Antimalarial activity of cecropin antimicrobial peptides derived from Anopheles mosquitoes.

The emergence of clinically drug-resistant malaria parasites requires the urgent development of new drugs. Mosquitoes are vectors of multiple pathogens and have developed resistance mechanisms against them, which often involve antimicrobial peptides (AMPs). An-cecB is an AMP of the malaria-transmitting mosquito genus Anopheles, and we herein report its antimalarial activity against Plasmodium falciparum 3D7, the artemisinin-resistant strain 803, and the chloroquine-resistant strain Dd2 in vitro. We also demonstrate its anti-parasite activity in vivo, using the rodent malaria parasite Plasmodium berghei (ANKA). We show that An-cecB displays potent antimalarial activity and that its mechanism of action may occur through direct killing of the parasite or through interaction with infected red blood cell membranes. Unfortunately, An-cecB was found to be cytotoxic to mammalian cells and had poor antimalarial activity in vivo. However, its truncated peptide An-cecB-1 retained most of its antimalarial activity and avoided its cytotoxicity in vitro. An-cecB-1 also showed better antimalarial activity in vivo. Mosquito-derived AMPs may provide new ideas for the development of antimalarial drugs against drug-resistant parasites, and An-cecB has potential use as a template for antimalarial peptides.

A novel antibacterial approach of Cecropin-B peptide loaded on chitosan nanoparticles against MDR Klebsiella pneumoniae isolates.

Egypt has witnessed the emergence of multidrug-resistant (MDR) Klebsiella pneumoniae, which has posed a serious healthcare challenge. The proper treatment choice for MDR-KP infections is not well determined which renders the problem more complicated, thus making the control of such infections a serious challenge for healthcare professionals. This study aims to encapsulate the cationic antimicrobial peptide; Cecropin-B (Cec-B), to increase its lifetime, drug targeting, and efficacy and study the antimicrobial effect of free and encapsulated recombinant rCec-B peptide on multidrug-resistant K. pneumoniae (MDR-KP) isolates. Fifty isolates were collected from different clinical departments at Theodore Bilharz Research Institute. Minimal inhibitory concentrations (MICs) of rCec-B against MDR-KP isolates were determined by the broth microdilution test. In addition, encapsulation of rCec-B peptide into chitosan nanoparticles and studying its bactericidal effect against MDR-KP isolates were also performed. The relative expression of efflux pump and porin coding genes (ArcrB, TolC, mtdK, and Ompk35) was detected by quantitative PCR in treated MDR-KP bacterial isolates compared to untreated isolates. Out of 60 clinical MDR isolates, 50 were MDR-KP. 60% of the isolates were XDR while 40% were MDR. rCec-B were bactericidal on 21 isolates, then these isolates were subjected to treatment using free nanocapsule in addition to the encapsulated peptide. Free capsules showed a mild cytotoxic effect on MDR-KP at the highest concentration. MIC of encapsulated rCec-B was higher than the free peptide. The expression level of genes encoding efflux and porin (ArcrB, TolC, mtdK, and Ompk35) was downregulated after treatment with encapsulated rCec-B. These findings indicate that encapsulated rCec-B is a promising candidate with potent antibacterial activities against drug-resistant K. pneumoniae.

Fighting Pseudomonas aeruginosa Infections: Antibacterial and Antibiofilm Activity of D-Q53 CecB, a Synthetic Analog of a Silkworm Natural Cecropin B Variant.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium responsible for severe nosocomial infections and is considered a critical pulmonary pathogen for both immunocompromised and cystic fibrosis patients. Planktonic cells of P. aeruginosa possess intrinsic and acquired resistances, inactivating several classes of conventional antibiotics. Additionally, this bacterium can grow, forming biofilms, and complex structures, further hampering the action of multiple antibiotics. Here, we report the biological properties of D-Q53 CecB, an all-D enantiomer of the silkworm natural peptide Q53 CecB. Compared to the L-variant, D-Q53 CecB was resistant to in vitro degradation by humans and P. aeruginosa elastases and showed an enhanced bactericidal activity against P. aeruginosa planktonic bacteria. D-Q53 CecB was thermostable and maintained its antimicrobial activity at high salt concentrations and in the presence of divalent cations or fetal-bovine serum, although at reduced levels. Against different types of human cells, D-Q53 CecB showed cytotoxic phenomena at concentrations several folds higher compared to those active against P. aeruginosa. When L- and D-Q53 CecB were compared for their antibiofilm properties, both peptides were active in inhibiting biofilm formation. However, the D-enantiomer was extremely effective in inducing biofilm degradation, suggesting this peptide as a favorable candidate in an anti-Pseudomonas therapy.

Novel antimicrobial anionic cecropins from the spruce budworm feature a poly-L-aspartic acid C-terminus.

Cecropins form a family of amphipathic α-helical cationic peptides with broad-spectrum antibacterial properties and potent anticancer activity. The emergence of bacteria and cancer cells showing resistance to cationic antimicrobial peptides (CAMPs) has fostered a search for new, more selective and more effective alternatives to CAMPs. With this goal in mind, we looked for cecropin homologs in the genome and transcriptome of the spruce budworm, Choristoneura fumiferana. Not only did we find paralogs of the conventional cationic cecropins (Cfcec+ ), our screening also led to the identification of previously uncharacterized anionic cecropins (Cfcec- ), featuring a poly-l-aspartic acid C-terminus. Comparative peptide analysis indicated that the C-terminal helix of Cfcec- is amphipathic, unlike that of Cfcec+ , which is hydrophobic. Interestingly, molecular dynamics simulations pointed to the lower conformational flexibility of Cfcec- peptides, relative to that of Cfcec+ . Phylogenetic analysis suggests that the evolution of distinct Cfcec+ and Cfcec- peptides may have resulted from an ancient duplication event within the Lepidoptera. Finally, we found that both anionic and cationic cecropins contain a BH3-like motif (G-[KQR]-[HKQNR]-[IV]-[KQR]) that could interact with Bcl-2, a protein involved in apoptosis; this observation is congruent with previous reports indicating that cecropins induce apoptosis. Altogether, our observations suggest that cecropins may provide templates for the development of new anticancer drugs. We also estimated the antibacterial activity of Cfcec-2 and a ∆Cfce-2 peptide as AMPs by testing directly their ability in inhibiting bacterial growth in a disk diffusion assay and their potential for development of novel therapeutics.

Novel Antimicrobial Peptides from a Cecropin-Like Region of Heteroscorpine-1 from Heterometrus laoticus Venom with Membrane Disruption Activity.

The increasing antimicrobial-resistant prevalence has become a severe health problem. It has led to the invention of a new antimicrobial agent such as antimicrobial peptides. Heteroscorpine-1 is an antimicrobial peptide that has the ability to kill many bacterial strains. It consists of 76 amino acid residues with a cecropin-like region in N-terminal and a defensin-like region in the C-terminal. The cecropin-like region from heteroscorpine-1 (CeHS-1) is similar to cecropin B, but it lost its glycine-proline hinge region. The bioinformatics prediction was used to help the designing of mutant peptides. The addition of glycine-proline hinge and positively charged amino acids, the deletion of negatively charged amino acids, and the optimization of the hydrophobicity of the peptide resulted in two mutant peptides, namely, CeHS-1 GP and CeHS-1 GPK. The new mutant peptide showed higher antimicrobial activity than the native peptide without increasing toxicity. The interaction of the peptides with the membrane showed that the peptides were capable of disrupting both the inner and outer bacterial cell membrane. Furthermore, the SEM analysis showed that the peptides created the pore in the bacterial cell membrane resulted in cell membrane disruption. In conclusion, the mutants of CeHS-1 had the potential to develop as novel antimicrobial peptides.

Identification and characteristics of a novel cecropin from the armyworm, Mythimna separata.

BACKGROUND: The recent emergence of antibiotic-resistant strains of bacteria has increased the need to develop effective alternatives to antibiotics. Antimicrobial peptides have been considered as a promising product with several advantages. RESULTS: In this present study, we identified a novel cecropin from the armyworm, Mythimna separata (armyworm cecropin 1, AC-1) by transcriptome sequencing and multi-sequence alignment analysis. The AC-1 precursor comprised 63 amino acid residues, containing a conserved cleavage site of the signal peptide, Ala23-Pro24, while the mature AC-1 included 39 amino acid residues. Chemically synthesized AC-1 exhibited low hemolytic activity against chicken red blood cells, low cytotoxicity against swine testis cells, and effective antimicrobial activity against Salmonella, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa. Its antimicrobial activity against Salmonella remained after incubation for 1 h at 100 °C or in 250 mM NaCl, KCl, or MgCl2 solution, implying good thermal- and salt-resistant stabilities. The bactericidal effect of AC-1 on E. coli gradually increased with increasing AC-1 concentration, resulting in deformation, severe edema, cytolysis, cell membrane damage, and reducing intracellular electron density. Additionally, recombinant AC-1 protein expressed in E. coli was digested by enterokinase protease to obtain AC-1, which showed similar antimicrobial activity against E. coli to chemically synthesized AC-1. CONCLUSIONS: This study identified a novel antimicrobial peptide that may represent a potential alternative to antibiotics.

Cathelicidins enhance protection of channel catfish, Ictalurus punctatus, and channel catfish ♀ × blue catfish, Ictalurus furcatus ♂ hybrid catfish against Edwarsiella ictaluri infection.

Cathelicidins are a class of antimicrobial peptides (AMPs) known to possess rapid and direct antimicrobial activities against a variety of microorganisms. Recently identified cathelicidins derived from alligator and sea snake were found to be more effective in inhibiting microbial growth than other AMPs previously characterized. The ability of these two cathelicidins along with the peptides, cecropin and pleurocidin, to protect channel catfish (Ictalurus punctatus, Rafinesque) and hybrid catfish (I. punctatus ♀ × blue catfish, Ictalurus furcatus, Valenciennes ♂) against Edwardsiella ictaluri, one of the most prevalent pathogens affecting commercial catfish industry, was investigated. Cathelicidin-injected fish (50 µg ml-1  fish-1 ) that were simultaneously challenged with E. ictaluri through bath immersion at a concentration of ~1 × 106 CFU/ml had increased survival rates compared with other peptide treatments and the infected control. Bacterial numbers were also reduced in the liver and kidney of channel catfish and hybrid catfish in the cathelicidin treatments 24 hr post-infection. After 8 days of challenge, serum was collected to determine immune-related parameters such as bactericidal activity, lysozyme, serum protein, albumin and globulin. These immune-related parameters were significantly elevated in fish injected with the two cathelicidins as compared to other peptide treatments. These results indicate that cathelicidins derived from alligator and sea snake can stimulate immunity and enhance the resistance to E. ictaluri infection in channel catfish and hybrid catfish.

Studies on the interactions of neutral Galleria mellonella cecropin D with living bacterial cells.

Cecropins constitute an important family of insect antimicrobial peptides involved in humoral innate immune response. In comparison with the highly basic cecropins A and B, cecropins D are less cationic and more hydrophobic. Interestingly, cecropins D were described only in lepidopteran insects, e.g., the greater wax moth Galleria mellonella. In the present study, interactions of neutral cecropin D (pI 6.47) purified from hemolymph of G. mellonella with living Escherichia coli cells were investigated. Fluorescence lifetime imaging microscopy using fluorescein isothiocyanate-labeled cecropin D revealed very fast binding of the peptide to E. coli cells. Fourier transform infrared spectroscopy analyses showed that G. mellonella cecropin D interacted especially with E. coli LPS and probably other lipid components of the bacterial cell envelope and exhibited an ordering effect with regard to lipid chains. This effect is consistent with the peptide binding mechanism based upon its incorporation into the lipid phase of the cell membrane. The interaction resulted in permeabilization of the bacterial cell membrane. Upon cecropin D binding, the cells lost characteristic surface topography, which was accompanied by altered nanomechanical properties, as revealed by atomic force microscopy. The interaction of the peptide with the bacterial cells also led to intracellular damage, i.e., loss of the cell envelope multilayer structure, formation of membrane vesicles, and enlargement of periplasmic space, which eventually caused death of the bacteria. In summary, it can be concluded that amphipathic character of α-helices, exposure of small positively charged patches on their polar surfaces and hydrophobic interactions are important physicochemical characteristics related to effective binding to E. coli cells and antibacterial activity of neutral G. mellonella cecropin D.

Expression of a recombinant hybrid antimicrobial peptide magainin II-cecropin B in the mycelium of the medicinal fungus Cordyceps militaris and its validation in mice.

BACKGROUND: Antibiotic residues can cause antibiotic resistance in livestock and their food safety-related issues have increased the consumer demand for products lacking these residues. Hence, developing safe and effective antibiotic alternatives is important to the animal feed industry. With their strong antibacterial actions, antimicrobial peptides have potential as antibiotic alternatives. RESULTS: We investigated the antibacterial and immunomodulatory activities and the mechanisms of action of an antimicrobial peptide. The hybrid antimicrobial peptide magainin II-cecropin B (Mag II-CB) gene was transformed into the medicinal Cordyceps militaris fungus. Recombinant Mag II-CB exhibited broad-spectrum antibacterial activity in vitro and its antibacterial and immunomodulatory functions were evaluated in BALB/c mice infected with Escherichia coli (ATCC 25922). Histologically, Mag II-CB ameliorated E. coli-related intestinal damage and maintained the integrity of the intestinal mucosal barrier by up-regulating tight junction proteins (zonula occludens-1, claudin-1 and occludin). The intestinal microbial flora was positively modulated in the Mag II-CB-treated mice infected with E. coli. Mag II-CB treatment also supported immune functioning in the mice by regulating their plasma immunoglobulin and ileum secreted immunoglobulin A levels, by attenuating their pro-inflammatory cytokine levels, and by elevating their anti-inflammatory cytokines levels. Moreover, directly feeding the infected mice with the C. militaris mycelium producing Mag II-CB further proofed the antibacterial and immunomodulatory functions of recombinant hybrid antimicrobial peptide. CONCLUSION: Our findings suggest that both purified recombinant AMPs and C. militaris mycelium producing AMPs display antibacterial and immunomodulatory activities in mice. And C. militaris producing AMPs has the potential to become a substitute to antibiotics as a feed additive for livestock in future.

Synergistic Efficacy of Aedes aegypti Antimicrobial Peptide Cecropin A2 and Tetracycline against Pseudomonas aeruginosa.

The increasing prevalence of antibiotic resistance has created an urgent need for alternative drugs with new mechanisms of action. Antimicrobial peptides (AMPs) are promising candidates that could address the spread of multidrug-resistant bacteria, either alone or in combination with conventional antibiotics. We studied the antimicrobial efficacy and bactericidal mechanism of cecropin A2, a 36-residue α-helical cationic peptide derived from Aedes aegypti cecropin A, focusing on the common pathogen Pseudomonas aeruginosa The peptide showed little hemolytic activity and toxicity toward mammalian cells, and the MICs against most clinical P. aeruginosa isolates were 32 to 64 µg/ml, and its MICs versus other Gram-negative bacteria were 2 to 32 µg/ml. Importantly, cecropin A2 demonstrated synergistic activity against P. aeruginosa when combined with tetracycline, reducing the MICs of both agents by 8-fold. The combination was also effective in vivo in the P. aeruginosa/Galleria mellonella model (P < 0.001). We found that cecropin A2 bound to P. aeruginosa lipopolysaccharides, permeabilized the membrane, and interacted with the bacterial genomic DNA, thus facilitating the translocation of tetracycline into the cytoplasm. In summary, the combination of cecropin A2 and tetracycline demonstrated synergistic antibacterial activity against P. aeruginosain vitro and in vivo, offering an alternative approach for the treatment of P. aeruginosa infections.

Characterization and functional study of a Cecropin-like peptide from the Chinese oak silkworm, Antheraea pernyi.

In present study, a Cecropin-like peptide from Antheraea pernyi (ApCec) was cloned and characterized. The full-length ApCec cDNA encoded a protein with 64 amino acids including a putative 22-amino-acid signal peptide, a 4-amino-acid propeptide, and a 38-amino-acid mature peptide. ApCec gene was highly expressed in Malpighian tubules of A. pernyi after induction for 24 h by Escherichia coli in PBS. Pro-ApCec (including propeptide and mature peptide) and M-ApCec (just mature peptide) were synthesized chemically and analyzed by HPLC and mass spectroscopy. The antibacterial activity of M-ApCec is more potent than pro-ApCec against E. coli K12 or B. subtilus in both minimum inhibitory concentration and inhibition zone assays. Hemolytic assay results showed M-ApCec possessed a low cytotoxicity to mammalian cells. The secondary structure of M-ApCec forms α-helical structure, shown by circular dichroism spectroscopy. Transmission electron microscopy analysis suggested that M-ApCec killed bacteria by disrupting bacterial cell membrane integrity. Our results indicate ApCec may play an important role in defending from pathogenic bacteria in A. pernyi, and it may be as a potential candidate for applications in antibacterial drug development and agriculture.

Fusion expression of cecropin B-like antibacterial peptide in Pichia GS115 and its antibacterial mechanism.

OBJECTIVES: To establish an efficient expression system for a fusion protein of glutathione S-transferase and cecropin B (GST-CB) and to clarify the antibacterial mechanism of CB. RESULTS: The optimal incubation time and methanol concentration for induced expression of CB were 36 h and 1 % w/v, respectively. The yield of GST-CB was 2.2 g/l. The minimum inhibitory concentrations of GST-CB towards Staphylococcus aureus subsp. saprophyticus (ATCC 15305) and Escherichia coli strain CFT073 were 250 and 125 µg/ml, respectively. Notably, mutations of proline 24 (P24) in CB produced a polypeptide without antimicrobial activity. CONCLUSION: The fusion protein GST-CB, which has a broad spectrum antimicrobial activity, can be abundantly expressed in Pichia pastoris GS115, and P24 may be an important amino acid for the antimicrobial activity of GST-CB.

Insect-derived cecropins display activity against Acinetobacter baumannii in a whole-animal high-throughput Caenorhabditis elegans model.

The rise of multidrug-resistant Acinetobacter baumannii and a concomitant decrease in antibiotic treatment options warrants a search for new classes of antibacterial agents. We have found that A. baumannii is pathogenic and lethal to the model host organism Caenorhabditis elegans and have exploited this phenomenon to develop an automated, high-throughput, high-content screening assay in liquid culture that can be used to identify novel antibiotics effective against A. baumannii. The screening assay involves coincubating C. elegans with A. baumannii in 384-well plates containing potential antibacterial compounds. At the end of the incubation period, worms are stained with a dye that stains only dead animals, and images are acquired using automated microscopy and then analyzed using an automated image analysis program. This robust assay yields a Z' factor consistently greater than 0.7. In a pilot experiment to test the efficacy of the assay, we screened a small custom library of synthetic antimicrobial peptides (AMPs) that were synthesized using publicly available sequence data and/or transcriptomic data from immune-challenged insects. We identified cecropin A and 14 other cecropin or cecropin-like peptides that were able to enhance C. elegans survival in the presence of A. baumannii. Interestingly, one particular hit, BR003-cecropin A, a cationic peptide synthesized by the mosquito Aedes aegypti, showed antibiotic activity against a panel of Gram-negative bacteria and exhibited a low MIC (5 µg/ml) against A. baumannii. BR003-cecropin A causes membrane permeability in A. baumannii, which could be the underlying mechanism of its lethality.

Cecropin-P17, an analog of Cecropin B, inhibits human hepatocellular carcinoma cell HepG-2 proliferation via regulation of ROS, Caspase, Bax, and Bcl-2.

Cecropin-P17 is a peptide derived from Cecropin B. In this study, we investigated the effects and relative mechanisms of Cecropin-P17 in a human liver cancer cell line (HepG-2) in vitro and in vivo. A cell viability assay, Annexin V/propidium iodide assay, western blot, flow cytometry, quantitative real-time polymerase chain reaction, and a tumor-xenograft model were applied to elucidate the mechanism exerted by Cecropin-P17 on HepG-2 cells. Cecropin-P17 significantly inhibited the proliferation of HepG-2 cells and demonstrated low cytotoxicity to normal liver cells in vitro. The apoptotic rate of HepG-2 cells was increased after Cecropin-P17 treatment together with increased production of reactive oxygen species. Moreover, Cecropin-P17 stimulated caspase-3, caspase-9, and Bax and inhibited Bcl-2 on both the transcriptional and translational levels. Finally, Cecropin-P17 significantly suppressed tumor growth in a HepG-2-bearing nude mouse model. All of these results indicated that Cecropin-P17 could be a potential agent for the treatment of liver cancer.

Expression and purification of an active cecropin-like recombinant protein against multidrug resistance Escherichia coli.

Lucilin is a 36 residue cecropin antimicrobial peptide identified as a partial genetic sequence in Lucilia sericata maggots. The antimicrobial spectrum and toxicity profile of Lucilin is unknown. We first report the expression of Lucilin as an active recombinant fusion protein with a cysteine protease domain (CPD) tag. The fusion protein, GWLK-Lucilin-CPD-His8, showed maximum overexpression in Escherichia coli BL21 cells after 12h induction with 0.5mM IPTG (isopropyl beta-d-thiogalactoside) and growth conditions were 37 °C and 150 rpm shaking. The fusion protein was expressed as a soluble form and was purified by Ni-IMAC. The purified protein was active against E. coli ATCC 35218 with a MIC of 0.68 µM, and a clinical isolate of E. coli with extended spectrum beta-lactamase (ESBL) with a MIC of 0.8 µM. The recombinant GWLK-Lucilin-CPD-His8 was not toxic against human erythrocytes or Vero cells with a therapeutic index >63. The results suggest that GWLK-Lucilin-CPD-His8 represents a potential candidate for therapy against multidrug resistant Gram-negative bacteria.

CecropinXJ, a silkworm antimicrobial peptide, induces cytoskeleton disruption in esophageal carcinoma cells.

Antimicrobial peptides exist in the non-specific immune system of organism and participate in the innate host defense of each species. CecropinXJ, a cationic antimicrobial peptide, possesses potent anticancer activity and acts preferentially on cancer cells instead of normal cells, but the mechanism of cancer cell death induced by cecropinXJ remains largely unknown. This study was performed to investigate the cytoskeleton-disrupting effects of cecropinXJ on human esophageal carcinoma cell line Eca109 using scanning electron microscopy observation, fluorescence imaging, cell migration and invasion assays, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. The electronic microscope and fluorescence imaging observation suggested that cecropinXJ could result in morphological changes and induce damage to microtubules and actin of Eca109 cells in a dose-dependent manner. The cell migration and invasion assays demonstrated that cecropinXJ could inhibit migration and invasion of tumor cells. Western blot and qRT-PCR analysis showed that there was obvious correlation between microtubule depolymerization and actin polymerization induced by cecropinXJ. Moreover, cecropinXJ might also cause decreased expression of α-actin, ß-actin, γ-actin, α-tubulin, and ß-tubulin genes in concentration- and time-dependent manners. In summary, this study indicates that cecropinXJ triggers cytotoxicity in Eca109 cells through inducing the cytoskeleton destruction and regulating the expression of cytoskeleton proteins. This cecropinXJ-mediated cytoskeleton-destruction effect is instrumental in our understanding of the detailed action of antimicrobial peptides in human cancer cells and cecropinXJ might be a potential therapeutic agent for the treatment of cancer in the future.

Transmission electron microscopic morphological study and flow cytometric viability assessment of Acinetobacter baumannii susceptible to Musca domestica cecropin.

Multidrug-resistant (MDR) Acinetobacter baumannii infections are difficult to treat owing to the extremely limited armamentarium. Expectations about antimicrobial peptides' use as new powerful antibacterial agents have been raised on the basis of their unique mechanism of action. Musca domestica cecropin (Mdc), a novel antimicrobial peptide from the larvae of Housefly (Musca domestica), has potently active against Gram-positive and Gram-negative bacteria standard strain. Here we evaluated the antibacterial activity of Mdc against clinical isolates of MDR-A. baumannii and elucidate the related antibacterial mechanisms. The minimal inhibitory concentration (MIC) of Mdc was 4 µg/mL. Bactericidal kinetics of Mdc revealed rapid killing of A. baumannii (30 min). Flow cytometry using viability stain demonstrated that Mdc causes A. baumannii membrane permeabilization in a concentration- and time-dependent process, which correlates with the bactericidal action. Moreover, transmission electron microscopic (TEM) examination showed that Mdc is capable of disrupting the membrane of bacterial cells, resulting in efflux of essential cytoplasmic components. Overall, Mdc could be a promising antibacterial agent for MDR-A. baumannii infections.

Molecular characterization and antimicrobial activity of cecropin family in Hermetia illucens.

Antimicrobial peptides are potential alternatives to traditional antibiotics in the face of increasing bacterial resistance. Insects possess many antimicrobial peptides and have become a valuable source of novel and highly effective antimicrobial peptides. Hermetia illucens as a resource insect, for example, has the highest number of antimicrobial peptides of any dipteran. However, most antimicrobial peptides, especially cecropin, have not been comprehensively identified and have not been evaluated for their antimicrobial ability. In this study, we analyzed the localization and gene structure of 33 cecropin molecules in the H. illucens genome and evaluated their activity against common human pathogens. The results showed that 32 cecropin molecules were concentrated on 1 chromosome, most with 2 exons. More importantly, most of the cecropins had a good antibacterial effect against Gram-negative bacteria, and were not hemolytic. The minimum inhibitory concentration (MIC) of the cecropin designated H3 against E. coli was 4 µg/mL. The toxicity, killing time kinetics, and anti-biofilm activity of H3 were further investigated and confirmed its antimicrobial ability. Overall, H3 is a potential candidate for the development of new antimicrobials to treat severe infections caused by Gram-negative pathogens such as E. coli.

Expression, purification and characterization of cecropin antibacterial peptide from Bombyx mori in Saccharomyces cerevisiae.

CecropinXJ is a cationic antimicrobial peptide originally isolated from the larvae of Bombyx mori. In this study, an antibacterial peptide gene of cecropinXJ was cloned into the pYES2/CT/α Factor expression vector and expressed in the Saccharomyces cerevisiae INVSc1 strain. Following an induction of recombinant protein expression in yeast for 120 h, the maximum amount of total secreted protein was 1.437 g/L. The percentage of recombinant cecropinXJ was estimated to be 79.45% of the total protein. After purification with Ni-NTA agarose column, recombinant cecropinXJ was noted to exert strong antimicrobial activities against a broad-spectrum of microorganisms, including Gram-negative and Gram-positive bacteria. Its minimal inhibitory concentration (MIC) against Escherichia coli ATCC25922 was 0.81 µM. In addition, transmission electron microscopy (TEM) analysis indicated that the surfaces of the treated pathogens underwent obvious morphological changes compared with the untreated controls, suggesting that this antimicrobial peptide exerts its action by directly disrupting membranes of microorganisms. CecropinXJ had a small hemolytic effect on red blood cells even with a peptide concentration of 200 µM. Thus, cecropinXJ acts selectively on bacterial membranes. Purified recombinant antibacterial peptide, cecropinXJ, retained a high stability against E. coli ATCC25922 over a temperature range from 4 °C to 100 °C and a pH range from pH 2.0 to 12.0. Taken together, this study demonstrates that recombinant cecropinXJ can be produced in large quantities in yeast with genetic engineering methods, and that it has strong and rapid antimicrobial activities against all of microorganisms tested. Our results suggest that cecropinXJ is a potential candidate for therapy.