Myeloid differentiation factor 2 (MD2), the TLR4 coreceptor, has been shown to possess opsonic activity and has been implicated in phagocytosis and intracellular killing of Gram-negative bacteria. However, any MD2 protein segment involved in phagocytosis of Gram-negative bacteria is not yet known. A short synthetic MD2 segment, MD54 (amino acid regions 54 to 69), was shown to interact with a Gram-negative bacterial outer membrane component, LPS, earlier. Furthermore, the MD54 peptide induced aggregation of LPS and facilitated its internalization in THP-1 cells. Currently, it has been investigated if MD2-derived MD54 possesses any opsonic property and role in phagocytosis of Gram-negative bacteria. Remarkably, we observed that MD54 facilitated agglutination of Gram-negative bacteria, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC BAA-427), but not of Gram-positive bacteria, Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC 25923). The MD54-opsonized Gram-negative bacteria internalized within PMA-treated THP-1 cells and were killed over a longer incubation period. However, both internalization and intracellular killing of the MD54-opsonized Gram-negative bacteria within THP-1 phagocytes were appreciably inhibited in the presence of a phagocytosis inhibitor, cytochalasin D. Furthermore, MD54 facilitated the clearance of Gram-negative bacteria E. coli (ATCC 25922) and P. aeruginosa (ATCC BAA-427) from the infected BALB/c mice whereas an MD54 analog, MMD54, was inactive. Overall, for the first time, the results revealed that a short MD2-derived peptide can specifically agglutinate Gram-negative bacteria, act as an opsonin for these bacteria, and facilitate their phagocytosis by THP-1 phagocytes. The results suggest that the MD54 segment could have a crucial role in MD2-mediated host–pathogen interaction involving the Gram-negative bacteria.

中文翻译：

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促进 THP-1 巨噬细胞介导的革兰氏阴性菌吞噬作用的骨髓分化因子 2 衍生肽的表征

骨髓分化因子 2 (MD2) 是 TLR4 的辅助受体，已被证明具有调理活性，并参与革兰氏阴性菌的吞噬作用和细胞内杀伤作用。然而，任何参与革兰氏阴性菌吞噬作用的 MD2 蛋白片段尚不清楚。较早的研究表明，一个短的合成 MD2 片段 MD54（氨基酸区域 54 至 69）可与革兰氏阴性细菌外膜成分 LPS 相互作用。此外，MD54 肽诱导 LPS 聚集并促进其在 THP-1 细胞中的内化。目前，人们正在研究MD2衍生的MD54是否具有任何调理特性以及在革兰氏阴性菌的吞噬作用中的作用。值得注意的是，我们观察到 MD54 促进革兰氏阴性菌、大肠杆菌(ATCC 25922) 和铜绿假单胞菌(ATCC BAA-427) 的凝集，但不促进革兰氏阳性菌、枯草芽孢杆菌(ATCC 6633) 和金黄色葡萄球菌(ATCC 25923) 的凝集。 ）。MD54 调理的革兰氏阴性细菌内化于 PMA 处理的 THP-1 细胞内，并在较长的潜伏期内被杀死。然而，在吞噬作用抑制剂细胞松弛素 D 的存在下，THP-1 吞噬细胞内 MD54 调理的革兰氏阴性菌的内化和细胞内杀伤均受到明显抑制。此外，MD54 促进了革兰氏阴性菌大肠杆菌的清除。 ATCC 25922) 和铜绿假单胞菌(ATCC BAA-427) 来自受感染的 BALB/c 小鼠，而 MD54 类似物 MMD54 则无活性。总体而言，结果首次揭示了短的 MD2 衍生肽可以特异性凝集革兰氏阴性细菌，充当这些细菌的调理素，并促进 THP-1 吞噬细胞的吞噬作用。结果表明，MD54 片段可能在涉及革兰氏阴性菌的 MD2 介导的宿主-病原体相互作用中发挥至关重要的作用。