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Influence of disulfide bonds in human beta defensin-3 on its strain specific activity against Gram-negative bacteria.
Biochimica et Biophysica Acta (BBA) - Biomembranes ( IF 2.8 ) Pub Date : 2020-03-19 , DOI: 10.1016/j.bbamem.2020.183273 Christian Nehls 1 , Arne Böhling 1 , Rainer Podschun 2 , Sabine Schubert 2 , Joachim Grötzinger 3 , Andra Schromm 1 , Henning Fedders 4 , Matthias Leippe 4 , Jürgen Harder 5 , Yani Kaconis 1 , Sabine Gronow 6 , Thomas Gutsmann 1
Biochimica et Biophysica Acta (BBA) - Biomembranes ( IF 2.8 ) Pub Date : 2020-03-19 , DOI: 10.1016/j.bbamem.2020.183273 Christian Nehls 1 , Arne Böhling 1 , Rainer Podschun 2 , Sabine Schubert 2 , Joachim Grötzinger 3 , Andra Schromm 1 , Henning Fedders 4 , Matthias Leippe 4 , Jürgen Harder 5 , Yani Kaconis 1 , Sabine Gronow 6 , Thomas Gutsmann 1
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Antimicrobial peptides (AMPs) play an important role in the host defense against various microbes. One of the most efficient human AMPs is the human beta defensin-3 (hBD-3) which is produced by, e.g. keratinocytes and lung epithelial cells. However, the structure-function relationship for AMPs and in particular for defensins with their typical three disulfide bonds is still poorly understood. In this study the importance of the three disulfide bonds for the activity of the AMPs is investigated with biological assays and with biophysical experiments utilizing different membrane reconstitution systems. The activities of natural hBD-3, hBD-3-c (cyclic variant with one disulfide bond), and hBD-3-l (linear variant without disulfide bonds) and fragments thereof were tested against specific Gram-negative bacteria. Furthermore, hemolytic and cytotoxic activities were analyzed as well as the potency to neutralize immune cell stimulation of lipopolysaccharide (LPS). Experiments using reconstituted lipid matrices composed of phospholipids or LPS purified from the respective Gram-negative bacteria, showed that the membrane activity of all three hBD-3 peptides is decisive for their capability to kill bacteria and to neutralize LPS. In most of the test systems the linear hBD-3-l showed the highest activity. It was also the only peptide significantly active against polymyxin B-resistant Proteus mirabilis R45. However, the stability of hBD-3 against protease activity decreases with decreasing number of disulfide bonds. This study demonstrates that the refining of AMP structures can generate more active compounds against certain strains.
中文翻译：
人β防御素3中的二硫键对其菌株针对革兰氏阴性细菌的比活性的影响。
抗菌肽（AMP）在宿主抵抗各种微生物的过程中起着重要作用。最有效的人AMP之一是人β防御素3（hBD-3），它是由例如角质形成细胞和肺上皮细胞产生的。然而，对于AMP，尤其是具有典型的三个二硫键的防御素的结构-功能关系仍然知之甚少。在这项研究中，通过生物测定和利用不同膜重构系统的生物物理实验研究了三个二硫键对AMPs活性的重要性。测试了天然hBD-3，hBD-3-c（带有一个二硫键的环状变异体）和hBD-3-l（没有二硫键的线性变异体）及其片段对特定革兰氏阴性细菌的活性。此外，分析了溶血和细胞毒性活性，以及中和脂多糖（LPS）免疫细胞刺激的能力。使用由从各自革兰氏阴性细菌中纯化的磷脂或LPS组成的重构脂质基质进行的实验表明，所有三种hBD-3肽的膜活性都决定了它们杀死细菌和中和LPS的能力。在大多数测试系统中，线性hBD-3-l表现出最高的活性。它也是唯一对多粘菌素B耐药的变形杆菌R45具有显着活性的肽。但是，hBD-3对蛋白酶活性的稳定性随二硫键数量的减少而降低。这项研究表明，AMP结构的细化可以针对某些菌株产生更多的活性化合物。
更新日期：2020-03-19
中文翻译：
人β防御素3中的二硫键对其菌株针对革兰氏阴性细菌的比活性的影响。
抗菌肽（AMP）在宿主抵抗各种微生物的过程中起着重要作用。最有效的人AMP之一是人β防御素3（hBD-3），它是由例如角质形成细胞和肺上皮细胞产生的。然而，对于AMP，尤其是具有典型的三个二硫键的防御素的结构-功能关系仍然知之甚少。在这项研究中，通过生物测定和利用不同膜重构系统的生物物理实验研究了三个二硫键对AMPs活性的重要性。测试了天然hBD-3，hBD-3-c（带有一个二硫键的环状变异体）和hBD-3-l（没有二硫键的线性变异体）及其片段对特定革兰氏阴性细菌的活性。此外，分析了溶血和细胞毒性活性，以及中和脂多糖（LPS）免疫细胞刺激的能力。使用由从各自革兰氏阴性细菌中纯化的磷脂或LPS组成的重构脂质基质进行的实验表明，所有三种hBD-3肽的膜活性都决定了它们杀死细菌和中和LPS的能力。在大多数测试系统中，线性hBD-3-l表现出最高的活性。它也是唯一对多粘菌素B耐药的变形杆菌R45具有显着活性的肽。但是，hBD-3对蛋白酶活性的稳定性随二硫键数量的减少而降低。这项研究表明，AMP结构的细化可以针对某些菌株产生更多的活性化合物。
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