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A novel cecropin B-derived peptide with antibacterial and potential anti-inflammatory properties.

ABSTRACT: Cecropins, originally found in insects, are a group of cationic antimicrobial peptides. Most cecropins have an amphipathic N-terminal segment and a largely hydrophobic C-terminal segment, and normally form a helix-hinge-helix structure. In this study, we developed the novel 32-residue cecropin-like peptide cecropin DH by deleting the hinge region (Alanine-Glycine-Proline) of cecropin B isolated from Chinese oak silk moth, Antheraea pernyi. Cecropin DH possesses effective antibacterial activity, particularly against Gram-negative bacteria, with very low cytotoxicity against mammalian cells. Interactions between cecropin DH and the highly anionic lipopolysaccharide (LPS) component of the Gram-negative bacterial outer membrane indicate that it is capable of dissociating LPS micelles and disrupting LPS aggregates into smaller assemblies, which may play a vital role in its antimicrobial activity. Using LPS-stimulated mouse macrophage RAW264.7 cells, we found that cecropin DH exerted higher potential anti-inflammatory activity than cecropin B, as demonstrated by the inhibition of pro-inflammatory cytokines nitric oxide production and secretion of tumor necrosis factor-?. In conclusion, cecropin DH has potential as a therapeutic agent for both antibacterial and anti-inflammatory applications.

SUBMITTER: Wang J

PROVIDER: S-EPMC6064198 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A novel cecropin B-derived peptide with antibacterial and potential anti-inflammatory properties.

Wang Jiarong J Ma Kun K Ruan Maosen M Wang Yujuan Y Li Yan Y Fu Yu V YV Song Yonghong Y Sun Hongbin H Wang Junfeng J

PeerJ 20180725

Cecropins, originally found in insects, are a group of cationic antimicrobial peptides. Most cecropins have an amphipathic N-terminal segment and a largely hydrophobic C-terminal segment, and normally form a helix-hinge-helix structure. In this study, we developed the novel 32-residue cecropin-like peptide cecropin DH by deleting the hinge region (Alanine-Glycine-Proline) of cecropin B isolated from Chinese oak silk moth, <i>Antheraea pernyi</i>. Cecropin DH possesses effective antibacterial act ...[more]

PMID: 30065898

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Project description: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.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-?B) 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.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-? (TNF-?), interferon-1? (IL-1?) and interferon-6 (IL-6) by blocking the activation of MAPKs and NF-?B signaling pathways. It mainly adopted an ?-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.These results demonstrated that in addition to the bactericidal capacity, SibaCec can function as immune regulator, inhibiting host secretion of inflammatory factors.

Project description:BACKGROUND:It has been reported that there are more than ten antimicrobial peptides (AMPs) belonging to the cecropin family in Musca domestica; however, few of them have been identified, and the functions of the other molecules are poorly understood. METHODS:Sequences of the M. domestica cecropin family of genes were cloned from cDNA template, which was reverse-transcribed from total mRNA isolated from third-instar larvae of M. domestica that were challenged with pathogens. Sequence analysis was performed using DNAMAN comprehensive analysis software, and a molecular phylogenetic tree of the cecropin family was constructed using the Neighbour-Joining method in MEGA v.5.0 according to the mature peptide sequences. Antibacterial activity of the synthetic M. domestica cecropin protein was detected and the minimum inhibitory concentration (MIC) values were determined using broth microdilution techniques. Time-killing assays were performed on the Gram-negative bacteria, Acinetobacter baumannii, at the logarithmic or stabilizing stages of growth, and its morphological changes when treated with Cec4 were assessed by scanning electron microscopy (SEM) and detection of leakage of 260 nm absorbing material. RESULTS:Eleven cecropin family genes, namely Cec01, Cec02 and Cec1-9, show homology to the Cec form in a multigene family on the Scaffold18749 of M. domestica. In comparing the encoded cecropin protein sequences, most of them have the basic characteristics of the cecropin family, containing 19 conservative amino acid residues. To our knowledge, this is the first experimental demonstration that most genes in the Cec family are functional. Cec02, Cec1, Cec2, Cec5 and Cec7 have similar antibacterial spectra and antibacterial effects against Gram-negative bacteria, while Cec4 displays a more broad-spectrum of antimicrobial activity and has a very strong effect on A. baumannii. Cec4 eliminated A. baumannii in a rapid and concentration-dependent manner, with antibacterial effects within 24 h at 1× MIC and 2× MIC. Furthermore, SEM analysis and the leakage of 260 nm absorbing material detection indicated that Cec4 sterilized the bacteria through the disruption of cell membrane integrity. CONCLUSIONS:Although there are more than ten cecropin genes related to M. domestica, some of them have no preferred antibacterial activity other than Cec4 against A. baumannii.

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Project description:Melanoma is a common malignant skin tumor, which is the only fatal skin tumor at present. Melanoma has a high degree of malignancy and metastasis. The activity of modified Temporin-La (T-La) peptides from bullfrog skin were evaluated for antitumor activity and improved targeting in melanoma cells. The amino acid sequence of T-La was modified, resulting in the antitumor peptide, T-La (FS). T-La and T-La (FS) were coupled to the RGD small molecule polypeptide to form the chimeric peptides RGD-T-La and RGD-T-La (FS), respectively. The secondary structures for the peptides, evaluated using circular dichroism, were found to be α-helical. The structure of T-La was evaluated using bioinformatics. In addition, the antitumor effects of the modified peptide and the targeting of RGD chimeric peptide to the tumor in vivo and in vitro were analyzed. Antitumor activity was measured in vitro using the MTT assay. Tumor cells with high integrin αvβ3 expression were detected using flow cytometry, and tumor cells were screened for sensitivity to RGD-T-La (FS) to establish a tumor model in nude mice. The effects of the peptides on tumor cells were measured using laser confocal microscopy in real-time. The mechanism of the peptide antitumor activity in tumor cells was evaluated with scanning electron microscopy. B16 melanoma cells were the most sensitive to the peptides, for which the cell survival rate was 24.65% for 10 µg/ml RGD-T-La (FS). RGD-La (FS) had a rapid effect on tumor cells. RGD chimeric polypeptides exhibited site-targeting cytotoxic effects in tumor cells. In the B16 melanoma mouse model, the peptides exhibited antitumor effects against early melanoma development and induced tumor apoptosis, possibly by inhibiting VEGF and promoting caspase-3 expression. Overall, the present study provides a scientific basis for the application of small molecule antimicrobial peptides as targeted antitumor agents and lays the foundation for the clinical application of these peptides as antitumor drugs.

Dataset Information

A novel cecropin B-derived peptide with antibacterial and potential anti-inflammatory properties.

Publications

A novel cecropin B-derived peptide with antibacterial and potential anti-inflammatory properties.

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