Philippe Roch

Protein analysis of earthworm coelomic fluid: 1) Polymorphic system of the natural hemolysin of Eisenia fetida andrei

Abstract The results described here imply the existence of a protein polymorphism in earthworms. Using analytical isoelectric focusing (IEF) the natural hemolysin of the coelomic fluid of Eisenia fetida andrei appears to be constituted by 4 lipoproteins or lipoprotein-bound substances, referred to as isoforms, which are characterized by their different isoelectric points, ranging from 5.90 to 6.30. Each isoform possesses a natural lytic activity against sheep erythrocytes. All animals possess either 2 or 3 isoforms, with one isoform (pH = 6.00) invariably present. The polymorphism determined here is based on the combinations of the 3 other isoforms. Among all the populations tested, only 6 different hemolytic patterns were found. A presumptive genotype based on the hemolytic patterns of some F1 and F2 descendants is proposed.

Antibacterial activity of the hemolytic system from the earthworm Eisenia fetida andrei

Abstract The coelomic fluid and the cocoon albumen of the earthworm Eisenia fetida andrei are demonstrated to possess an antibacterial activity. The antibacterial activity and the already known hemolytic activity are due, in fact, to the same lipoproteic molecules. The antibacterial activity (bacteriostatic effect) is only directed against the highly pathogenic soil bacteria. Only these pathogenic bacteria strains possess at least one common antigen with the sheep red blood cells.

Protein analysis of earthworm coelomic fluid—II. Isolation and biochemical characterization of the Eisenia fetida andrei factor (EFAF)

Abstract The hemolytic/bacteriostatic factor naturally present in the fluid of all the Eisenia fetida andrei is: 1. 1. Constituted by 2 different molecules with apparent mol. wt of 40,000 and 45,000, 2. 2. Of proteinic composition including an associated glycolipid, 3. 3. Floculable by 55°C heating, 4. 4. Isolable by preparative gel filtration. The EFAF hemolytic activity is mediated by the binding of the factor molecules on the erythrocyte membrane. This binding, which does not concern the Forssman antigens, is inhibited by the N- acetyl- d -glucosamine and the α-methyl- d -mannopyranoside (α-MM).

Formation of brown bodies in the coelomic cavity of the earthworm Eisenia fetida andrei and attendant changes in shape and adhesive capacity of constitutive cells

The formation of brown bodies in the coelomic cavity may result from an aggregation of coelomocytes around offending foreign cells such as bacteria, gregarines, incompatible graft fragments, and altered self structures such as setae or necrotic muscle cells. The initial nodule rapidly increases in volume by aggregation of new coelomocytes and various waste particles. When a brown body has reached a diameter of 1-2 mm, its external cells flatten and lose their adhesiveness toward free coelomocytes or waste particles and its pigment rapidly darkens. Brown bodies play an important role in homeostasis.

Interactions between earthworm hemolysins and sheep red blood cell membranes

The hemolytic activity exhibited by the coelomic fluid of the Annelid Eisenia fetida andrei is mediated by two lipoproteins of mass 40 and 45 kDa, each of them capable of hemolysis. Such an activity is not inhibited by zymosan, inulin or lipopolysaccharide (LPS), nor by hydrazine or methylamine, suggesting that earthworm hemolysins are not related to C3 or C3b complement components. Among the membrane lipids tested (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin and cholesterol) only sphingomyelin inhibited hemolysis. The analysis of E.f. andrei proteins bound to sphingomyelin microvesicles, as well as to sheep red blood cell (SRBC) membranes, revealed a polymerization of E.f. andrei 40 kDa and/or 45 kDa hemolysins. Consequently, sphingomyelin appears a likely candidate for hemolytic complex receptor. Electron microscopy observations suggested that the polymerization causes an open channel through the lipid bilayer. As demonstrated using metal ions, heparin, chondroitin sulfate, poly(L-lysine) and protamine chloride, the mode of action of earthworm hemolytic complex is not analogous to that of C9 or perforine.

Evidence for concanavalin a-receptors and their redistribution on lumbricid leukocytes

Abstract Fluorescent and radioactive concanavalin A (Con A) were used to demonstrate the existence of Con A-receptors on earthworm leukocyte membranes. Inhibition of binding by methyl-α- d -mannose, methyl-α- d - glucose and d -fructose suggested that Con A can bind to specific sites. Differences of labelling between two types of leukocytes support the existence of physiological heterogeneity among the leukocytic population. Native distribution of Con A-receptors was uniform on the whole cell membrane. After 30 minutes at room temperature, patches of Con A-binding site complexes were formed. Capping occurred in 30 % of the small-spheric-cells which represented 20 % of the non-adherent leukocytes, after 1 hour at room temperature. The con A molecules were then internalized by endocytosis of the cap membrane.

Antibacterial activity of Eisenia fetida andrei coelomic fluid: III--Relationship within the polymorphic hemolysins.

The antibacterial activity exhibited by 10 different hemolytic, genetic families was established by measuring the inhibition of spontaneous in vitro growth by cell-free coelomic fluid toward 2 bacteria which are pathogenic for the earthworm: Bacillus megaterium (Gram +) and Aeromonas hydrophila (Gram -). Only two families (B and K) displayed potent inhibitory activities. This finding is consistent with the fact that the B family occurs most frequently in both natural as well as in industrial breedings. Nevertheless, evidence of a poor antibacterial defense in some frequent families suggests the existence of alternative antibacterial mechanisms.

Protein analysis of earthworm coelomic fluid. III. Isolation and characterization of several bacteriostatic molecules from Eisenia fetida andrei

A bacteriostatic activity in Eisenia fetida andrei cell free coelomic fluid is described. This activity is detected by growth inhibition of a bacteria Bacillus megaterium. Gel filtration analysis revealed eleven coelomic fluid protein fractions designated A, B,..J. Antibacterial activity was mainly found within fractions B and C. Chromatofocusing resolved fractions B-C into five different peaks named alpha BC, beta BC,... epsilon BC. Antibacterial activity appeared mediated by three different proteins characterized by their molecular weights (20,000, 40,000 and 45,000) and their isoelectric points (4.9, 5.75 and 6.0). These bacteriostatic proteins possess either hemolysis or hemagglutination activities. The polymorphic aspect of this humoral antibacterial defense is discussed.

Isolation of agglutinins from lysisn in earthworth coelomic fluid by gel filtration followed by chromtofocusing

Agglutinin, hemolysin and bacteriostatin activities have been detected in several fractions of earthworm coelomic fluid separated by gel filtration. Chromatofocusing performed on fractions A (pH gradient 7.4–4.5), B (pH gradient 7.5–5.0) and C (pH gradient 6.2–4.0) have yielded several single proteins characterized by sodium dodecyl sulphate—polyacrylamide gel electrophoresis and analytical isoelectric focusing. Four different molecules are capable of agglutination: MW 11,500 (pI 7.5); MW 20,000 (pI 4.8); MW 32,000 (pI 1.48) and MW 40,000 (pI 6.3). Three molecules have an hemolytic activity: MW 40,000 (pI 6.3); MW 40,000 (pI 5.9) and MW 45,000 (pI 6.0). The molecule having MW 40,000 (pI 6.3) appears to be a multivalent lytic protein also capable of agglutination.

Evidence and cellular localization of an oxidative activity in the coelomic fluid of the earthworm Eisenia fetida andrei

Abstract An oxidative activity was evidenced in the coelomic fluid of the earthworm Eisenia fetida andrei, when l -DOPA was used as substrate. Oxidative activity, measured by optical density due to dopachrome formation, was found to be optimal at basic pH (9–10). Changes in the rate of substrate conversion observed after the addition of hydrogen peroxide or catalase suggest that oxidation of l -DOPA by E. fetida andrei coelomic fluid is mediated by both oxidasic and peroxidasic systems. Microscopy studies, using substrates for the determination of both phenoloxidase and peroxidase activities, demonstrated that the enzyme(s) is localized in chloragocyte vesicles called chloragosomes. The persistence of a remnant staining after incubation with phenoloxidase inhibitors as well as with peroxidase inhibitors strengthened the hypothesis of a synergistic activity of two oxidative systems in E. fetida andrei coelomic fluid.