Mode of action of diptericin A,a bactericidal peptide induced in the hemolymph of Phormia terranovae larvae

Diptericin A is a member of a multigenic family of antibacterial peptides that are synthesized by larvae of Phormia terranovae (Diptera) in response to a bacterial injection or to injury. The 82-residue peptide is active only against a limited range of Gram-negative bacteria. Data presented suggest that the primary action of diptericin A is on the cytoplasmic membrane of growing bacteria.     

The typology and topography of the tarsal chemoreceptors of the blow flies Calliphora vicina Robineau-Desvoidy and Phormia terranovae Robineau-Desvoidy and the Housefly Musca domestica L

A comparative morphological study concerning typology and topography of chemoreceptors on the prothoracic legs of Calliphora vicina, Phormia terranovae and Musca domestica has been carried out. The typological criteria of Grabowski and Dethier ('54) and Hansen and Heumann ('71) were used. A single criterion, the shape of the tip, was used to define the different types of chemoreceptors. A-hairs have a rhombic pore at the side of the tip; B-hairs have an oval pore at the tip apex and D-hairs have a rectangular pore under an undulated, cap-like structure at the hair tip. A-, B-and D-hairs were found in the tarsomeres of Phormia; in Musca and Calliphora only B- and D-hairs were found. An opening and closing mechanism may operate on the pores of the tips of the chemoreceptors. Chemoreceptors were counted and a topographical map was completed, using SEM-techniques. Topographical maps are of value in electrophysiological and behavioural research, where only a limited optical magnification is possible.     

Cytochemical localization of phosphodiesterase: axonal mitochondria and microtubules.

Phosphodiesterase activity has been found associated with axonal microtubules in the sensory nerves of the blowfly, Phormia regina, taste receptors. A specific reaction product for this enzyme was also observed within axonal mitochondria, but not within the mitochondria of the surrounding glial cells. A possible explanation for these data is discussed.     

Localization of adenylate cyclase in the blowfly labellar chemoreceptors

Adenylate cyclase activity has been demonstrated in tissue of the blowfly, Phormia regina, chemoreceptor sensillium and in labellar epidermal cells. Sensillar tissue included sensory neurons innervating the sensillum and associated supportive cells. Epidermal cells which surround the internal labellar apophysis and those below the external cuticles both displayed enzyme activity. Possible explanations for these data are discussed.     

Identification of up-regulated proteins potentially involved in the antagonism mechanism of Bacillus amyloliquefaciens G1

The use of Bacillus probiotics has been demonstrated as a promising method in the biocontrol of bacterial diseases in aquaculture. However, the molecular antibacterial mechanism of Bacillus still remains unclear. In order to explore the antibacterial mechanism of the potential antagonistic Bacillus amyloliquefaciens strain G1, comparative proteomics between B. amyloliquefaciens strain G1 and its non-antagonistic mutant strain was investigated. The 2-dimensional electrophoresis gel maps of their total extracted proteins were described and 42 different proteins were found to be highly expressed in strain G1 in comparison with those in the mutant strain. 35 of these up-regulated proteins were successfully identified using MALDI-TOF-TOF MS and databank analysis, and their biological functions were analyzed through the KEGG database. The increased expression of these proteins suggested that high levels of energy metabolism, biosynthesis and stress resistance could play important roles in strain G1’s antagonism. To our knowledge, this is the first report on the proteins involved in the antagonism mechanism of B. amyloliquefaciens using a proteomic approach and the proteomic data also contribute to a better understanding of the molecular basis for the antagonism of B. amyloliquefaciens.     

Molecular forms of four aldolases from larval and adult stages of the blowfly Phormia regina (Meigen)

Fructose diphosphate ldolases in crude muscle extracts of larval and adult Phormia regina are indistinguishable by polyacrylamide gel electrophoresis (PAGE) in several buffer systems. By contrast, larval and adult fat body aldolases differ in electrophoretic mobility, a difference shown to be due to the inequality of their net charge. Phormia regina larval muscle and fat body isozymes are clearly distinguishable by PAGE, and by pH optima and Michaelis constants.     

Identification of a novel antibacterial protein from hemolymph of freshwater zooplankton Mesocyclops leuckarti

Bacterial infections are the most important problem of health care worldwide. The hemolymph antibacterial proteins of Mesocyclops leuckarti was isolated for the first time and its antibacterial efficacy was evaluated against four different human pathogenic microbes viz., Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia and Shigella flexneri. The antibacterial potential of the antimicrobial proteins of hemolymph samples from plankton cultured in water enriched with Cow Urine Distillate (CUD) was compared with normal ones. The results indicated that the hemolymph proteins were more potential against Gram negative bacteria than Gram positive bacteria. Klebsiella pneumonia was more susceptible to the hemolymph proteins exhibiting a zone of inhibition measuring 27 mm. The supplement of CUD to the culture media further enriched the antibacterial activity of the hemolymph proteins (29 mm). The SDS-PAGE analysis indicated two different types of clear bands representing proteins of 53 kDa and 19 kDa. Overall, this investigation signified that the microcrustaceans have a defence mechanism hemolymph of Mesocyclops leuckarti have a potential agent for novel antibiotics.     

An isotonic saline for the adult blowfly, Phormia regina, and its application to perfusion experiments

Measurements have been made of Na (119 mM), K (5·6 mM), and Ca (2·4 mM) concentrations and total osmotic pressure (487 mOs/kg) of blood of adult Phormia regina. From these data an isotonic saline has been constructed which can be coupled successfully with a newly described total perfusion technique. Using this method it has been shown that adult male Phormia 5 days after emergence recover 50 per cent of their blood trehalose within 12 hr after total perfusion, and are back to normal with reference to this carbohydrate between 24 and 48 hr.     

Antibacterial effect and proteomic analysis of graphene-based silver nanoparticles on a pathogenic bacterium Pseudomonas aeruginosa

Graphene-based silver nanoparticles (Ag NPs–GE) material has been developed and demonstrated antibacterial effect against Escherichia coli and Pseudomonas aeruginosa. In this study, the antibacterial activity and mechanism on P. aeruginosa were investigated. The experiments results showed the minimum bactericidal concentration of Ag NPs–GE to P. aeruginosa is 20 μg/ml. When P. aeruginosa were exposed to 20 μg/ml Ag NPs–GE for 1 h, the cell wall was breakdown. In order to study the mechanism of antibacterial effect of Ag NPs–GE, two-dimensional electrophoresis was carried out to compare the protein expressional profiles of P. aeruginosa exposed to 5 μg/ml Ag NPs–GE or 5 μg/ml AgNO₃ with the untreated bacteria. Identification of differentially expressed protein was performed by MALDI–TOF/TOF MS. The change of proteomic profile induced by Ag NPs–GE was distinct from that induced by AgNO₃. Seven identified proteins were found induced and nine proteins were suppressed by Ag NPs–GE. Five identified proteins were found induced and twenty proteins were suppressed by AgNO₃. In addition, either Ag NPs–GE or AgNO₃ suppressed the expression of eight proteins, amidotransferase, 30S ribosomal protein S6, bifunctional proline dehydrogenase/pyrroline-5-carboxylate dehydrogenase, arginyl-tRNA synthetase, nitroreductase, acetolactate synthase 3, methionyl-tRNA synthetase and periplasmic tail-specific protease. Furthermore, gene ontology analysis and KEGG pathway analysis were used to characterize the functions of those proteins.     

Role of Phote-HrTH (Phormia terraenovae hypertrehalosemic hormone) in modulating the supercontractile muscles of the crop of adult Phormia regina Meigen

Phote-HrTH (Phormia terraenovae hypertrehalosemic hormone) has been demonstrated in the Diptera to be involved in flight metabolism, reproduction, and diapause. Each of these events needs the hormone’s action and requirement for carbohydrates is the common denominator. In Diptera, carbohydrates are taken up during feeding by action of the cibarial pump and are then stored in the crop. Using adult Phormia regina, both a bioassay and electrophysiological recordings show that Phote-HrTH slows down or inhibits the crop lobe muscles (P5) and, at the same time, stimulates the muscles of the pump 4 (P4) involved in pushing fluids out of the crop and up into the midgut for digestion. The EC₅₀ for P4 was in the nanomolar range while the IC₅₀ for P5 was 1.4–75.1 pM. The effect of Phote-HrTH on P4/5 suggests that the peptide is important in coordinating the two pumps, which are involved in moving carbohydrates up into the midgut for digestion. The adult crop organ is an essential storage organ for carbohydrates and now should be considered an important structure capable of delivering nutrients to the midgut for digestion.     

Regulatory mechanisms and the role of calcium and potassium channels controlling supercontractile crop muscles in adult Phormia regina

Bioassays and electrophysiological recordings were conducted in the adult blowfly Phormia regina to provide new insights into the regulatory mechanisms governing the crop filling and emptying processes of the supercontractile crop muscles.     

Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria

Disulfide bonds are important for the stability and function of many secreted proteins. In Gram-negative bacteria, these linkages are catalyzed by thiol-disulfide oxidoreductases (Dsb) in the periplasm. Protein oxidation has been well studied in these organisms, but it has not fully been explored in Gram-positive bacteria, which lack traditional periplasmic compartments. Recent bioinformatics analyses have suggested that the high-GC-content bacteria (i.e., actinobacteria) rely on disulfide-bond-forming pathways. In support of this, Dsb-like proteins have been identified in Mycobacterium tuberculosis, but their functions are not known. Actinomyces oris and Corynebacterium diphtheriae have recently emerged as models to study disulfide bond formation in actinobacteria. In both organisms, disulfide bonds are catalyzed by the membrane-bound oxidoreductase MdbA. Remarkably, unlike known Dsb proteins, MdbA is important for pathogenesis and growth, which makes it a potential target for new antibacterial drugs. This review will discuss disulfide-bond-forming pathways in bacteria, with a special focus on Gram-positive bacteria.     

Screening & analysis of anionic peptides from Foeniculum vulgare Mill by mass spectroscopy

Fennel (Foeniculum vulgare Mill.) member from the family Umbelliferae (Apiaceae) and has been used in Saudi Arabia as an medicine as of the from the tradition. Our previous work with seed extracts of this plant generated DEAE-ion exchange purified proteins that exhibited antibacterial properties. The current study moves this work forward by using 2-D gel separation and MALDI TOF/TOF to identify proteins in this active extract. Fourteen protein spots were excised, digested, and identified. Several putative functions were identified, including: a copper-trans locating ATPase PAA1 chloroplastic-like isoform X1; a cytosolic enolase; a putative pentatricopeptide repeat-containing protein; an NADP-requiring isocitrate dehydrogenase; two proteins annotated as being encoded downstream from Son-like proteins; three probable nuclear proteins 5–1; and four predicted/ unidentified proteins. Future efforts will further characterize their relevant antimicrobial properties with the aim of cloning and high throughput synthesis of the antimicrobial element(s).     

Characterization of six antibacterial response genes from the European corn borer (Ostrinia nubilalis) larval gut and their expression in response to bacterial challenge

Six cDNAs encoding putative antibacterial response proteins were identified and characterized from the larval gut of the European corn borer (Ostrinia nubilalis). These antibacterial response proteins include four peptidoglycan recognition proteins (PGRPs), one β-1,3-glucanase-1 (βglu-1), and one lysozyme. Tissue-specific expression analysis showed that these genes were highly expressed in the midgut, except for lysozyme. Analysis of expression of these genes in different developmental stage showed that they were expressed in larval stages, but little or no detectable expression was found in egg, pupa and adult. When larvae were challenged with Gram-negative bacteria (Enterobacter aerogenes), the expression of all six genes was up-regulated in the fatbodies. However, when larvae were challenged with Gram-positive bacteria (Micrococcus luteus), only PGRP-C and lysozyme genes were up-regulated. This study provides additional insights into the expression of antibacterial response genes in O. nubilalis larvae and helps us better understand the immune defense response in O. nubilalis.     

Cellular and humoral aspects of innate immunity in the shark

Survival of many species depends, to a great extent, on their innate immunity. Innate immunity in the nurse shark (Ginglymostoma cirratum), a primitive elasmobranch, has been shown to consist of components, both humoral and cellular, which are in some respects similar to those found in mammals and other vertebrates. Innate immune factors present in the shark include complement (a complex system of serum proteins) and antibacterial proteins and enzymes, such as lysozyme. Shark complement, although opsonic and lytic in nature, differs from classical mammalian complement in the number of functionally distinct components involved in the activation sequence. Functional and structural analogues of several mammalian complement proteins have been isolated from the shark, and activation of shark serum by lipopolysaccharide or zymosan produces anaphylatoxin-like ligand(s) inducing mammalian smooth muscle contraction and chemotaxis of human leucocytes in vitro. Lysozyme activity has been recovered from shark leucocyte lysates, which also contain antibacterial peptides, distinct from lysozyme. The composition and antibacterial activity of shark leucocyte granules, the putative source of the activity, is under investigation. Cellular aspects of the inflammatory response which is an integral component of innate immunity, are leucocyte phagocytosis and chemotaxis. Both processes are functions of two distinct shark cell types, the granulocyte and the monocyte-macrophage. It should be noted that the innate resilience of the nurse shark is also augmented by a large pool of serum natural antibodies, which can account for as much as 45% of the total serum protein.     

Insect immunity: Galleria mellonella and other lepidoptera have cecropia-P9-like factors active against gram negative bacteria

The humoral immune system of diapausing pupae of Hyalophora cecropia was recently found to contain two small, basic antibacterial proteins, P9A and P9B, which are active against several Gram negative bacteria (Hultmarket al., 1980). These factors could be separated by polyacrylamide gel electrophoresis at pH 4 and afterwards visualized by seeding the gel with viable Escherichia coli. We have now used this technique to demonstrate that immune haemolymph from larvae of Galleria mellonella contains antibacterial proteins acting against Gram negative bacteria. Immunization with either live Enterobacter cloacae or heat killed Pseudomonas aeruginosa induced a potent antibacterial activity against both E. coli and P. aeruginosa. Cation exchange chromatography of immune haemolymph from G. mellonella was used to separate two antibacterial factors (designated G4 and G5) from the lysozyme activity. Several properties were found to be similar for Galleria factors G4 and G5 and the proteins P9A and P9B from Cecropia. It is concluded that the humoral immune systems of H. cecropia and G. mellonella are very similar. Both forms of P9 as well as G4 and G5 were shown to act on P. aeruginosa. Antibacterial factors against E. coli were demonstrated also in six other species of Lepidoptera.     

Analysis of sex chromosome homologies in representatives of the family calliphoridae

The distribution of sequences homologous to the Calliphora erythrocephala Mg. sex chromosome was studied in Protophormia terranovae R-D and Lucilia sp. The chromatin structure was found to be similar in regions containing homologous DNA sequences.     

A potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin

A new potent antibacterial protein, for which we propose the name royalisin, was found in royal jelly of the honeybee Apis mellifera L. and purified to homogeneity for the first time by acid extraction, gel filtration, and reverse-phase high pressure liquid chromatography. The primary structure of royalisin was determined to consist of 51 residues, with three intramolecular disulfide linkages, having a calculated molecular mass of 5523 Da. Royalisin is an amphipathic protein, with the C-terminal half of the molecule being rich in charged amino acids; and it showed extensive sequence homology to two other antibacterial proteins, sapecin from embryonic Sarcophaga peregrina cells and phormicins from Phormia terranovae larvae. Royalisin was found to have potent antibacterial activity against Gram-positive bacteria at low concentrations, but not against Gram-negative bacteria. Royalisin may be involved in a defense system active against bacterial invasion of the honeybee.     

Multiple-site mutations of phage Bp7 endolysin improves its activities against target bacteria

The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7(Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a p ET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated(L99A, M102E) to construct p ET28a-Bp7Δe, with p ET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.