The Institute of Microbiology, Department of Biology, ETH Zürich, Zürich, Switzerland.
PLoS One. 2013 Jul 29;8(7):e69901. doi: 10.1371/journal.pone.0069901. Print 2013.
The C-type lectin RegIIIβ can kill certain Gram-positive and Gram-negative bacteria. The susceptibility of S. Typhimurium depends on the bacterial growth phase, i.e., bacteria from the logarithmic growth phase do bind RegIIIβ and are subsequently killed. Lipid A is one of the bacterial targets for RegIIIβ. However, at the molecular level, it is not understood how RegIIIβ interacts with and kills Gram-negative bacteria. Here, we show that RegIIIβ interacts with Gram-negative bacteria in two distinct steps. Initially, it binds to surface-exposed lipid A. The lipid A can be shielded by the O-antigen of lipopolysaccharide (LPS), as indicated by the exquisite susceptibility of wbaP mutants to RegIIIβ-mediated killing. Increased cell viability after incubation with an anti-lipid A antibody also supports this conclusion. This RegIIIβ-binding permeabilizes the outer membrane to hydrophobic dyes like Ethidium bromide or to bulky bacteriolytic enzymes like lysozyme. Conversely, compromising the outer membrane integrity by the mild detergent Triton X-100 enhances the antibacterial effect of RegIIIβ. Based on our observations, we conclude that RegIIIβ interacts with Gram-negative bacteria in two subsequent steps. Initially, it binds to the outer membrane thus leading to outer membrane permeabilization. This initial step is necessary for RegIIIβ to reach a second, still not well understood target site (presumably localized in the periplasm or the cytoplasmic membrane), thereby triggering bacterial death. This provides novel insights into the outer membrane-step of the bactericidal mechanism of RegIIIβ.
C 型凝集素 RegIIIβ 可以杀死某些革兰氏阳性和革兰氏阴性细菌。鼠伤寒沙门氏菌的易感性取决于细菌的生长阶段，即对数生长期的细菌会结合 RegIIIβ 并随后被杀死。脂 A 是 RegIIIβ 的细菌靶标之一。然而，在分子水平上，尚不清楚 RegIIIβ 如何与革兰氏阴性细菌相互作用并杀死它们。在这里，我们表明 RegIIIβ 通过两个不同的步骤与革兰氏阴性细菌相互作用。最初，它与表面暴露的脂 A 结合。脂 A 可以被脂多糖（LPS）的 O 抗原屏蔽，这表明 wbaP 突变体对 RegIIIβ 介导的杀伤非常敏感。用抗脂 A 抗体孵育后细胞活力增加也支持了这一结论。这种 RegIIIβ 结合使外膜对疏水性染料（如溴化乙锭）或大体积溶菌酶等细菌裂解酶具有通透性。相反，通过温和的去污剂 Triton X-100 破坏外膜完整性会增强 RegIIIβ 的抗菌作用。基于我们的观察，我们得出结论，RegIIIβ 通过两个后续步骤与革兰氏阴性细菌相互作用。最初，它与外膜结合，从而导致外膜通透性。这第一步是 RegIIIβ 到达第二个仍未完全理解的靶位点（推测位于周质或细胞质膜中）从而触发细菌死亡所必需的。这为 RegIIIβ 杀菌机制的外膜步骤提供了新的见解。
