S-layer protein 2 of Lactobacillus crispatus 2029, its structural and immunomodulatory characteristics and roles in protective potential of the whole bacteria against foodborne pathogens
S-layer protein 2 of Lactobacillus crispatus 2029, its structural and immunomodulatory characteristics and roles in protective potential of the whole bacteria against foodborne pathogens
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DOI：
10.1016/j.ijbiomac.2020.02.065
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发表时间：
2020-05-01
影响因子：
8.2
通讯作者：
Karlyshev, Andrey V.
中科院分区：
文献类型：
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作者：
Abramov, Vyacheslav M.;Kosarev, Igor V.;Karlyshev, Andrey V.
我们之前已经证明，人阴道卷曲乳杆菌 2029 (LC2029) 菌株与宫颈阴道上皮细胞高度粘附，对泌尿生殖病原体表现出拮抗活性，并表达表面层蛋白 (Slp)。本研究的目的是阐明 Slp 的结构和免疫调节特性及其在整个阴道 LC2029 细菌针对食源性病原体的保护特性中的作用。使用肠Caco-2和结肠HT-29细胞系作为人肠上皮层的体外模型。 LC2029菌株具有两个同源表面层（S层）基因，slpl和slp2。虽然我们没有发现 sfpl 在所用生长条件下表达的证据，但检测到 sip2 基因的表达水平非常高。 Slp2蛋白的C端部分氨基酸序列与猪肠分离的卷曲乳杆菌Zj001的SIpA蛋白和鸡肠分离的CbsA蛋白(AL.crispatus CM5810)的C端保守区域高度相似，并且在N端和中间区域存在显着差异。SIpA和CbsA的氨基酸序列同一性高达84%，而这些蛋白的同一性水平较高。发现不能产生 Slp2 的 LC2029 细胞在模拟胃液和肠液中的存活率分别为 49% 和 50%。不表达 Slp2 的阴道 L. ciispatus 1385 (LC1385) 菌株对胃和肠应激也非常敏感。 LC2029 乳酸杆菌定植 24 小时后，未检测到对 Caco-2 或 HT-29 上皮细胞的毒性或损伤。 Caco-2 和 HT-29 细胞中的 NF-κ B 激活，但不诱导先天免疫介质 11-8,11-10 的表达，TNF-α 和 LC2029 抑制 MALP-2 诱导的 Caco-2 和 HT-29 细胞中 11-8 的产生，并增加抗炎细胞因子 11-6 的产生，从而抑制 CXCL1 和 RANTES 的产生。 Caco-2 细胞在 Slp2 存在的情况下培养 Caco-2 和 HT-29 细胞，在与 Caco-2 和 HT-29 细胞结合后，Slp2 蛋白和 LC2029 乳酸杆菌被 Toll 样受体 (TLR) 2/6 识别。本研究中使用的食源性病原体空肠弯曲杆菌、肠炎沙门氏菌和大肠杆菌 O157:H 的强共聚合因子 Slp2 负责 LC2029 共聚合这些肠道病原体的能力，并抑制食源性病原体诱导的 caspase-9 和 caspase-3 激活。此外，Slp2 和 51p2 阳性 LC2029 菌株降低了测试的病原菌对 Caco-2 和 HT-29 细胞的粘附，51p2 阳性 LC2029 菌株 buL 单独对 fooclbome 病原体具有杀菌作用。这些结果表明了一系列涉及抑制细菌生长、活力和细胞粘附特性的机制。产生 LC2029 的 Slp2 致病性变形菌，可能有助于治疗新生儿坏死性小肠结肠炎 (NEC) 以及儿童和成人的食源性传染病，增加定植抵抗力并维持肠道稳态 (C) 2020 Elsevier B.V. 保留所有权利。
We have previously demonstrated that human vaginal Lactobacillus crispulus 2029 (LC2029) strain is highly adhesive to cervicovaginal epithelial cells, exhibits antagonistic activity against genitourinary pathogens and expresses surface-layer protein (Slp). The aims of the present study were elucidation of Slp structural and immunomodulatory characteristics and its roles in protective properties of the whole vaginal LC2029 bacteria against foodborne pathogens. Enteric Caco-2 and colon HT-29 cell lines were used as the in vitro models of the human intestinal epithelial layer. LC2029 strain has two homologous surface-layer (S-layer) genes, slpl and slp2. Whilst we found no evidence for the expression of sfpl under the growth conditions used, a very high level of expression of the sip2 gene was detected. C-terminal part of the amino sequence of Slp2 protein was found to be highly similar to that of the conserved C-terminal region of SIpA protein of L. crispatus Zj001 isolated from pig intestines and CbsA protein (AL. crispatus CM5810 isolated from chicken intestines, and was substantially variable at the N-terminal and middle regions. The amino acid sequence identity between SIpA and CbsA was as high as 84%, whilst the identity levels of these sequences with that of Slp2 were only 49% and 50% (respectively). LC2029 strain was found to be both acid and bile tolerant. Survival in simulated gastric and intestinal juices of LC2029 cells unable to produce Slp2 was reduced by 2-3 logs. Vaginal L. ciispatus 1385 (LC1385) strain not expressing Slp was also very sensitive to gastric and intestinal stresses. Slp2 was found to be non-covalently bound to the surface of the bacterium, acting as an adhesin and facilitating interaction of LC2029 lactobacilli with the host immature or fully differentiated Caco-2 cells, as well as HT-29 cells. No toxicity to or damage of Caco-2 or HT-29 epithelial cells were detected after 24 h of colonization by LC2029 lactobacilli. Both Slp2 protein and LC2029 cells induced NF-kappa B activation in Caco-2 and HT-29 cells, but did not induce expression of innate immunity mediators 11-8,11-10, and TNF-alpha. Slp2 and LC2029 inhibited 11-8 production in Caco-2 and HT-29 cells induced by MALP-2 and increased production of anti-inflammatory cytokine 11-6. Slp2 inhibited production of CXCL1 and RANTES by Caco-2 cells during differentiation and maturation process within 15 days. Culturing Caco-2 and HT-29 cells in the presence of Slp2 increased adhesion of bifidobacteria BLI-2780 to these enterocytes. Upon binding to Caco-2 and HT-29 cells, Slp2 protein and LC2029 lactobacilli were recognized by toll-like receptors (TLR) 2/6. It was shown that LC2029 strain is a strong co-aggregator of foodbome pathogens Campylobacter jejuni, Salmonella enteritidis and Escherichia colt O157:H used in this study. The Slp2 was responsible for the ability of LC2029 to co-aggregate these enteropathogens. Slp2 and intact LC2029 lactobacilli inhibited foodborne pathogen-induced activation of caspase-9 and caspase-3 as apoptotic biomarkers in Caco-2 and HT-29 cells. In addition, Slp2 and 51p2-positive LC2029 strain reduced adhesion of tested pathogenic bacteria to Caco-2 and HT-29 cells. 51p2-positive LC2029 strain buL not Slp2 alone provided bactericidal effect on fooclbome pathogens.These results suggest a range of mechanisms involved in inhibition of growth, viability, and cell-adhesion properties of pathogenic Proteobacteria by the Slp2 producing LC2029, which may be useful in treatment of necrotizng enterocolitis (NEC) in newborns and foodbome infectious diseases in children and adults, increasing the colonization resistance and maintaining the intestinal homeostasis. (C) 2020 Elsevier B.V. All rights reserved.