Characterization of hemolytic and cytotoxic Gallysins: a relationship with arylphorins.

Gallysin-1, an inducible effector protein in the protective response of Galleria mellonella larvae is a 75 kDa component of hemolytically active material (HAM) isolated from immune cell-free hemolymph. The sequence of the first 20 N-terminal amino acids of the antibacterial protein Gallysin-1 is identical to the predicted sequence of the first 20 amino acids of the Galleria arylphorin Lhp76 (larval hemolymph protein 76). A murine monoclonal antibody to the 20 amino acid N-terminal peptide of Gallysin-1 (GYPQYHYDVETRKLDPSLVN) provides additional evidence for a link between Gallysin-1 and Lhp76, and is used to characterize HAM further. HAM, initially characterized as a mixture of two proteins, Gallysin-1 and a 69 kDa component is now identified as a 450-500 kDa heteromultimer, designated Gallysin. In vivo levels of Gallysin rise during the effector phase of an induced immune response. The monoclonal antibody inhibits the hemolytic activity of Gallysin. In addition to a hemolytic activity for mammalian erythrocytes, Gallysin possesses a cytotoxic activity for the human tumor cell line, K562. Lipopolysaccharides (LPS) and a Pseudomonas aeruginosa vaccine induce a cytotoxic activity which reaches its maximum levels in the hemolymph early (2 hours post-vaccination) in the protective response. The partially purified cytotoxic material (Cyt-M) obtained from cell-free hemolymph collected 2 hours after vaccination has hemolytic activity and shows structural similarities to Gallysin and Lhp76. The previously established role of Gallysin-1 as an effector protein in the protective response of Galleria mellonella indicates that arylphorins may play a role in insect immune responses.

Identification of antigenic sites on staphylococcal enterotoxin B and toxoid.

The staphylococcal enterotoxins (SEs) are capable of causing both food poisoning and a toxic shock-like illness in man. In addition, SEs are known to act as superantigens, stimulating T-cells according to their T-cell receptor Vbeta type. Relatively little is known of their antigenic determinants and how these may relate to the structure and function of the toxins. As a step in the study of these relationships, the entire molecule of SEB was synthesized in duplicate as a series of octapeptides overlapping by seven residues. This series thus represented all the potential linear epitopes of eight residues or less. The reactivity of the octapeptide series with antisera raised to purified SEB and to formaldehyde-inactivated SEB has been used to locate several antigenic sites on native SEB and to identify antigenic differences in the toxoid. Three antigenic peptides identified from the antigenic profile were synthesized and characterized. These represented amino acids 21-32, 93-107 and 202-217 of SEB. None of these peptides affected SEB-induced T-cell proliferation. However, the occurrence or absence of cross-reactivity of these peptides with antibodies to native SEB corresponds to the degree of exposure and/or the rigidity of these regions within SEB.

Gallysin-1, an antibacterial protein isolated from hemolymph of Galleria mellonella.

An inducible hemolysin with antibacterial properties was isolated from the hemolymph of immune Galleria mellonella larvae. The Galleria-derived lysin, named Gallysin-1, was shown to have an apparent molecular weight of 75,000 and to be relatively heat stable at 56 degrees C. Although Gallysin alone was not bactericidal it caused sufficient damage of the outer cell membranes of Pseudomonas aeruginosa RP4 and Escherichia coli K176 to release beta-lactamase from the periplasm. In the presence of either purified Galleria lysozyme or egg white lysozyme Gallysin-1 had potent antibacterial activity against gram-negative bacteria. Gallysin-1 killed osmotically shocked P. aeruginosa and E. coli that suggests that it can also attack exposed inner cell membranes of gram-negative bacteria. The identification of Gallysin-1 recognizes another distinct member of the bactericidins involved in insect immunity.

Cane sugar factor as an inducing agent of immunity in Galleria mellonella.

The injection of cane sugar factor (CSF) into Galleria mellonella larvae results in an immune response similar to that produced by a formalized Pseudomonas aeruginosa vaccine. Vaccination with CSF is followed by: an increase in the LD50 of Pseudomonas aeruginosa; an in vivo protective response to P. aeruginosa the development of which can be inhibited by cobra venom factor (CVF); an antibacterial activity in hemolymph 24 h after the injection of CSF; the development of a transferrable immune response in hemolymph of donor larvae capable of protecting recipient larvae against a lethal challenge of Pseudomonas aeruginosa; an increase in extracellular lysozyme equal to that induced by Pseudomonas vaccine; a reduction in total hemocyte count during the period of protective immunity; and the presence in hemolymph of new basic proteins, with electrophoretic mobilities and appearance times after the CSF injection, identical to those induced by the formalized vaccine. CSF was shown to be composed primarily of glucose.

Effects of selected carbohydrates and the contribution of the prophenoloxidase cascade system to the adhesion of strains of Pseudomonas aeruginosa and Proteus mirabilis to hemocytes of nonimmune larval Galleria mellonella.

The adhesion of strains of Pseudomonas aeruginosa and Proteus mirabilis to the plasmatocytes and granular cells of nonimmune larval Galleria mellonella was influenced by and varied with the type of carbohydrate. Laminarin enhanced prophenoloxidase activation and bacterial adhesion to the hemocytes whereas sucrose suppressed both activities. For all other sugars there was no correlation between bacterial adhesion to the hemocytes and phenoloxidase activity. It is proposed that bacterial adhesion to the hemocytes may be mediated by both lectinlike binding and components of the prophenoloxidase activating system acting like opsonins.

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.

A cobra venom factor (CVF)-induced C3 convertase activity in the hemolymph of Galleria mellonella.

A cobra venom factor (CVF)-induced C3 convertase has been generated from the hemolymph of Galleria mellonella. CVF was immobilized on Sepharose 4B and treated with cell-free hemolymph obtained from either unvaccinated G. mellonella larvae or larvae immunized with formalized Pseudomonas aeruginosa. The C3-cleaving activity was detected by the ability to cleave the alpha-chain of bovine C3 in a manner analogous to the CVF-induced mammalian C3 convertase, CVF,Bb. The insect-derived C3 convertase formed at 28 degrees C but not at 37 degrees C, then once formed was active at both 28 degrees C and 37 degrees C. EDTA did not inhibit the formation and action of the insect derived C3 cleaving activity.

Reactions of hemocytes of immune and non-immune Galleria mellonella larvae to Proteus mirabilis.

Immune larval Galleria mellonella removed live Proteus mirabilis from the hemolymph less effectively than did non-immune larvae. This was attributed to a decline in total hemocyte counts, levels of plasmatocytes and granulocytes and hemocyte adhesion capacity. Immune serum possessed factors which reduced bacterial adhesion to the hemocytes. This was not due to altering bacterial surfaces but rather to irreversible suppression of hemocyte activity.

Structural alterations to Proteus mirabilis as a result of exposure to haemolymph from the larvae of Galleria mellonella.

Scanning and transmission electron microscopy was used to examine Proteus mirabilis, strain 11-1A, for morphologic alterations following exposure to normal and immune cell-free haemolymph obtained from Galleria mellonella larvae. It was found that both normal and immune haemolymph exerted effects on the bacterial cells, with immune haemolymph affecting a much higher percentage of cells than normal haemolymph. The morphological damage observed by scanning electron microscopy (SEM) included abnormal shapes, blebbing of the cell wall, surface erosion and lysis of the cell. Transmission electron microscopy showed evidence of damage to the cell wall and supported the results obtained from SEM.

The in vitro generation of an antibacterial activity from the fat body and hemolymph of non-immunized larvae of Galleria mellonella.

A state of immunity in Galleria mellonella against the pathogen Pseudomonas aeruginosa is known to be induced by the injection of lipopolysaccharide (LPS), isolated from the homologous organism. An in vitro mixture of the LPS and whole or cell-free hemolymph from non-immunized larvae is not antibacterial. In vitro mixtures of fat body and cell-free hemolymph from non-immunized larvae, incubated at 25 degrees C for 20 hours generated a proteinaceous antibacterial activity. The generation of this activity was enhanced by the presence in the incubation mixture of LPS and/or hemocytes from non-immunized larvae. It is suggested that LPS causes the release of a hemocyte factor(s) which acts in conjunction with or directly on the fat body resulting in an enhanced production of antibacterial factors.

Transfer of immunity against Pseudomonas aeruginosa P11-1 in Galleria mellonella 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.

Effects of hemolymph from immune and non-immune larvae of Galleria mellonella on the ultra-structure of Pseudomonas aeruginosa.

The ultrastructure of Pseudomonas aeruginosa, a pathogen of Galleria mellonella is rapidly altered after in vitro 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 degrees 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.

Time and dose studies of the effect of cobra venom factor on the in vivo immune response in Galleria mellonella to Pseudomonas aeruginosa.

The induced resistance of wax moth larvae vaccinated with formolized Pseudomonas aeruginosa 18 hr prior to challenge with the live organism was significantly inhibited by 0.2 to 1.0 units of cobra venom factor (CVF) per insect given 6 hr after vaccination. Approximately 90% inhibition of the protective response was caused by 1.0 units CVF/insect. Administration of this dose 6 hr before vaccination, with the vaccine, and 6 hr before challenge had no significant effect. The CVF effect was inhibited by heating the CVF preparation at 70 degrees for 30 min.

The pathogenicity of rough strains of Pseudomonas aeruginosa for Galleria mellonella.

Rough mutants of two strains of Pseudomonas aeruginosa have been isolated by selection for resistance to virulent lipopolysaccharide-specific phages. The rough mutants fell into two classes on the basis of colonial morphology and agglutination by acriflavine and NaCl. The pigment and exoenzyme-synthesizing properties of these derivatives were identical with those of the parental cultures. The rough strains were 8- to 62-fold less pathogenic for the larvae of the wax moth (Galleria mellonella) than the smooth wild-type cells.