Activation of the Host Immune Response in Hyphantria cunea (Drury) (Lepidoptera: Noctuidae) Induced by Serratia marcescens Bizio

Host–pathogen interactions are essential to our understanding of biological pesticides. Hyphantria cunea (Drury) is an important forest pest worldwide. The immune mechanism of the interaction between H. cunea and Serratia marcescens Bizio (SM1) is unclear. First, transcriptome sequencing and quantitative real-time PCR (qRT-PCR) analysis described the H. cunea immune response to SM1. A total of 234 immune-related differentially expressed genes (DEGs) were found. Many immune regulatory genes in three classical pathways were found. Antimicrobial peptides, including attacin B, cecropin A, gloverin, lebocin and diapausin, are involved in defending against SM1 challenge, and are mainly produced by Toll and immune deficiency (IMD) pathways. Some melanization genes were changed in H. cunea, which suggested that H. cunea melanization was activated by SM1. Furthermore, phagocytosis, autophagolysosome and apoptosis pathways in cellular immunity were activated in H. cunea against SM1. Finally, the expression patterns of 10 immune genes were analyzed systematically by qRT-PCR, and most of the genes were upregulated compared to the control. Our studies provide useful information about the immune response of H. cunea under the stress of SM1, which is important to understand how SM1 affects the immune system of H. cunea and provides new ideas to control H. cunea by using SM1.

Combating Algae Blooms

Microcystis aeruginosa is a common freshwater cyanobacterium that can form toxic algal blooms that harm other species and the environment. This project studied the effects of the antimicrobial peptide Cecropin A on the growth of M. aeruginosa to assess Cecropin A’s effectiveness as a tool to combat algal blooms and limit their environmental impacts. In this study, different concentrations of Cecropin A were tested on M. aeruginosa, the growth of which was then measured using a plate count. Each concentration of Cecropin A tested resulted in a significant decrease in M. aeruginosa growth compared to the control group, indicating the effectiveness of this peptide at inhibiting M. aeruginosa. Because Cecropin A is a peptide, bacteria can be genetically engineered to produce it for anti-algal applications. This study also analyzed the effects of Cecropin A on the non-pathogenic E. coli K12 in order to study development of antibiotic resistance in this bacterium and determine its feasibility for anti-algal applications such as producing or distributing Cecropin A. The effects of Cecropin A were tested on successive generations to determine if this strain of bacterium can build up a resistance to Cecropin A that would make it a suitable candidate to produce large quantities of this peptide. The results over three 24-hour periods of exposure to Cecropin A seem to indicate a development of resistance to Cecropin A by E. coli K12, suggesting that this bacterium may be suitable for production and/or distribution of Cecropin A for anti-bloom control efforts.

Influence of Supplementation of Lactoferrin, Melittin and Cecropin A to Rat Diet on Changes in Faecal Ammonia Concentrations, Short-Chain Fatty Acid Concentrations and Activities of Bacterial Enzymes

We hypothesised that the dietary addition of the bioactive antimicrobial protein lactoferrin (LF) and peptides melittin (MT) or cecropin A (CR) at a dosage of 100 mg/kg to the diet of Wistar rats would result in strong modulatory effects on faecal microbial enzymatic activity, short-chain fatty acid and ammonia concentrations. To date, the changes in bacterial extracellular and intracellular enzymatic activities upon addition of dietary AMPs have not yet been studied. This experiment lasted 15 days; during the first 5 day period, the rats were fed the control diet (S) and diets supplemented with LF, MT or CR. On days 6–15, all rats were fed the control S diet. The faecal fermentation processes were substantially stopped after two days of treatment, on average, in all rats receiving LF and two AMPs. The deepest suppression effect was observed on the last day of treatment (day 5) and persisted through days 5–8. The highest decreases in faecal bacterial β-glucosidase and β-glucuronidase activities as well as in SCFA and ammonia concentrations were observed in the rats fed the CR diet. Only in the CR animals did the mechanism of suppressed microbial fermentation involve diminished enzyme release from bacterial cells to the digesta.

OMN6 a novel bioengineered peptide for the treatment of multidrug resistant Gram negative bacteria

New antimicrobial agents are urgently needed, especially to eliminate multidrug resistant Gram-negative bacteria that stand for most antibiotic-resistant threats. In the following study, we present superior properties of an engineered antimicrobial peptide, OMN6, a 40-amino acid cyclic peptide based on Cecropin A, that presents high efficacy against Gram-negative bacteria with a bactericidal mechanism of action. The target of OMN6 is assumed to be the bacterial membrane in contrast to small molecule-based agents which bind to a specific enzyme or bacterial site. Moreover, OMN6 mechanism of action is effective on Acinetobacter baumannii laboratory strains and clinical isolates, regardless of the bacteria genotype or resistance-phenotype, thus, is by orders-of-magnitude, less likely for mutation-driven development of resistance, recrudescence, or tolerance. OMN6 displays an increase in stability and a significant decrease in proteolytic degradation with full safety margin on erythrocytes and HEK293T cells. Taken together, these results strongly suggest that OMN6 is an efficient, stable, and non-toxic novel antimicrobial agent with the potential to become a therapy for humans.

Inhibition of defensin A and cecropin A responses to dengue virus 1 infection in Aedes aegypti

Introduction: It is essential to determine the interactions between viruses and mosquitoes to diminish dengue viral transmission. These interactions constitute a very complex system of highly regulated pathways known as the innate immune system of the mosquito, which produces antimicrobial peptides that act as effector molecules against bacterial and fungal infections. There is less information about such effects on virus infections.Objective: To determine the expression of two antimicrobial peptide genes, defensin A and cecropin A, in Aedes aegypti mosquitoes infected with DENV-1.Materials and methods: We used the F1 generation of mosquitoes orally infected with DENV-1 and real-time PCR analysis to determine whether the defensin A and cecropin A genes played a role in controlling DENV-1 replication in Ae. aegypti. As a reference, we conducted similar experiments with the bacteria Escherichia coli.Results: Basal levels of defensin A and cecropin A mRNA were expressed in uninfected mosquitoes at different times post-blood feeding. The infected mosquitoes experienced reduced expression of these mRNA by at least eightfold when compared to uninfected control mosquitoes at all times post-infection. In contrast with the behavior of DENV-1, results showed that bacterial infection produced up-regulation of defensin and cecropin genes; however, the induction of transcripts occurred at later times (15 days).Conclusion: DENV-1 virus inhibited the expression of defensin A and cecropin A genes in a wild Ae. aegypti population from Venezuela.

Exploring Galleria mellonella larval model to evaluate antibacterial efficacy of Cecropin A (1-7)- Melittin against multi-drug resistant enteroaggregative Escherichia coli

High throughput in vivo laboratory models is need for screening and identification of effective therapeutic agents to overcome microbial drug-resistance. This study was undertaken to evaluate in vivo antimicrobial efficacy of short-chain antimicrobial peptide- Cecropin A (1–7)-Melittin (CAMA) against three multi- drug resistant enteroaggregative Escherichia coli (MDR-EAEC) field isolates in a Galleria mellonella larval model. The minimum inhibitory concentration (MIC; 2.0 mg/L) and minimum bactericidal concentration (MBC; 4.0 mg/L) of CAMA were determined by microdilution assay. CAMA was found to be stable at high temperatures, physiological concentration of cationic salts and proteases; safe with sheep erythrocytes, secondary cell lines and commensal lactobacilli at lower MICs; and exhibited membrane permeabilisation. In vitro time-kill assay revealed concentration- and time-dependent clearance of MDR-EAEC in CAMA-treated groups at 30 min. CAMA- treated G. mellonella larvae exhibited an increased survival rate, reduced MDR-EAEC counts, immunomodulatory effect and proved non-toxic which concurred with histopathological findings. CAMA exhibited either an equal or better efficacy than the tested antibiotic control, meropenem. This study highlights the possibility of G. mellonella larvae as an excellent in vivo model for investigating the host-pathogen interaction, including the efficacy of antimicrobials against MDR-EAEC strains.

Design, Synthesis and Anticancer Activity of Site Specific Short Chain Cationic Peptide

Background: In spite of current progress in treatment methods, cancer is a major source of morbidity and death rate all over the world. Traditional chemotherapeutic agents aim to divide cancerous cells, are often associated with deleterious side effects to healthy cells and tissues. Host defense peptides Cecropin A and B obtained from insects are capable to lyses various types of human cancer cells at peptide concentrations which are not fatal to normal eukaryotic cells. Methods: In the present work we have designed short chain α-helical linear and cyclic peptide from cecropin A having same cationic charge, hydrophobicity and helicity. Synthesis of designed novel short chain linear (10) and cyclic compound (12) was accomplished by using solution phase method. All the coupling reactions were carried out by using dicyclohexylcarbodiimide (DCC) as the coupling reagent at room temperature in the presence of N-methylmorpholine (NMM) as the base. The Structure of newly synthesized peptidse were elucidated by 1H-NMR, 13C-NMR, FT-IR, FABMS and elemental analysis data.Cytotoxicity of synthesized compound was tested against Dalton’s Lymphoma Ascites (DLA), Ehrlich’s Ascites Carcinoma (EAC) and MCF-7 cell lines by using MTT assay and 5-FU as reference compound. Results: From biological assessment,it was found that short chain cyclicpeptide12 showed high level of cytotoxic activity against DLA and EAC cell lines. Conclusion: By utilizing a structure-based rational approach to anticancer peptide design from cecropin A, we were able to develop short chain linear and cyclic peptides having same charge, hydrophobicity and with improved activity. Systematically removing amino acids, we were able to retaining peptide charge and hydrophobicity/hydrophilicity in linear and cyclic peptide which results to optimize the anticancer activity against DLA and EAC cell lines.

Dichloroacetate-induced metabolic reprogramming improves lifespan in a Drosophila model of surviving sepsis

Sepsis is the leading cause of death in hospitalized patients and beyond the hospital stay and these long-term sequelae are due in part to unresolved inflammation. Metabolic shift from oxidative phosphorylation to aerobic glycolysis links metabolism to inflammation and such a shift is commonly observed in sepsis under normoxic conditions. By shifting the metabolic state from aerobic glycolysis to oxidative phosphorylation, we hypothesized it would reverse unresolved inflammation and subsequently improve outcome. We propose a shift from aerobic glycolysis to oxidative phosphorylation as a sepsis therapy by targeting the pathways involved in the conversion of pyruvate into acetyl-CoA via pyruvate dehydrogenase (PDH). Chemical manipulation of PDH using dichloroacetic acid (DCA) will promote oxidative phosphorylation over glycolysis and decrease inflammation. We tested our hypothesis in a Drosophila melanogaster model of surviving sepsis infected with Staphylococcus aureus. Drosophila were divided into 3 groups: unmanipulated, sham and sepsis survivors, all treated with linezolid; each group was either treated or not with DCA for one week following sepsis. We followed lifespan, measured gene expression of Toll, defensin, cecropin A, and drosomycin, and levels of lactate, pyruvate, acetyl-CoA as well as TCA metabolites. In our model, metabolic effects of sepsis are modified by DCA with normalized lactate, TCA metabolites, and was associated with improved lifespan of sepsis survivors, yet had no lifespan effects on unmanipulated and sham flies. While Drosomycin and cecropin A expression increased in sepsis survivors, DCA treatment decreased both and selectively increased defensin.