Pathogenicity of microsclerotia from Metarhizium robertsii against Aedes aegypti larvae and antimicrobial peptides expression by mosquitoes during fungal-host interaction.

Aedes aegypti is a vector of various disease-causing arboviruses. Chemical insecticide-based methods for mosquito control have increased resistance in different parts of the world. Thus, alternative control agents such as the entomopathogenic fungi are excellent candidates to control mosquitoes as part of an ecofriendly strategy. There is evidence of the potential of entomopathogenic fungal conidia and blastospores for biological control of eggs, larval and adult stages, as well as the pathogenicity of fungal microsclerotia against adults and eggs. However, there are no studies on the pathogenicity of microsclerotia against either aquatic insects or insects that develop part of their life cycle in the water, such as the A. aegypti larvae. In this study, we assayed the production of microsclerotia and their pathogenicity against A. aegypti larvae of two isolates of Metarhizium robertsii, i.e., CEP 423 isolated in La Plata, Argentina, and the model ARSEF 2575. Both isolates significantly reduced the survival of A. aegypti exposed to their microsclerotia. The fungus-larva interaction resulted in a delayed response in the host. This was evidenced by the expression of some humoral immune system genes such as defensins and cecropin on the 9th day post-infection, when the fungal infection was consolidated as a successful process that culminates in larvae mortality. In conclusion, M. robertsii microsclerotia are promising propagules to be applied as biological control agents against mosquitoes since they produce pathogenic conidia against A. aegypti larvae.

New Insect Host Defense Peptides (HDP) From Dung Beetle (Coleoptera: Scarabaeidae) Transcriptomes.

The Coleoptera Scarabaeidae family is one of the most diverse groups of insects on the planet, which live in complex microbiological environments. Their immune systems have evolved diverse families of Host Defense Peptides (HDP) with strong antimicrobial and immunomodulatory activities. However, there are several peptide sequences that await discovery in this group of organisms. This would pave the way to identify molecules with promising therapeutic potential. This work retrieved two sources of information: 1) De-novo transcriptomic data from two species of neotropical Scarabaeidae (Dichotomius satanas and Ontophagus curvicornis); 2) Sequence data deposited in available databases. A Blast-based search was conducted against the transcriptomes with a subset of sequences representative of the HDP. This work reports 155 novel HDP sequences identified in nine transcriptomes from seven species of Coleoptera: D. satanas (n = 76; 49.03%), O. curvicornis (n = 23; 14.83%), (Trypoxylus dichotomus) (n = 18; 11.61%), (Onthophagus nigriventris) (n = 10; 6.45%), (Heterochelus sp) (n = 6; 3.87%), (Oxysternon conspicillatum) (n = 18; 11.61%), and (Popillia japonica) (n = 4; 2.58%). These sequences were identified based on similarity to known HDP insect families. New members of defensins (n = 58; 37.42%), cecropins (n = 18; 11.61%), attancins (n = 41; 26.45%), and coleoptericins (n = 38; 24.52%) were described based on their physicochemical and structural characteristics, as well as their sequence relationship to other insect HDPs. Therefore, the Scarabaeidae family is a complex and rich group of insects with a great diversity of antimicrobial peptides with potential antimicrobial activity.

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis.

BACKGROUND: Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. METHODS: AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. RESULTS: AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. CONCLUSIONS: The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.

Expression of antimicrobial peptide Cecropin P1 in Saccharomyces cerevisiae and its antibacterial and antiviral activity in vitro

BACKGROUND: Cecropin P1, acting as an antimicrobial, has a broad-spectrum antibacterial activity with some antiviral and antifungal properties. It is a promising natural alternative to antibiotics which is originally isolated from the pig intestinal parasitic nematode Ascaris suum. Many studies have shown that Cecropin P1 is helpful for the prevention or treatment of clinical diseases. Therefore, it is very necessary to establish a safe, nontoxic, and efficient expression method of Cecropin P1. RESULTS: The results indicated that the recombinant protein was about 5.5 kDa showed by Tricine­SDS­ PAGE and Western blot. And Cecropin P1 was efficiently secreted and expressed after 12 h of induction, with an increasing yield over the course of the induction. Its maximum concentration was 7.83 mg/L after concentration and purification. In addition, in vitro experiments demonstrated that Cecropin P1 not only exerted a strong inhibitory effect on Escherichia coli, Salmonella sp., Shigella sp., and Pasteurella sp., but also displayed an antiviral activity against PRRSV NADC30-Like strain. CONCLUSIONS: Collectively, the strategy of expressing Cecropin P1 in Saccharomyces cerevisiae is harmless, efficient, and safe for cells. In addition, the expressed Cecropin P1 has antiviral and antibacterial properties concurrently.

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis

Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. Results AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. Conclusions The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.(AU)

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis

Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. Results AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. Conclusions The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.(AU)

A Novel Cecropin D-Derived Short Cationic Antimicrobial Peptide Exhibits Antibacterial Activity Against Wild-Type and Multidrug-Resistant Strains of Klebsiella pneumoniae and Pseudomonas aeruginosa.

Infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa and Klebsiella pneumoniae are a serious worldwide public health concern due to the ineffectiveness of empirical antibiotic therapy. Therefore, research and the development of new antibiotic alternatives are urgently needed to control these bacteria. The use of cationic antimicrobial peptides (CAMPs) is a promising candidate alternative therapeutic strategy to antibiotics because they exhibit antibacterial activity against both antibiotic susceptible and MDR strains. In this study, we aimed to investigate the in vitro antibacterial effect of a short synthetic CAMP derived from the ΔM2 analog of Cec D-like (CAMP-CecD) against clinical isolates of K pneumoniae (n = 30) and P aeruginosa (n = 30), as well as its hemolytic activity. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of CAMP-CecD against wild-type and MDR strains were determined by the broth microdilution test. In addition, an in silico molecular dynamic simulation was performed to predict the interaction between CAMP-CecD and membrane models of K pneumoniae and P aeruginosa. The results revealed a bactericidal effect of CAMP-CecD against both wild-type and resistant strains, but MDR P aeruginosa showed higher susceptibility to this peptide with MIC values between 32 and >256 µg/mL. CAMP-CecD showed higher stability in the P aeruginosa membrane model compared with the K pneumoniae model due to the greater number of noncovalent interactions with phospholipid 1-Palmitoyl-2-oleyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG). This may be related to the boosted effectiveness of the peptide against P aeruginosa clinical isolates. Given the antibacterial activity of CAMP-CecD against wild-type and MDR clinical isolates of P aeruginosa and K pneumoniae and its nonhemolytic effects on human erythrocytes, CAMP-CecD may be a promising alternative to conventional antibiotics.

The plant-based chimeric antimicrobial protein SlP14a-PPC20 protects tomato against bacterial wilt disease caused by Ralstonia solanacearum.

Cecropin-B (CecB) is a peptide with well-established antimicrobial properties against different phytopathogenic bacteria. Despite modest action against Ralstonia solanacearum, its animal source limits the acceptance in transgenic applications. To overcome this, we selected eight alpha-helical (AH) cationic peptides derived from plant protein sequences and investigated their antimicrobial properties against R. solanacearum. Remarkably, PPC20 (a linear AH-peptide present in phosphoenolpyruvate carboxylase) has a three-fold lower lethal dose on R. solanacearum than CecB and lower toxicity to human intestinal epithelial cells. Linking PPC20 to SlP14a (part of a pathogenesis-related protein) established an apoplast-targeted protein providing a means of secreting and stabilizing the antimicrobial peptide in the plant compartment colonized by the pathogen. SlP14a is also a potential antimicrobial, homologous to a human elastase which likely targets outer membrane proteins in Gram-negative bacteria. Recombinant SlP14a-PPC20 showed antibacterial activity against R. solanacearum in vitro, making it a promising candidate for plant protection. This was confirmed with genetically-modified tomato plants engineered to express SlP14a-PPC20, in which bacterial populations in stems were reduced compared to inoculated wild-type control plants. Disease symptoms were also markedly less severe in SlP14a-PPC20-expressing plants, demonstrating a viable strategy to improve resistance against bacterial wilt in tomato.

Bactericidal Activity of a Cationic Peptide on Neisseria meningitidis.

BACKGROUND: The increasing prevalence of antibiotic resistant bacteria has raised an urgent need for substitute remedies. Antimicrobial peptides (AMPs) are considered promising candidates to address infections by multidrug-resistant bacteria through new mechanisms of action that require a careful evaluation of their performance. OBJECTIVE: Identification of effective AMPs against Neisseria meningitidis, which represents a pathogen of great public health importance worldwide that is intrinsically resistant to some AMPs, such as polymyxin B. METHODS: A cationic 11-residue peptide (KLKLLLLLKLK), referred to as poly-Leu, was synthesized and its antimeningococcal activity was compared to cecropin A and poly-P (KLKPPPPPKLK) through a variety of assays. Flow cytometry was used to measure propidium iodide uptake by N. meningitidis serotype B as an indicator of the effectiveness of each peptide when added to cultures at different concentrations. RESULTS: The addition of the poly-Leu peptide led to a 90.3% uptake of the dye with an EC50 value of 7.9 µg mL-1. In contrast, uptake was <10% in cells grown in the absence of peptides or with an identical concentration of cecropin and poly-Pro peptides. Electron micrographs indicated that the integrity of the cellular wall and internal membrane was impacted in relation to peptide concentrations, which was confirmed by the detection of released alkaline phosphatase from the periplasmic space due to disruption of the external membrane. CONCLUSION: Poly-Leu peptide demonstrated definitive antimicrobial activity against N. meningitidis.

Co-supplementation of dietary seaweed powder and antibacterial peptides improves broiler growth performance and immune function

The feasibility of Laminaria japonica powder (LJP) combined with cecropin as a dietary supplement to enhance broiler growth performance and immune function was evaluated in this study. In total, 648 one-day-old Arbor Acres broiler chicks were randomly distributed into nine numerically-equal treatment groups: T1 (control group; fed a basal diet); T2 (fed the basal diet supplemented with 1% LJP);T3 (fed the basal diet supplemented with 300mg cecropin/kg); and T4,T5,T6,T7,T8 and T9, individually fed with the dietary supplemented with varying levels of LJP and cecropin). Compared with the control, dietary of LJP or cecropin supplementation slightly improved feed conversion ratio (FCR). However, the dietary supplementation of LJP combined with cecropin significantly improved broiler growth performance during the periods of 1-21,21-42, and 1-42 days (p<0.05).The dietary supplementation of 3% LJP combined with 300 mg/kg cecropin significantly increased FCR, and serum Newcastle disease antibody titers and lymphocyte numbers during the periods of 1-21, 21-42, and 1-42 days (p<0.05). Cecal microorganisms were cultivated and the number of Escherichia coli and Lactobacillus colonies were counted. The dietary supplementation of LJP combined with cecropin remarkably inhibited E. coli growth and increased Lactobacillus growth. The results of this study demonstrate the feasibility of using LJP and cecropin as feed supplement for improving the growth performance and enhancing the immune function of broilers.(AU)

Co-supplementation of dietary seaweed powder and antibacterial peptides improves broiler growth performance and immune function

The feasibility of Laminaria japonica powder (LJP) combined with cecropin as a dietary supplement to enhance broiler growth performance and immune function was evaluated in this study. In total, 648 one-day-old Arbor Acres broiler chicks were randomly distributed into nine numerically-equal treatment groups: T1 (control group; fed a basal diet); T2 (fed the basal diet supplemented with 1% LJP);T3 (fed the basal diet supplemented with 300mg cecropin/kg); and T4,T5,T6,T7,T8 and T9, individually fed with the dietary supplemented with varying levels of LJP and cecropin). Compared with the control, dietary of LJP or cecropin supplementation slightly improved feed conversion ratio (FCR). However, the dietary supplementation of LJP combined with cecropin significantly improved broiler growth performance during the periods of 1-21,21-42, and 1-42 days (p<0.05).The dietary supplementation of 3% LJP combined with 300 mg/kg cecropin significantly increased FCR, and serum Newcastle disease antibody titers and lymphocyte numbers during the periods of 1-21, 21-42, and 1-42 days (p<0.05). Cecal microorganisms were cultivated and the number of Escherichia coli and Lactobacillus colonies were counted. The dietary supplementation of LJP combined with cecropin remarkably inhibited E. coli growth and increased Lactobacillus growth. The results of this study demonstrate the feasibility of using LJP and cecropin as feed supplement for improving the growth performance and enhancing the immune function of broilers.

Mitochondrial inactivation by Anopheles albimanus cecropin 3: molecular mechanisms.

Cecropin 3 (Ccrp3) is an antimicrobial peptide from Anopheles albimanus, which is expressed during Plasmodium berghei infection. Here, we report that synthetic Ccrp3, aside from antibacterial activity, also shows cardio regulatory functions. In rats, Ccrp3 significantly diminishes blood pressure as well as the heartbeat frequency at nanomolar concentration. Ccrp3 affect the rat cardiac muscle mitochondria, inducing uncoupling of oxidative phosphorylation, oxygen consumption and transport of Ca(2). Ccrp3 treatment of the mitochondria causes mitochondrial damage promoting oxidative stress, causing overproduction of reactive oxygen species (ROS) and inhibition of superoxide dismutase. At nM concentration, Ccrp3 inhibits superoxide dismutase activity through direct interaction, diminishing by its enzymatic activity. Ccrp3 induces the release of the pro-apoptotic marker Bax from the mitochondria. Altogether, these results suggest that Ccrp3 pro-oxidative activity on cardiac muscle mitochondria could be responsible for triggering the heartbeat frequency and blood pressure lowering observed the Ccrp3 injected rats.

Intein-mediated expression of cecropin in escherichia coli

Different strategies have been used to overcome the difficulties to produce antimicrobial peptides. Here we used Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT-System, New England Biolabs) for the expression of the antimicrobial peptide cecropin to reduce its sensitivity to intracellular proteases and use its inducible self-cleaving capability to remove the carrier. Cecropin was cloned into suitable expression vector pTYB11, and expression induced by IPTG in Escherichia coli ER2566. The use of 22ºC induction allowed the expression of cecropin with its intein carrier in soluble form. Cell extracts were purified by chitin affinity chromatography and intein-mediated splicing of the target protein was achieved by thiol addition, obtaining a final yield of 2.5 mg cecropin/l. Cecropin cleaved from the intein had its proper biologically active form, showing a micromolar antimicrobial activity against Vibrio ordalii, Vibrio alginolyticus and Escherichia coli.