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首页> 外文期刊>BMC Biotechnology >Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus casei inhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells
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Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus casei inhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells

机译：霍乱毒素B亚基与干酪乳杆菌分泌的YVAD的重组融合蛋白抑制脂多糖诱导的caspase-1活化以及随后在Caco-2细胞中分泌IL-1 beta

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Background Lactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB–YVAD). YVAD is a tetrapeptide (tyrosine–valine–alanine–aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1β, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB–YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs. Results We constructed the rCTB–YVAD secretion vector pSCTB–YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB–YVAD was generated by transformation with pSCTB–YVAD. Both the culture supernatant of pSCTB–YVAD-transformed L. casei and purified rCTB–YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB–YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB–YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1β secretion in Caco-2 cells, without affecting cell viability. Conclusions The rCTB protein fused to a functional peptide secreted by L. casei can bind to GM1 ganglioside, like rCTB, and recombinant YVAD secreted by L. casei may exert anti-inflammatory effects in the intestine. Therefore, rCTB secreted by L. casei has potential utility as a vector for the delivery of YVAD to IECs.
机译：背景乳杆菌属细菌被用作细菌载体以将功能性肽递送至肠，因为它们是活体递送至肠，定居在粘膜表面并继续产生所需的蛋白质。以前，我们产生了重组霍乱毒素B亚单位（CTB）的干酪乳杆菌，该菌可以通过GM1神经节苷脂转移到肠道上皮细胞（IEC）中。由于重组CTB（rCTB）的高细胞渗透能力，CTB与功能性肽的重组融合蛋白已被用作将这些肽传递至IEC的载体。然而，没有关于rCTB与乳杆菌属物种表达或分泌的肽融合的报道。在本研究中，我们构建了分泌带有CTB与YVAD的重组融合蛋白（rCTB–YVAD）的干酪乳杆菌。 YVAD是一种四肽（酪氨酸-缬氨酸-丙氨酸-天冬氨酸），可特异性抑制caspase-1，该酶催化无活性前体产生炎性细胞因子白介素（IL）-1β。在这里，我们检查了干酪乳杆菌分泌的rCTB-YVAD是否与GM1神经节苷脂结合并抑制了用作IEC模型的Caco-2细胞中的caspase-1活化。结果我们通过修饰rCTB分泌载体pSCTB构建了rCTB-YVAD分泌载体pSCTB-YVAD。干酪乳杆菌分泌rCTB-YVAD是通过用pSCTB-YVAD转化产生的。 pSCTB–YVAD转化的干酪乳杆菌的培养上清液和纯化的rCTB–YVAD均与GM1神经节苷脂结合，pSCTB–转化的干酪乳杆菌和纯化的rCTB的培养上清液也是如此。有趣的是，尽管纯化的rCTB-YVAD和rCTB都转移到Caco-2细胞中，无论脂多糖（LPS）如何，但只有纯化的rCTB-YVAD而非rCTB抑制LPS诱导的caspase-1激活以及随后Caco-2中IL-1β的分泌。细胞，而不影响细胞活力。结论与干酪乳杆菌分泌的功能性肽融合的rCTB蛋白可与rCTB一样与GM1神经节苷脂结合，干酪乳杆菌分泌的重组YVAD可在肠道发挥抗炎作用。因此，干酪乳杆菌分泌的rCTB作为将YVAD递送至IEC的载体具有潜在的实用性。

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