ABSTRACT

The probiotic bacterial strain Enterococcus faecium SF68 has been shown to alleviate symptoms of intestinal inflammation in human clinical trials and animal feed supplementation studies. To identify factors involved in immunomodulatory effects on host cells, E. faecium SF68 and other commensal and clinical Enterococcus isolates were screened using intestinal epithelial cell lines harboring reporter fusions for NF-κB and JNK(AP-1) activation to determine the responses of host cell innate immune signaling pathways when challenged with bacterial protein and cell components. Cell-free, whole-cell lysates of E. faecium SF68 showed a reversible, inhibitory effect on both NF-κB and JNK(AP-1) signaling pathway activation in intestinal epithelial cells and abrogated the response to bacterial and other Toll-like receptor (TLR) ligands. The inhibitory effect was species-specific, and was not observed for E. avium, E. gallinarum, or E. casseliflavus. Screening of protein fractions of E. faecium SF68 lysates yielded an active fraction containing a prominent protein identified as arginine deiminase (ADI). The E. faecium SF68 arcA gene encoding arginine deiminase was cloned and introduced into E. avium where it conferred the same NF-_κB inhibitory effects on intestinal epithelial cells as seen for E. faecium SF68. Our results indicate that the arginine deiminase of E. faecium SF68 is responsible for inhibition of host cell NF-κB and JNK(AP-1) pathway activation, and is likely to be responsible for the anti-inflammatory and immunomodulatory effects observed in prior clinical human and animal trials. The implications for the use of this probiotic strain for preventive and therapeutic purposes are discussed.

摘要

在人体临床试验和动物饲料补充研究中，益生菌菌株粪肠球菌SF68 已被证明可缓解肠道炎症症状。为了确定与宿主细胞免疫调节作用有关的因素，使用含有 NF-κB 和 JNK(AP-1) 激活报告基因融合的肠上皮细胞系筛选粪肠球菌SF68 和其他共生和临床肠球菌分离株，以确定宿主的反应当受到细菌蛋白质和细胞成分的挑战时，细胞先天免疫信号通路。粪肠球菌的无细胞全细胞裂解物SF68 对肠上皮细胞中的 NF-κB 和 JNK(AP-1) 信号通路激活显示出可逆的抑制作用，并消除了对细菌和其他 Toll 样受体 (TLR) 配体的反应。抑制作用是物种特异性的，在鸟肠杆菌、鸡肠杆菌或卡氏肠杆菌中没有观察到。筛选E. faecium SF68 裂解物的蛋白质级分产生了一种活性级分，其中含有一种被鉴定为精氨酸脱亚胺酶 (ADI) 的突出蛋白质。将编码精氨酸脱亚氨酶的粪肠球菌SF68 arcA基因克隆并引入鸟粪肠球菌，在那里它赋予相同的 NF- _κB 对肠上皮细胞的抑制作用，如E. faecium SF68 所示。我们的研究结果表明，粪肠球菌SF68 的精氨酸脱亚氨酶负责抑制宿主细胞 NF-κB 和 JNK(AP-1) 通路的激活，并且可能负责先前临床中观察到的抗炎和免疫调节作用。人类和动物试验。讨论了将该益生菌菌株用于预防和治疗目的的意义。