Abstract
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 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.ResultsSibaCec 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.ConclusionsThese results demonstrated that in addition to the bactericidal capacity, SibaCec can function as immune regulator, inhibiting host secretion of inflammatory factors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1176-8) contains supplementary material, which is available to authorized users.
Highlights
Several antimicrobial peptides (AMPs) belonging to the cecropin family have been identified from the salivary glands of different black fly species, the immunological functions for these molecules were poorly understood
After Edman degradation, the initial 22 N-terminal amino acid residues of SibaCec was identified with the following sequence: GKLTKDKLKRGAKKALNVASKV
Due to the majority of insect cecropin-like peptides containing a C-terminally amidated residue, we presumed that the same structure feature exists in the SibaCec
Summary
Several antimicrobial peptides (AMPs) belonging to the cecropin family have been identified from the salivary glands of different black fly species, the immunological functions for these molecules were poorly understood. There was relatively limited information available on pharmacologically active compounds in black fly salivary glands, until when the salivary transcriptomes were published for three black fly species (S. vittatum, S. nigrimanum and S. guianense) [6,7,8]. In these studies, some transcripts related to immunity, including six AMPs of cecropin family, have been revealed by sequence similarities with known peptides/proteins from other organisms. Other immune-related molecules including lectins, prophenoloxidase and antimicrobial peptides/proteins have been identified from black flies [9,10,11]
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