fusion induced by Burkholderia thailandensis[J]. Mol Biol Cell, 2022, 33(8): ar70. doi: 10.1091/mbc.E22-02-0056 URL
[13]	Qin L, Wang XQ, Gao YL, et al. Roles of EvpP in Edwardsiella piscicida- macrophage interactions[J]. Front Cell Infect Microbiol, 2020, 10: 53. doi: 10.3389/fcimb.2020.00053 URL
[14]	Logan SL, Thomas J, Yan JY, et al. The Vibrio cholerae type VI secretion system can modulate host intestinal mechanics to displace gut bacterial symbionts[J]. Proc Natl Acad Sci USA, 2018, 115(16): E3779-E3787.
[15]	Coyne MJ, Zitomersky NL, McGuire AM, et al. Evidence of extensive DNA transfer between bacteroidales species within the human gut[J]. mBio, 2014, 5(3): e01305-e01314.
[16]	García-Bayona L, Comstock LE. Streamlined genetic manipulation of diverse Bacteroides and Parabacteroides isolates from the human gut microbiota[J]. mBio, 2019, 10(4): e01762-19.
[17]	Ito T, Gallegos R, Matano LM, et al. Genetic and biochemical analysis of anaerobic respiration in Bacteroides fragilis and its importance in vivo[J]. mBio, 2020, 11(1): e03238-19.
[18]	Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11[J]. Mol Biol Evol, 2021, 38(7): 3022-3027. doi: 10.1093/molbev/msab120 pmid: 33892491
[19]	Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees[J]. Mol Biol Evol, 1987, 4(4): 406-425. doi: 10.1093/oxfordjournals.molbev.a040454 pmid: 3447015
[20]	Want EJ, Wilson ID, Gika H, et al. Global metabolic profiling procedures for urine using UPLC-MS[J]. Nat Protoc, 2010, 5(6): 1005-1018. doi: 10.1038/nprot.2010.50 pmid: 20448546
[21]	Dunn WB, Broadhurst D, Begley P, et al. Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry[J]. Nat Protoc, 2011, 6(7): 1060-1083. doi: 10.1038/nprot.2011.335 pmid: 21720319
[22]	Rosenfeldt V, Benfeldt E, Valerius NH, et al. Effect of probiotics on gastrointestinal symptoms and small intestinal permeability in children with atopic dermatitis[J]. J Pediatr, 2004, 145(5): 612-616. doi: 10.1016/j.jpeds.2004.06.068 URL
[23]	Haroun E, Kumar PA, Saba L, et al. Intestinal barrier functions in hematologic and oncologic diseases[J]. J Transl Med, 2023, 21(1): 233. doi: 10.1186/s12967-023-04091-w pmid: 37004099
[24]	Chen Y, Yang B, Ross RP, et al. Orally administered CLA ameliorates DSS-induced colitis in mice via intestinal barrier improvement, oxidative stress reduction, and inflammatory cytokine and gut microbiota modulation[J]. J Agric Food Chem, 2019, 67(48): 13282-13298. doi: 10.1021/acs.jafc.9b05744 URL
[25]	Martín R, Chamignon C, Mhedbi-Hajri N, et al. The potential probiotic Lactobacillus rhamnosus CNCM I-3690 strain protects the intestinal barrier by stimulating both mucus production and cytoprotective response[J]. Sci Rep, 2019, 9(1): 5398. doi: 10.1038/s41598-019-41738-5
[26]	Sofi MH, Wu YX, Ticer T, et al. A single strain of Bacteroides fragilis protects gut integrity and reduces GVHD[J]. JCI Insight, 2021, 6(3): e136841. doi: 10.1172/jci.insight.136841 URL
[27]	Jenke A, Ruf EM, Hoppe T, et al. Bifidobacterium septicaemia in an extremely low-birthweight infant under probiotic therapy[J]. Arch Dis Child Fetal Neonatal Ed, 2012, 97(3): F217-F218. doi: 10.1136/archdischild-2011-300838 URL
[28]	Mater DDG, Langella P, Corthier G, et al. A probiotic Lactobacillus strain can acquire vancomycin resistance during digestive transit in mice[J]. J Mol Microbiol Biotechnol, 2008, 14(1-3): 123-127.
[29]	Wegh CAM, Geerlings SY, Knol J, et al. Postbiotics and their potential applications in early life nutrition and beyond[J]. Int J Mol Sci, 2019, 20(19): 4673. doi: 10.3390/ijms20194673 URL
[30]	Russell AB, Wexler AG, Harding BN, et al. A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism[J]. Cell Host Microbe, 2014, 16(2): 227-236. doi: S1931-3128(14)00261-3 pmid: 25070807
[31]	Le NH, Pinedo V, Lopez J, et al. Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector[J]. Proc Natl Acad Sci USA, 2021, 118(40): e2106555118. doi: 10.1073/pnas.2106555118 URL
[32]	Wexler AG, Bao YQ, Whitney JC, et al. Human symbionts inject and neutralize antibacterial toxins to persist in the gut[J]. Proc Natl Acad Sci USA, 2016, 113(13): 3639-3644. doi: 10.1073/pnas.1525637113 pmid: 26957597
[33]	Ting SY, Martínez-García E, Huang S, et al. Targeted depletion of bacteria from mixed populations by programmable adhesion with antagonistic competitor cells[J]. Cell Host Microbe, 2020, 28(2): 313-321.e6. doi: 10.1016/j.chom.2020.05.006 URL