A cecropin-like antimicrobial peptide with anti-inflammatory activity from the black fly salivary glands.
A cecropin-like antimicrobial peptide with anti-inflammatory activity from the black fly salivary glands.
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来自黑蝇唾液腺的具有抗炎活性的类天蚕素抗菌肽
DOI：
10.1186/s13071-015-1176-8
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发表时间：
2015-10-24
影响因子：
3.2
通讯作者：
Wei L
中科院分区：
文献类型：
--
作者：
Wu J;Mu L;Zhuang L;Han Y;Liu T;Li J;Yang Y;Yang H;Wei L
背景从不同种类的果蝇唾液腺中分离到了抗菌肽（Cecropin）家族的抗菌肽（Amps），但其免疫功能尚不清楚。通过自动Edman降解和cDNA测序的组合方法确定SibaCec的氨基酸序列。扫描电镜观察SibaCec对革兰氏阴性菌大肠杆菌的作用。进行定量PCR（qPCR）以分析诱导型NO合酶（iNOS）和促炎细胞因子的mRNA表达。一氧化氮（NO）的产生进行了检查，使用Griess测定和促炎细胞因子的分泌通过酶联免疫吸附测定（ELISA）确定。Western blotting法检测细胞外信号调节激酶（ERK）、p38的活化和核因子-κ B（NF-κB）的核转位。圆二色性（CD）光谱用于评估SibaCec在溶剂环境中的二级结构。使用异硫氰酸荧光素（FITC）缀合的脂多糖（LPS）聚集体研究SibaCec与LPS的相互作用。通过显色鲎试剂（LAL）试验测定SibaCec对LPS的中和作用。结果SibaCec对革兰氏阴性菌具有较强的抗菌活性，对哺乳动物细胞的细胞毒性较低。SEM分析表明SibaCec通过破坏细胞膜的完整性来杀灭细菌。此外，SibaCec通过阻断MAPK和NF-κB信号通路的激活，显著抑制脂多糖（LPS）诱导的NO和促炎细胞因子如肿瘤坏死因子-α（TNF-α）、干扰素-1 β（IL-1β）和干扰素-6（IL-6）的产生。SibaCec在拟膜环境中主要以α-螺旋构象存在，能与LPS相互作用并中和LPS。黑蝇感染细菌引起的表达ofSibaCec.ConclusionsThese结果表明，除了杀菌能力，SibaCec可以作为免疫调节剂，抑制宿主分泌的炎症因子的上调。
BackgroundSeveral antimicrobial peptides (AMPs) belonging to the cecropin family have been identified from the salivary glands of different black fly species, however, the immunological functions for these molecules were poorly understood.MethodsA novel cecropin-like antimicrobial peptide (SibaCec) was purified using reverse phase high-performance liquid chromatography (RP-HPLC) from the salivary glands of the black flySimulium bannaense. The amino acid sequence ofSibaCec was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-negative bacteriaEscherichia colitreated withSibaCec were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the mRNA expression of the inducible NO synthase (iNOS) and pro-inflammatory cytokines. Nitric oxide (NO) generation was examined using a Griess assay and the secretion of pro-inflammatory cytokines was determined by an enzyme-linked immunosorbent assay (ELISA). The activation of extracellular signal-regulated kinase (ERK), p38, and the nuclear translocation of nuclear factor-kappaB (NF-κB) were assessed by Western blotting analysis. Circular dichroism (CD) spectroscopy was performed to evaluate the secondary structure ofSibaCec in solvent environment. Interaction ofSibaCec with lipopolysaccharide (LPS) was studied using fluorescein isothiocyanate (FITC)- conjugated LPS aggregates. Neutralization of LPS bySibaCec was assayed with the chromogenic limulus amebocyte lysate (LAL) test. qPCR was also used to analyze the expression ofSibaCec mRNA in the salivary glands of insects after oral infection with the bacteriaE.coli.ResultsSibaCec possessed potent antimicrobial activity against Gram-negative bacteria, and showed low cytotoxicity toward mammalian cells. SEM analysis indicated thatSibaCec killed bacteria through the disruption of cell membrane integrity. Furthermore,SibaCec significantly inhibited lipopolysaccharide (LPS)-induced production of NO and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-1β (IL-1β) and interferon-6 (IL-6) by blocking the activation of MAPKs and NF-κB signaling pathways. It mainly adopted an α-helix conformation in membrane-mimetic environments.SibaCec could interact and neutralize LPS. Infection of black flies with bacteria caused an upregulation of the expression ofSibaCec.ConclusionsThese results demonstrated that in addition to the bactericidal capacity,SibaCec can function as immune regulator, inhibiting host secretion of inflammatory factors.