A cecropin-like antimicrobial peptide with anti-inflammatory activity from the black fly salivary glands
- Jing Wu†1,
- Lixian Mu†1,
- Li Zhuang3,
- Yi Han1,
- Tong Liu1,
- Jun Li1,
- Yuan Yang1,
- Hailong Yang1Email author and
- Lin Wei2Email author
© Wu et al. 2015
Received: 30 August 2015
Accepted: 20 October 2015
Published: 24 October 2015
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-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.
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.
These results demonstrated that in addition to the bactericidal capacity, SibaCec can function as immune regulator, inhibiting host secretion of inflammatory factors.
Black flies (Diptera: Simuliidae) are annoying biting pests of humans and animals. They serve as obligate vectors for serious diseases such as human onchocerciasis (river blindness) and numerous arboviruses of livestock . A number of salivary peptides/proteins have been characterized from hematophagous arthropods such as horseflies [2, 3], mosquitoes  and ticks . 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) [6–8]. 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. However, there have been very few subsequent efforts to establish the functions of these molecules in black flies. In addition, other immune-related molecules including lectins, prophenoloxidase and antimicrobial peptides/proteins have also been identified from black flies [9–11].
AMPs, including cecropins, defensins, and cathelicidins, are a unique and diverse group of effector molecules that play an important role in humoral immunity in all living organisms . Cecropin was the first insect AMP isolated from the bacteria-challenged Hyalophora cecropia pupa , and then a number of cecropin-like peptides have been identified from different species of insect orders (Diptera, Lepidoptera, Hymenoptera, Coleoptera and Isoptera) [14, 15] and other organisms including mammals . In insects, cecropin-like peptides have been shown to have effects on bacteria , fungi , parasites  and viruses .
The immune-stimulatory/modulatory functions and mechanisms of vertebrate AMPs, especially mammalian AMPs, such as defensins (i.e., hBD-2, hBD-3, and hBD-4) and cathelicidins (i.e., LL-37 and PR-39), have been well studied [21–23]. These AMPs are involved in modulating chemokine and cytokine production in immune cells, altering gene expression in host cells, limiting sepsis, improving wound healing and angiogenesis in vitro and in vivo [24, 25]. Some insect AMPs, such as the AMPs from blood-sucking triatomine bug and midges, have been shown to be involved in the immune responses [26–28]. In fact, there are relatively few studies that focus on the anti-inflammatory functions for these AMPs. To date, two AMPs of cecropin family (papiliocin and cecropin A) with anti-inflammatory activity have been characterized from the swallowtail butterfly Papilio xuthus  and cecropia moth Hyalophora cecropia . Additionally, several hybrid peptides that are composed of cecropin A and other AMPs also showed the same activity [31–33].
We report herein the purification and characterization of a novel cecropin-like peptide with both antimicrobial and anti-inflammatory activities from the salivary glands of the hematophagous insect black fly S. bannaense.
Salivary gland dissection
Adult S. bannaense were collected near streams in Xishuangbanna, Yunnan, China. As our previous report , the black fly salivary glands were dissected in ice cold HEPES saline (10 mM HEPES pH 7.2, 150 mM NaCl) using fine entomological needles under a stereomicroscope, and stored in liquid nitrogen until use.
The study was approved by the Animal Care and Use Ethics Committee of Kunming Medical University.
MALDI-TOF MS analysis
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to analyze the purified peptide. AXIMA CFR mass spectrometer (Kratos Analytical) was operated in linear and positive ion mode. Polypeptide mass standard (Kratos Analytical) is used as an external calibration.
cDNA library construction and cDNA cloning
As our previous report , the In-Fusion SMARTerTM Directional cDNA Library Construction Kit (Takara, Japan) was used to prepare cDNAs. A sense degenerate primer (SibaCec-F1, according to the sequence determined by Edman degradation) was coupled with a 3’ PCR primer (the adaptor sequence of 3’ In-Fusion SMARTer CDS Primer provided in the kit) to screen the 3’ fragment of cDNA encoding SibaCec. The full length cDNA was finally obtained from primers of SibaCec-R1(antisense primer, according to the 3’-coding region of cDNA) and 5’ PCR primer from the library kit. The PCR conditions were: 95 °C for 5 min and 30 cycles of 95 °C (30 s), 56 °C (30 s), 72 °C (60 s) followed by an extension step at 72 °C for 8 min. Primers used in this research are shown in Additional file 1: Table S1.
The theoretical isoelectric point (pI) and molecular weight of SibaCec were analyzed through Bioinformatics Resource Portal (http://www.expasy.org/tools/) . Multi-sequence alignment was performed using ClustalW (http://embnet.vital-it.ch/software/ClustalW.html) . The phylogenetic tree was constructed by the neighbor-joining method in Mega 5 package.
The microbicidal activity of SibaCec was tested as described in our previous paper . Standard and clinically isolated drug resistance bacteria were obtained from the First Affiliated Hospital of Kunming Medical University. Minimal inhibitory concentration (MIC) was defined as the lowest concentration at which no visible growth of microorganisms occurred.
Bacteria E. coli ATCC 25922 were incubated with SibaCec (1 × MIC) diluted in phosphate buffered saline (PBS) at 37 °C for 45 min. Bacteria incubated with PBS was used as negative control. After centrifugation, bacteria pellets were fixed with 2.5 % glutaraldehyde solution for 2 h at 4 °C and then postfixed in 1 % osmium tetroxide for 2 h. Dehydration was carried out with a graded series of alcohols. Bacteria were mounted onto aluminium stubs and sputtered with gold. Images were visualized in a Hitachi S-4800 electron microscope.
Cytotoxic and hemolytic assay
The cytotoxic activity was carried out as described in our previous work . Briefly, 2 ml of 3 % (w/v) Brewer thioglycollate medium was injected into the peritoneal cavity of C57BL/6 mice. Three days later, peritoneal macrophages were harvested and cultured in 96-well plates (2 × 104 cells/well) in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 2 % fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. SibaCec dissolved in serum-free RPMI 1640 medium were added to wells, and the serum-free RPMI 1640 medium without SibaCec was used as control. After incubation at 37 °C for 24 h, 20 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 mg/ml) was added to each well, and the cells were further incubated for 4 h at 37 °C. The cells were dissolved in 200 μl of Me2SO, and the absorbance at 570 nm was measured on a microplate reader (Epoch Etock, BioTek, USA).
Hemolytic assay was conducted as previously reported . Serial dilutions of SibaCec were incubated with the washed human erythrocytes at 37 °C for 30 min. After centrifugation, the absorbance of supernatant was measured at 540 nm. 1 % (v/v) Triton X-100 was used to determine the maximal hemolysis and PBS was used as negative control.
Mouse peritoneal macrophages were incubated either with LPS (100 ng/ml, from Escherichia coli 0111:B4, Sigma-Aldrich, USA) and SibaCec (0, 5, 10, and 20 μg/ml) dissolved in serum-free RPMI 1640 medium or incubated with SibaCec (10 μg/ml) alone in 24-well plates (2.5 × 105 cells/well) for 24 h. The cells incubated with serum-free RPMI 1640 and 100 ng/ml LPS were used as control. The culture medium was harvested to detect NO production. NO production was determined by detecting the nitrite level using Griess reagent (Beyotime, China) according to the manufacturer’s instructions.
Mouse peritoneal macrophages were cultured in 6-well plates (2 × 106 cells/well) with RPMI 1640 (2 % FBS). The cells were incubated either with LPS (100 ng/ml) and SibaCec (0, 5, 10, and 20 μg/ml) dissolved in serum-free RPMI 1640 medium or incubated with SibaCec (10 μg/ml) alone. The cells incubated with serum-free RPMI 1640 and 100 ng/ml LPS were used as control. After treatment for 6 h, the cells were collected and total RNA was isolated. PrimeScript® Reverse Transcriptase (Takara, Japan) and SYBR green master mix (Takara, Japan) were used following the manufacturer’s instructions. qPCR was performed on a Realplex Mastercycler real-time PCR system (Eppendorf, Germany). The cycle counts of the target genes were normalized to the GAPDH gene, and accordingly the fold changes of the target genes were calculated. The primers used for qPCR are listed in Additional file 1: Table S1.
Pro-inflammatory cytokine determination
Mouse peritoneal macrophages were incubated either with LPS (100 ng/ml) and SibaCec (0, 5, 10, and 20 μg/ml) dissolved in serum-free RPMI 1640 medium or incubated with SibaCec (10 μg/ml) alone in 24-well plates (2.5 × 105 cells/well). The cells incubated with serum-free RPMI 1640 and 100 ng/ml LPS were used as control. After treatment for 6 h, the cell culture supernatants were collected and assessed for TNF-α, IL-1β, and IL-6 by using ELISA kits (Dakewei, China).
Western blot analysis
Mouse peritoneal macrophages were incubated either with LPS (100 ng/ml) and SibaCec (0, 5, 10, and 20 μg/ml) dissolved in serum-free RPMI 1640 medium or incubated with SibaCec (10 μg/ml) alone in 6-well plates (2 × 106 cells/well). The cells incubated with serum-free RPMI 1640 and 100 ng/ml LPS were used as control. After incubation for 30 min, the cells were washed twice with ice-cold PBS and lysed with RIPA lysis buffer (Beyotime, China). Then the cytoplasmic or nuclear proteins were extracted for Western blot analysis according to our previously described method . Primary antibodies of phospho-ERK/ERK, phospho-p38/p38, NF-κB p65 (1:2000, Cell Signaling Technology, USA), GAPDH/Lamin B (1:5000, Santa Cruz Biotechnology, USA), and secondary antibody (1:5000, Cell Signaling Technology, USA) were used in Western blot analysis.
Circular dichroism spectroscopy
CD spectroscopy was performed using a Jasco J-715 spectrophotometer (Jasco, Japan) to evaluate the secondary structure of SibaCec in solvent environment. SibaCec was dissolved in sodium dodecyl sulfate (SDS)/H2O solutions (0, 5, 10, 20, 40 mM) or LPS/H2O solution (0, 50, 100, 200, 400 ng/ml) to an ultimate concentration of 0.2 mg/ml. The spectra were measured at 298 K between 190 and 250 nm using 0.1 cm path-length cell with 1 nm bandwidth, 1 s response time, and a scan speed of 100 nm/min. Three consecutive scans were performed and averaged, followed by subtraction of the solvent signal.
Dissociation of FITC-LPS aggregates
The ability of SibaCec to dissociate LPS micelles was studied using FITC-conjugated LPS (1 μg/ml, Sigma-Aldrich, USA). FITC-LPS was excited at 480 nm and the change in the emission of FITC at 515 nm was monitored with different concentrations of SibaCec (0, 50, 100, 200 μg/ml) dissolved in PBS. PBS was used as control. The fluorescence experiment was performed using a Cary Eclipse fluorescence spectrophotometer (Varian, Inc., USA).
LPS neutralization assay
The ability of SibaCec to neutralize LPS was assayed with the chromogenic LAL test which was performed according to the manufacturer’s instruction (GenScript, Nanjing, China). Briefly, different concentrations of SibaCec (0, 25, 50, 100 μg/ml) dissolved in PBS were incubated with LPS at 37 °C for 30 min. PBS was used as control. After incubation, 100 μl of LAL solution was added to LPS-peptide mixtures (100 μl) in a pyrogen-free tube and incubated for 10 min followed by addition of pre-warmed substrate. After incubation of 6 min for the reaction, the absorbance was recorded at 545 nm on a microplate reader. The percentage of LPS neutralization was calculated as (A blank − A sample)/A blank × 100, where A blank represents the absorbance of blank control (50 μl of LAL water + 50 μl of LPS solution).
Bacterial feed experiment was carried out as previously described . In brief, the collected S. bannaense (~250 flies) were fed with 70 % sucrose solution ad libitum. After starving for 12 h, black flies were fed through cotton wool with 20 % sucrose solution (OD600 = 0.2) containing Gram-negative bacteria E.coli ATCC 25922. Total RNA was extracted from the salivary glands of immune stimulated or naive insects (sugar fed controls) at 12, 24, 36, 48 and 72 h after feeding. qPCR was performed to analyze the expression of SibaCec mRNA as described above, with the housekeeping gene β-actin as an endogenous control.
Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) and Stata 10.0 software (StataCorporation, College Station, TX, USA). Data were presented as mean ± standard errors of mean, and compared using two-tailed equal variance Student’s t-test. *P < 0.05 and **P < 0.01 were considered as statistical significance.
Characterization of SibaCec
As illustrated in Fig. 1a, the fractions with antimicrobial activity (marked by A1) were collected, lyophilized, and further purified by C18 RP-HPLC. One peptide was purified from this step (Fig. 1b), and it was designated SibaCec. 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. After carboxypeptidase Y treatment, no free amino acid can be detected by HPLC (data not shown). The result indicated that the C-terminal end of SibaCec was amidated, which was further confirmed by mass spectrometry analysis. MALDI-TOF MS analysis (Fig. 1c) indicated that SibaCec with the C-terminal amidation had a measured molecular mass of 3431.21 Da, matching well with the calculated molecular mass 3432.16 Da.
Multi-sequence alignment of the known cecropin-like peptide precursors from four black fly species (Fig. 2b) indicated that the putative signal peptide of these sequences are divergent, and nine amino acids residues are highly conserved within the mature peptide (Lys28, Asp29, Lys30, Leu31, Gla34, Ala35, Lys37, Ala38 and Leu39).
Phylogenetic analysis of SibaCec
Antimicrobial activity of SibaCec
Antimicrobial activity of SibaCec
Escherichia coli ATCC 25922
E. coli clinical strain 1
E. coli clinical strain 2
E. coli clinical strain 3
Pseudomonas aeruginosa ATCC 9027
Salmonella typhimurium ATCC 14028
Acinetobacter baumannii ATCC 17978
Staphylococcus aureus ATCC 6538
Bacillus subtilis ATCC 6633
Micrococcus luteus ATCC 4698
SibaCec alters the morphology of E. coli
SibaCec shows low cytotoxicity and hemolytic activity
SibaCec exhibited very low cytotoxicity toward the mouse peritoneal macrophages. At concentrations up to 200 μg/ml (58.27 μM), which is almost 67-fold higher than the MIC value of SibaCec against E.coli ATCC 25922, SibaCec induced cell death percentages as low as 4.26 %. As to hemolytic activity, SibaCec yielded a hemolysis of 3.25 % at the same concentration of 200 μg/ml.
SibaCec reduces LPS-induced iNOS transcription and NO production
SibaCec inhibits LPS-induced pro-inflammatory cytokine production
SibaCec inhibits LPS-induced inflammatory response pathways
Secondary structure of SibaCec
Secondary structural components of SibaCec in different solutions
SibaCec dissociates FITC-LPS aggregates
SibaCec neutralizes LPS
The chromogenic LAL assay was used to determine whether SibaCec is active in neutralization of endotoxin. In parallel with the dissociation of FITC-LPS aggregates assay, SibaCec exhibited LPS neutralizing activity in a concentration dependent manner (Fig. 9b). At the concentrations of 25, 50, 100 μg/ml, SibaCec inhibited 23.8, 33.2 and 49.4 % of LPS, respectively.
Transcript levels of SibaCec increase after infection with E.coli
Black flies are blood-sucking insects that can secrete various immunomodulatory molecules to suppress the host’s inflammatory and immunologic reactions, and to contribute to efficient transmission of fly-borne pathogens . The salivary gland extract of black fly S. vittatum, has been shown to contain immunomodulatory activities that reduces expression of I-A (mouse MHC class II), IL-5 and IL-10 in splenocytes [40, 41], and inhibits mitogen-stimulated mouse splenocyte proliferation . However, the component(s) responsible for immunomodulation has not been characterized from black flies so far.
The current work identified a novel cationic cecropin-like peptide (SibaCec) from the salivary glands of the black fly S. bannaense. The structural organization of SibaCec precursor (Fig. 2a) is similar to other insect cecropin precursors, comprising a putative signal peptide and the mature peptide. The alanine residue (Ala22) in the precursor is cleaved to release the mature peptide, which is consistent with most cecropins isolated from Diptera . SibaCec presents a canonical feature of most characterized insect cecropins (an amidated C-terminus), but it is devoid of another feature (a tryptophan residue at position 1 or 2) . The first two amino acid residues at the N-terminus of SibaCec are glycine and lysine. The absence of tryptophan residue within the N-terminal domain was also reported in some mosquito cecropins [17, 43].
The phylogenetic tree of cecropin precursors (Fig. 3) showed that the black fly cecropins appear in one branch and other insect cecropins are grouped in another branch. This suggests the cecropins occurred in the insects before the divergence of the Diptera, Lepidoptera and Coleoptera. The result also supports that the cecropin molecules have evolved independently between these insect taxa .
SibaCec, like other cationic AMPs, is a highly basic peptide with the net charge of +8, which implies that it would be readily attracted and adhered to the negative-charged bacterial surface. As expected, SibaCec has a broad spectrum of antibacterial activity (Table 1), and it is more effective against Gram-negative bacteria (MICs ranging from 0.87 to 2.33 μM) than against Gram-positive bacteria (MICs ranging from 14.56 to 43.7 μM). SEM analysis indicated that such activities are carried out with a lytic effect on the bacterial membranes (Fig. 4). Additionally, CD spectroscopy indicated that SibaCec mainly adopt an α-helical conformation in membrane-mimetic solutions (Fig. 8), which contributes to the ability of cationic peptide to kill bacteria. These results confirm that the microbial membrane is a key target for SibaCec.
Extensive research has established that MAPK signal transduction pathways are involved in regulating the transcriptions of cytokine genes [29, 37]. In this study, SibaCec significantly inhibited the transcription and production of pro-inflammatory factors induced by LPS in mouse peritoneal macrophages, including NO, TNF-α, IL-1β, and IL-6 (Figs. 5 and 6). Furthermore, Western blot analysis showed that SibaCec significantly suppressed the LPS induced activation of ERK, p38, and the translocation of NF-κB p65 subunit (Fig. 7). These data indicated that SibaCec executes the anti-inflammatory effect in LPS-stimulated murine macrophages by blocking the activation of MAPKs and NF-κB signaling pathways.
The analysis of interaction between SibaCec and LPS indicated that SibaCec can dissociate the aggregated form of LPS in a dose-dependent manner (Fig. 9a), and the LAL assay indicated that SibaCec also can neutralize LPS in a dose-dependent fashion (Fig. 9b). Since SibaCec is a membrane-targeting AMP with polycationic (basic-amino-acid-rich) and amphipathic α-helix structure, we rationally conclude SibaCec binds to negatively charged LPS mostly through electrostatic interaction and the amphiphilic helical structure . All these results implied that the neutralization of LPS by SibaCec prevents LPS from binding to the LPS-binding protein, and hence suppresses the production of cytokines induced by LPS. Gram-negative bacteria and/or their endotoxins (LPS) may trigger a systemic inflammatory response, leading to some life-threatening systemic diseases, such as sepsis and septic shock . The above results indicate that SibaCec is not only involved in suppressing Gram-negative bacteria growth but also that it attenuates inflammatory responses induced by LPS. These functions of SibaCec may favor resolution of infection and reverse potentially harmful inflammation.
The transcript levels of SibaCec in the salivary glands of insects increased after oral infection with Gram-negative bacteria E.coli, reaching maximum at 24 h and then slowly decreased from that time point (Fig. 10). These data suggested that SibaCec is involved in the innate humoral response of the black fly S. bannaense.
In conclusion, the black fly cecropin-like peptide (SibaCec) was identified in the present work by peptide purification and molecular cloning procedures. SibaCec possesses potent antimicrobial activity against Gram-negative bacteria, and shows low cytotoxicity toward mammalian cells. It can neutralize LPS and exhibit strong anti-inflammatory activity. All the amalgamated properties make SibaCec a potentially potent candidate for the treatment of inflammatory and infectious diseases.
This work was supported by Chinese National Natural Science Foundation (81373380, 81360253, 81260258, 81402830) and Jiangsu Province Foundation (BK20140362, 14KJD350003).
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