Immune Hemolymph Research Articles

Isolation and structure of cecropins, inducible antibacterial peptides, from the silkworm, Bombyx mori

1. 1. Cecropins, inducible antibacterial peptides, were purified by simple two step chromatography from immunized larval hemolymph of the silkworm, Bombyx mori. 2. 2. The cecropins were further separated into four major and two minor forms by reverse-phase HPLC. 3. 3. The four major cecropins were sequenced and divided into two types, A and B, whose structures were quite similar to cecropins A and B of Hyalophora cecropia. 4. 4. Three of them contained an unusual amino acid, δ-hydroxylysine. 5. 5. No remarkable difference in antibacterial activity against E. coli was detected among these cecropins.

Mass spectrometric identification of peptides present in immunized and parasitised hemolymph from honeybees without purification

We propose that a substance, identified using mass spectrometry, present in the hemolymph of the honeybee (Apis mellifera) as a result of stimulating the insect immune system corresponds with Apidaecin I, an antibacterial peptide recently described. The plasma desorption mass spectra indicate that several other higher molecule mass substances are synthesised as a result of bacterial or parasite infection.

Assessment of antibiotic potentials of insect antibacterial factors

Immune haemolymphs from the giant silkmoth, Hyalophora cecropia and, to a lesser extent, from the tsetse, Glossina morsitans morsitans possess antibacterial activities against several species of bacteria known to be pathogenic to man, animals and poultry, as demonstrated by in vitro bacterial growth inhibition assays. Cecropia immune haemolymph possesses a broader antibacterial spectrum and was found to be active against several Gram-positive and Gramnegative pathogenic bacteria. Mice injected with pre-determined lethal doses of Enterobacter cloacae, Klebsiella pneumoniae and Corynebacterium pseudotuberculosis and then treated with single intraperitoneal or subcutaneous injections of Cecropia immune haemolymph had much lower mortality than untreated controls. A possibility of developing a broad-spectrum antibiotic modelled on insect immune factors is discussed.

The hemolytic activity of Galleria mellonella hemolymph

A hemolytic activity was identified in the hemolymph of normal and immune Galleria mellonella larvae. The hemolysin was active against sheep, human, guinea pig, and rabbit erythrocytes. Hemolysis occurred in the presence of 0.04M EDTA. Vaccination of the larvae with formalized Pseudomonas aeruginosa increased the hemolytic activity. The increase, and subsequent decline of this activity paralleled the pattern of induced in vivo antibacterial activity that is characteristic of the insect's immune response. The hemolytic activity was distinct from induced phospholipase A-like and phospholipase C-like activities that were detected in immune hemolymph and which were inhibited by EDTA. The hemolytically active material (HAM) was partially purified (apparent molecular weight range 69,000 to 75,000) and was found not to be antibacterial for P. aeruginosa. The physiological role of the HAM is as yet unknown. It is possible that it may act together with other hemolymph components to produce an immune state.

The humoral defense system in tsetse: Differences in response due to age, sex and antigen types

Inoculation of live Escherichia coli into tsetse flies, Glossina morsitans morsitans , stimulated a higher antibacterial immune response in females than in males. It increased with age in females from emergence to approximately 2 weeks and thereafter declined. In males, there was also a significant decrease in immune response with aging. Inoculation of killed bacteria failed to stimulate antibacterial activity but stimulated a lysozyme response which was weaker than that stimulated by live bacteria. No antibacterial activity was present in the hemolymph of larvae from immunized pregnant tsetse. Inoculation of live Trypanosoma brucei brucei and T . congolense failed to induce production of antibacterial activity and lysozyme. Furthermore, tsetse inoculated with or naturally infected with T . b . brucei and T . congolense failed to show any evidence of immunosuppression when challenged with live E . coli . Various species of live bacteria stimulated different levels of antibacterial factors, with Enterobacter cloacae stimulating the highest level of antibacterial activity and E . coli the highest level of lysozyme. Saline in which certain species of bacteria and T . b . brucei were incubated inactivated tsetse immune hemolymph.

Insect immunity: Induction of cecropin and attacin-like antibacterial factors in the haemolymph of Glossina morsitans morsitans

Injections of live Escherichia coli into adult tsetse flies, Glossina morsitans morsitans induced an antibacterial activity in the haemolymph after a lag period of 6–18 hr. Peak activity occurred after 24–72 hr with a dose of 10 4 bacteria/fly. Acidic electrophoresis of immune haemolymph from G. m. morsitans followed by an antibacterial assay on the gel revealed the presence of cecropin- and attacin-like factors. The induction of antibacterial activity in tsetse was completely blocked by injection of cycloheximide, a known inhibitor of protein synthesis in eukaryotic organisms. Purified InA from Bacillus thuringiensis, a proteolytic enzyme with specificity for cecropins and attacins in haemolymph, inactivated the antibacterial activity in tesetse immune haemolymph. When tested against 10 different bacterial species, the spectrum was the same for the antibacterial activity in immune haemolymph from tsetse and Cecropia.

Bacteriophage-resistant mutants of Bacillus thuringiensis with decreased virulence in pupae of Hyalophora cecropia.

Starting from a crystal-negative parental strain of Bacillus thuringiensis, we isolated certain bacteriophage-resistant mutants which showed decreased virulence in pupae of the cecropia moth (Hyalophora cecropia). These strains (class I mutants) were highly pleiotropic and showed resistance to seven or eight different phages, sensitivity to methicillin, and loss of flagella. They were also more sensitive to cecropia immune hemolymph in vitro. In addition, the export of at least three proteins was reduced. Revertants (class II mutants) were sensitive to phages, virulent, and resistant to penicillin derivatives. One class II mutant was a complete revertant in all properties examined. The other class II mutant was an incomplete revertant still susceptible to immune hemolymph and with repressed export of proteins. Virulence was not coupled to phage resistance as such or to lack of flagella because other mutants affected in these properties were virulent. Other factors which could be excluded as causes of virulence were production of extracellular protease and hemolysin.

Antibacterial hemolymph proteins of Manduca sexta.

Exclusion column fractionated immune hemolymph of the M. sexta larva contains five peaks of anti-E. coli activity with molecular weights of greater than 140 kD and approximately 91, 54, 14 and 4 kD, plus one peak of lysozyme activity with a molecular weight of 17 kD. Purification of the 54 kD peak showed that this peak consists of the previously described M18 proteins which have monomeric weights of approximately 20 kD and had antibacterial activity against certain gram negative bacteria. Approximately 80% of the total hemolymph antibacterial activity was detected in the 14 and 4 kD peaks. These proteins, which kill both gram negative and gram positive bacteria, appeared to be directly analogous to the cecropins of H. cecropia. The greater than 140 and 91 kD peaks constituted only a minor part of the total antibacterial activity.

Preliminary characterization of the inducible humoral factor in the American cockroach (Periplaneta americana)

Previous studies have defined an inducible humoral immune response in the American cockroach that demonstrates specificity and memory. In addition, the response is affected by the age and gender of the animal. Preliminary studies have been initiated to determine which of the protein components found in immune hemolymph might be responsible for mediating this activity. Comparisons between hemolymphs from immune and control animals, using SDS-PAGE gel analysis, revealed that there was a unique band generated in immune animals with an approximate MW of 162K. In addition, several other bands, with MWs of 220K, 115K, 102K, 95K, 80K and 77K, appear to be enhanced in immune hemolymph. The 240K, 220K and 115K bands were found only in females, and so have been temporarily excluded from further study because they were genderdependent. When hemolymph samples were absorbed with HBT toxoid, the 162K band was no longer present in immune hemolymph, and the 115K, 102K, 95K and the 80/77K doublet were all greatly reduced as compared to controls. Analysis of supernatants from 24 hour cultures of cells from immune and BTS-injected animals, revealed that immune cells secreted the 162K, 102K, 95K and 80K bands. The 115K band was barely detectable, and the 77K band appeared to be missing. Thus, these studies have provided some valuable initial information that reduces the number of proteins that should be considered for further study as mediators of the roach inducible humoral response.

Insect immunity. The primary structure of the antibacterial protein attacin F and its relation to two native attacins from Hyalophora cecropia

The attacins are antibacterial proteins present in the hemolymph of the pupae of the silk moth Hyalophora cecropia after bacterial infection. We present the primary structure of one attacin, the F form. We show that this protein is derived by proteolysis from the native protein, attacin E. Using a method for rapid purification from the hemolymph of immunized pupae of the neutral attacin E and a basic attacin, both proteins were found in freshly collected immune hemolymph. We conclude that they are the native products of two attacin genes, the existence of which was inferred from the isolation of two cDNA clones as described in the accompanying paper. The two proteins, which differed in their pIs (7 and 9), were found to have similar mol. wts. (20 000) and closely related primary structures, displaying a total of 40 amino acid substitutions, 12 of which were of a non-conservative nature.

Characterization of inhibitor A, a protease from Bacillus thuringiensis which degrades attacins and cecropins, two classes of antibacterial proteins in insects.

The insect pathogen Bacillus thuringiensis produces an exoprotease, inhibitor A, at the beginning of the stationary growth phase. In vitro, the enzyme selectively destroys cecropins and attacins, two antibacterial proteins found in immune hemolymph from Hyalophora cecropia. The specificity of this enzyme was investigated using cecropin A(1-33) and HPLC for separation and characterization of the fragments obtained. A maximum of 12 different peptides were produced and their positions in the known sequence of cecropin A(1-33) were deduced from their amino acid compositions. The enzyme did not show a stringent requirement for a specific amino acid sequence at the cleavage site but prefers a hydrophobic residue on the C-terminal side. The specificity of the enzyme is explained in terms of the open structure of the cecropins and a pronounced inability of inhibitor A to attack globular proteins.

Transfer of immunity against [formula omitted] P11-1 in [formula omitted] larvae

Larvae of Galleria mellonella may be immunized against Pseudomonas aeruginosa by the transfer of whole hemolymph, cell-free hemolymph or hemocytes from insects previously immunized with the lipopolysaccharide of the homologous organisms. Whole immune hemolymph injected as early as 3 hours after vaccination confers protection which persists as long as 40 hours. Hemocytes alone confer good protection when transferred as early as 30 minutes after, and up to about 4 hours after immunization. Transferred protection does not appear to be due to excess immunogen in the hemolymph.

Insect Pathogenic Properties Of Serratia Marcescens. Passive And Active Resistance To Insect Immunity Studied With Protease-Deficient And Phage-Resistant Mutants

Many insects have a cell-free immune system in which small basic proteins called cecropins are the main defence against Gram-negative bacteria. We have earlier shown that an insect pathogenic strain of Serratia marcescens was resistant to insect immunity and that certain mutants resistant to phage ɸJ become sensitive to cecropins. We have found that protease-deficient mutants with and without resistance to ɸJ appear to be deficient in the mechanism of protease induction. Three different protease fractions exist and for two of the enzymes we describe a partial purification and characterization. The proteases show pronounced autodegradation which increases with the purity. Both enzymes are only partly affected by EDTA and they are highly toxic to Drosophila melanogaster. All three enzymes destroy cecropins in immune haemolymph from Cecropia pupae. However, in vivo experiments with different mutants indicate that in Serratia, passive resistance to insect immunity is more important for virulence in Drosophila than the production of proteases which can destroy cecropins.

Effects of Hemolymph from Immune and Non-Immune Larvae of GalleriaMellonella on the Ultra-Structure of PseudomonasAeruginosa

The ultrastructure of Pseudomonasaeruginosa, a pathogen of Galleriamellonella is rapidly altered after invitro exposure to the hemolymph of vaccinated larvae. The bacteria were treated with normal and immune hemolymph for periods of time ranging from 7 to 28 min at 28°C. In contrast to the apparent non-damaging effects of normal. hemolymph, the immune hemolymph caused progressive damage to the cells within 7 min. The initial attack was directed towards the cell wall. Complete degradation was observed after 14 to 28 min exposure to the immune hemolymph.

Interactions between insect immunity and an insect-pathogenic nematode with symbiotic bacteria

It is well known that some insect-pathogenic nematodes always live in symbiosis with Gram negative bacteria belonging to the genus Xenorhabdus (earlier referred to as Achromobacter ). We have here used diapausing pupae of Hyalophora cecropia as a model system and investigated the Mexican strain of Neoaplectana carpocapsae and its bacterium Xenorhabdus nematophilus for their ability to withstand insect immunity separately and in the symbiotic unit. We have prepared a reconstituted nematode containing a streptomycin-resistant mutant of X. nematophilus . In normal pupae LD 50 (the injected dose that causes death of 50% of the population) of the bacteria was about 500 cells; for immunized pupae it was 5 x 10 5 cells. Cecropia immunity did not affect the nematodes. Fewer than ten nematodes with bacteria were lethal, while LD 50 of axenic nematodes was about 500. Immune haemolymph caused lysis of X. nematophilus . Immune proteins P9A and P9B were identified as the active components in Cecropia immunity against X. nematophilus . It was found that the nematodes help the bacteria by excreting an immune inhibitor that selectively destroys both forms of P9 as well as immune protein P5. These results contribute to our understanding of the symbiotic relationship between nematodes and their bacteria and its survival value against induced insect immunity.

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 ( Hultmark et 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.

Insect pathogenic properties of Serratia marcescens: phage-resistant mutants with a decreased resistance to Cecropia immunity and a decreased virulence to Drosophila.

A non-pigmented strain of Serratia marcescens (Db10) was isolated from moribund Drosophila flies. From this strain were isolated spontaneous mutants resistant to streptomycin (Db11) and nalidixic acid (Db12). Mutant Db11 was used for the isolation of two phages, phi J and phi K, which grew on Db10, Db11 and Db12, but not on three reference strains of S. marcescens. Mutant Db11 was demonstrated to fulfil koch's postulates. Strain Db10 and its antibiotic-resistant derivatives were lethal to Drosophila whether given in the food or by injection. Evidence for toxin(s) was found only in sterile supernatants from 7 d cultures. Such extracts contained proteolytic activity and inactivated the antibacterial activity in immune haemolymph from Cecropia. Phages phi J and phi K were used to isolate phage-resistant mutants of Db11. Three such mutants and their parental strain were investigated for their susceptibility to immune haemolymph from Cecropia. The parental strain was resistant to incubation with 90% haemolymph for 2 h at 37 degrees C; all phage-resistant mutants were susceptible to the immune haemolymph with "killing times" (i.e. the time required to kill 90% of the viable cells) ranging from 15 to 55 min. When the same strains were compared for their virulence to Drosophila, the phage-resistant mutants had significantly reduced virulence. It is concluded that resistance to insect immunity plays an important role in the overall pathogenicity of S. marcescens.

Further studies on the effect and role of cobra venom factor on protective immunity in Galleria mellonella: Activity in the response against Proteus mirabilis

The inhibitory effect of cobra venom factor (CVF) on the in vivo protective immune response of Galleria mellonella to Proteus mirabilis is dose dependent; 89.6% reduction in LD 50 was achieved with a dose of 1.0 unit of CVF per larva. CVF administered to larvae at 6 or 12 hr after vaccination with homologous vaccine significantly reduced the protective immunity against this pathogen. Administration of CVF prior to, or concurrent with vaccine resulted in no significant diminution of LD 50. Immune hemolymph is bactericidal in vitro against Pr. mirabilis but this activity was reduced in the presence of CVF.

Induction of specific humoral immunity to soluble proteins in the American cockroach ( Periplaneta americana). I. Nature of the primary response

Adult male American cockroaches ( Periplaneta americana) generated a specific protective response when injected with soluble protein toxins. This response developed over a period of time, peaking within 2 weeks and then gradually subsiding by the fifth week. Specificity of this reactivity was demonstrated by the fact that immunized animals were only protected against the original immunizing toxin, and not to a heterologous toxin. Passive transfer studies revealed that protection could be transferred to virgin animals with cell-free immune hemolymph. Thus, this advanced invertebrate is capable of generating a specific adaptive humoral immune response to these soluble proteins.

Virulence factors in Bacillus thuringiensis: purification and properties of a protein inhibitor of immunity in insects.

We have previously shown that Bacillus thuringiensis subsp. alesti, serotype 3, produces two extracellular inhibitors of the immune system of Saturniid pupae (designated inhibitors A and B; Edlund et al., 1976). Starting from the culture supernatant of a new mutant of B. thuringiensis with a decreased extracellular proteolytic activity, we have now purified immune inhibitor A(InA). The procedure described consists of three steps: ultrafiltration, precipitation with ammonium sulphate and chromatography on hydroxylapatite. Purified InA gave a single band on polyacrylamide gel electrophoresis using either a gel concentration of 7.5% (w/v) and reducing and denaturing conditions or a gradient gel and native conditions. In both cases the apparent molecular weight was 78 000. A certain amount of proteolytic activity was always co-purified with InA but the two activities could be dissociated by heat or EDTA treatment. Antiserum against purified InA gave only one sharp precipitation band on immunodiffusion against InA with or without EDTA. InA inhibited the in vitro killing of Escherichia coli by immune haemolymph but did not affect the killing of Bacillus subtilis. InA was toxic for Drosophila when injected into the abdomen of adult male flies.