ABSTRACT

The respiratory tract is a major entry site for microbial pathogens. To combat bacterial infections, the immune system has various defense mechanisms at its disposal, including antimicrobial peptides (AMPs). To search for novel AMPs from the respiratory tract, a peptide library from human broncho-alveolar-lavage (BAL) fluid was screened for antimicrobial activity by radial diffusion assays allowing the efficient detection of antibacterial activity within a small sample size. After repeated testing-cycles and subsequent purification, we identified ß-2-microglobulin (B2M) in antibacterially active fractions. B2M belongs to the MHC-1 receptor complex present at the surface of nucleated cells. It is known to inhibit the growth of Listeria monocytogenes and Escherichia coli and to facilitate phagocytosis of Staphylococcus aureus. Using commercially available B2M we confirmed a dose-dependent inhibition of Pseudomonas aeruginosa and L. monocytogenes. To characterize AMP activity within the B2M sequence, peptide fragments of the molecule were tested for antimicrobial activity. Activity could be localized to the C-terminal part of B2M. Investigating pH dependency of the antimicrobial activity of B2M demonstrated an increased activity at pH values of 5.5 and below, a hallmark of infection and inflammation. Sytox green uptake into bacterial cells following the exposure to B2M was determined and revealed a pH-dependent loss of bacterial membrane integrity. TEM analysis showed areas of disrupted bacterial membranes in L. monocytogenes incubated with B2M and high amounts of lysed bacterial cells. In conclusion, B2M as part of a ubiquitous cell surface complex may represent a potent antimicrobial agent by interfering with bacterial membrane integrity.

摘要

呼吸道是微生物病原体的主要进入部位。为了抵抗细菌感染，免疫系统具有各种防御机制，包括抗菌肽（AMPs）。为了从呼吸道中搜索新的AMP，通过放射扩散分析筛选了来自人支气管肺泡灌洗液（BAL）的肽库的抗菌活性，从而可以在小样本量内有效检测抗菌活性。经过反复的测试周期和随后的纯化，我们在抗菌活性部分中鉴定出ß-2-微球蛋白（B2M）。B2M属于存在于有核细胞表面的MHC-1受体复合物。已知抑制单核细胞增多性李斯特菌和大肠杆菌的生长并促进金黄色葡萄球菌的吞噬作用。使用市售的B2M，我们证实了铜绿假单胞菌和单核细胞增生李斯特菌的剂量依赖性抑制作用。为了表征B2M序列内的AMP活性，测试了分子的肽片段的抗微生物活性。活动可以定位到B2M的C端部分。pH对B2M抗菌活性的依赖性研究表明，在pH值低于5.5时，活性增强，这是感染和炎症的标志。确定暴露于B2M后细菌细胞中的Sytox绿色吸收，并揭示了pH依赖性细菌膜完整性的损失。TEM分析显示细菌膜破裂的区域单核细胞增生李斯特氏菌与B2M和大量裂解细菌细胞孵育。总之，B2M作为普遍存在的细胞表面复合物的一部分，可以通过干扰细菌膜的完​​整性来代表有效的抗菌剂。