Chileveru Haritha R, Lim Shion A, Chairatana Phoom, Wommack Andrew J, Chiang I-Ling, Nolan Elizabeth M
Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
Biochemistry. 2015 Mar 10;54(9):1767-77. doi: 10.1021/bi501483q. Epub 2015 Mar 2.
Human α-defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits broad-spectrum antimicrobial activity and contributes to innate immunity in the human gut and other organ systems. Despite many years of investigation, its antimicrobial mechanism of action remains unclear. In this work, we report that HD5ox, the oxidized form of this peptide that exhibits three regiospecific disulfide bonds, causes distinct morphological changes to Escherichia coli and other Gram-negative microbes. These morphologies include bleb formation, cellular elongation, and clumping. The blebs are up to ∼1 μm wide and typically form at the site of cell division or cell poles. Studies with E. coli expressing cytoplasmic GFP reveal that HD5ox treatment causes GFP emission to localize in the bleb. To probe the cellular uptake of HD5ox and subsequent localization, we describe the design and characterization of a fluorophore-HD5 conjugate family. By employing these peptides, we demonstrate that fluorophore-HD5ox conjugates harboring the rhodamine and coumarin fluorophores enter the E. coli cytoplasm. On the basis of the fluorescence profiles, each of these fluorophore-HD5ox conjugates localizes to the site of cell division and cell poles. These studies support the notion that HD5ox, at least in part, exerts its antibacterial activity against E. coli and other Gram-negative microbes in the cytoplasm.
人α-防御素5（HD5）是一种富含32个氨基酸残基的富含半胱氨酸的宿主防御肽，具有广谱抗菌活性，对人类肠道和其他器官系统的先天免疫有重要作用。尽管经过多年研究，其抗菌作用机制仍不清楚。在这项研究中，我们报告称，HD5的氧化形式HD5ox具有三个区域特异性二硫键，会导致大肠杆菌和其他革兰氏阴性微生物出现明显的形态变化。这些形态变化包括形成泡状结构、细胞伸长和聚集。泡状结构宽度可达约1μm，通常在细胞分裂部位或细胞两极形成。对表达细胞质绿色荧光蛋白（GFP）的大肠杆菌进行的研究表明，HD5ox处理会导致GFP荧光定位于泡状结构中。为了探究HD5ox的细胞摄取及随后的定位情况，我们描述了一种荧光团-HD5缀合物家族的设计与特性。通过使用这些肽，我们证明携带罗丹明和香豆素荧光团的荧光团-HD5ox缀合物进入了大肠杆菌细胞质。根据荧光分布情况，这些荧光团-HD5ox缀合物中的每一种都定位于细胞分裂部位和细胞两极。这些研究支持了这样一种观点，即HD5ox至少部分地在细胞质中对大肠杆菌和其他革兰氏阴性微生物发挥其抗菌活性。
