Moniruzzaman Md, Islam Md Zahidul, Sharmin Sabrina, Dohra Hideo, Yamazaki Masahito
Integrated Bioscience Section, Graduate School of Science and Technology, ‡Instrumental Research Support Office, Research Institute of Green Science and Technology, §Nanomaterials Research Division, Research Institute of Electronics, ∥Department of Physics, Graduate School of Science, Shizuoka University , Shizuoka 422-8529, Japan.
Biochemistry. 2017 Aug 22;56(33):4419-4431. doi: 10.1021/acs.biochem.6b01274. Epub 2017 Aug 9.
Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4-9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4-9) and lissamine rhodamine B red-labeled LfcinB (4-9) (Rh-LfcinB (4-9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4-9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4-9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4-9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4-9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4-9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4-9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4-9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.
乳铁蛋白B（LfcinB）及其较短的肽段具有抗菌活性。然而，这些肽与脂质膜和细菌相互作用的基本过程仍未得到充分理解。为了阐明其抗菌活性机制，我们研究了LfcinB（4-9）（其序列为RRWQWR）与大肠杆菌细胞和巨型单层囊泡（GUVs）的相互作用。LfcinB（4-9）和丽丝胺罗丹明B红色标记的LfcinB（4-9）（Rh-LfcinB（4-9））并未诱导膜不透性荧光探针SYTOX green从大肠杆菌细胞外部流入其细胞质，这表明其质膜未受到损伤。为了检测LfcinB（4-9）进入大肠杆菌细胞质的活性，我们使用共聚焦显微镜研究了Rh-LfcinB（4-9）与含有钙黄绿素的大肠杆菌单细胞的相互作用。我们发现Rh-LfcinB（4-9）进入了细胞质而没有钙黄绿素泄漏。接下来，我们使用单个GUV方法研究了Rh-LfcinB（4-9）与含有荧光探针Alexa Fluor 647酰肼（AF647）的二油酰磷脂酰甘油（DOPG）和二油酰磷脂酰胆碱（DOPC）混合物的单个GUVs的相互作用。结果表明，GUV外部的Rh-LfcinB（4-9）穿过GUV膜并进入其内腔而没有AF647泄漏。Rh-LfcinB（4-9）与DNA的相互作用极大地增加了其荧光强度。因此，我们可以得出结论，Rh-LfcinB（4-9）可以穿过大肠杆菌细胞质膜的脂质膜区域进入其细胞质而没有钙黄绿素泄漏，并且其抗菌活性不是由于其质膜受损。
