Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores ("carpet" mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 107 bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs.

中文翻译：

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抗菌肽与细菌细胞的结合：与不同物种，菌株和细胞成分的相互作用。

抗菌肽（AMPs）通过破坏细菌的细胞膜来选择性杀死细菌，并且是有望与耐药菌抗药的化合物。对模型膜的生物物理研究已经表征了AMP /膜的相互作用和双层扰动的机制，表明阳离子小分子分子在外部小叶中的积累导致了孔的形成（“地毯”机制）。但是，对真实细胞的类似定量研究极为有限。在这里，我们调查了丹酰化的PMAP23肽（DNS-PMAP23）与革兰氏阳性细菌的相互作用，表明杀死每个细胞需要107个结合的肽分子。这一结果与我们先前对革兰氏阴性菌株的发现一致，在该研究中，确定了类似的高致死阈值，证明了地毯模型与真实细菌的一般相关性。但是，在革兰氏阳性菌株的情况下，该分子数甚至超过了细菌膜上可用的总表面。DNS-PMAP23对革兰氏阳性细胞壁的阴离子回壁酸具有很高的亲和力，而对革兰氏阴性细菌的脂多糖则没有，这为这一发现提供了理论依据。为了更好地定义阴离子脂质在肽/细胞缔合中的作用，我们研究了DNS-PMAP23与缺乏磷脂酰甘油和/或心磷脂的大肠杆菌突变菌株的相互作用。令人惊讶地，这些菌株显示了与野生型相似的肽亲和力。通过观察发现这些细菌的其他阴离子脂质含量增加，可以合理地发现这一发现，因此保持总膜电荷基本恒定。最后，DNS-PMAP23与死亡细菌的关联研究表明，与活细胞相比，亲和力高一个数量级，表明与膜内扰动后可进入的细胞内组分的强肽结合。由于死细菌可以通过隔离大量肽分子来保护存活的细胞，因此这种作用可能在种群对AMP作用的抗性中发挥作用。总体而言，我们的数据表明，肽与细菌的缔合的定量研究可以导致对AMPs作用机理的更好理解。提示强肽与细胞内成分结合，这些成分在膜微扰后即可进入。由于死细菌可以通过隔离大量肽分子来保护存活的细胞，因此这种作用可能在种群对AMP作用的抗性中发挥作用。总体而言，我们的数据表明，肽与细菌的缔合的定量研究可以导致对AMPs作用机理的更好理解。提示强肽与细胞内成分结合，这些成分在膜微扰后即可进入。由于死细菌可以通过隔离大量肽分子来保护存活的细胞，因此这种作用可能在种群对AMP作用的抗性中发挥作用。总体而言，我们的数据表明，肽与细菌的缔合的定量研究可以导致对AMPs作用机理的更好理解。