A cecropin-like antimicrobial peptide with anti-inflammatory activity from the black fly salivary glands.
BACKGROUND
Several 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.


METHODS
A novel cecropin-like antimicrobial peptide (SibaCec) was purified using reverse phase high-performance liquid chromatography (RP-HPLC) from the salivary glands of the black fly Simulium bannaense. The amino acid sequence of SibaCec was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-negative bacteria Escherichia coli treated with SibaCec 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 of SibaCec in solvent environment. Interaction of SibaCec with lipopolysaccharide (LPS) was studied using fluorescein isothiocyanate (FITC)- conjugated LPS aggregates. Neutralization of LPS by SibaCec was assayed with the chromogenic limulus amebocyte lysate (LAL) test. qPCR was also used to analyze the expression of SibaCec mRNA in the salivary glands of insects after oral infection with the bacteria E.coli.


RESULTS
SibaCec possessed potent antimicrobial activity against Gram-negative bacteria, and showed low cytotoxicity toward mammalian cells. SEM analysis indicated that SibaCec 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 of SibaCec.


CONCLUSIONS
These results demonstrated that in addition to the bactericidal capacity, SibaCec can function as immune regulator, inhibiting host secretion of inflammatory factors.
School of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, 650500, Yunnan, China. wujing_205@163.com.
Jing
J
Wu
School of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, 650500, Yunnan, China. mulixian77@163.com.
Lixian
L
Mu
The Second Department of Internal Medicine, the Third Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China. kekzhuangli@163.com.
Li
L
Zhuang
School of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, 650500, Yunnan, China. kmhanyi@163.com.
Yi
Y
Han
School of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, 650500, Yunnan, China. 962620924@qq.com.
Tong
T
Liu
School of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, 650500, Yunnan, China. lijunall123@163.com.
Jun
J
Li
School of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, 650500, Yunnan, China. yangyuan1109@sina.com.
Yuan
Y
Yang
School of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, 650500, Yunnan, China. jxauyhl@aliyun.com.
Hailong
H
Yang
Jiangsu Key Laboratory of Infection and Immunity, Institute of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu, China. weilin1005@126.com.
Lin
L
Wei
D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015153:Blotting, Western; D054807:Cecropins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D016207:Cytokines / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000187:drug effects / Q000648:ultrastructure; D020869:Gene Expression Profiling; D007313:Insecta; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis; D011487:Protein Conformation; D060888:Real-Time Polymerase Chain Reaction; D012469:Salivary Glands / Q000737:chemistry; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D012843:Simuliidae / Q000502:physiology*
D000890:Anti-Infective Agents; D000893:Anti-Inflammatory Agents; D054807:Cecropins; D016207:Cytokines; D008070:Lipopolysaccharides; D052247:Nitric Oxide Synthase Type II