Cecropins as a marker of Spodoptera frugiperda immunosuppression during entomopathogenic bacterial challenge.

An antimicrobial peptide (AMP) of the cecropin family was isolated by HPLC from plasma of the insect pest, Spodoptera frugiperda. Its molecular mass is 3910.9 Da as determined by mass spectrometry. Thanks to the EST database Spodobase, we were able to describe 13 cDNAs encoding six different cecropins which belong to the sub-families CecA, CecB, CecC and CecD. The purified peptide identified as CecB1 was chemically synthesized (syCecB1). It was shown to be active against Gram-positive and Gram-negative bacteria as well as fungi. Two closely related entomopathogenic bacteria, Xenorhabdus nematophila F1 and Xenorhabdus mauleonii VC01(T) showed different susceptibility to syCecB1. Indeed, X. nematophila was sensitive to syCecB1 whereas X. mauleonii had a minimal inhibitory concentration (MIC) eight times higher. Interestingly, injection of live X. nematophila into insects did not induce the expression of AMPs in hemolymph. This effect was not observed when this bacterium was heat-killed before injection. On the opposite, both live and heat-killed X. mauleonii induced the expression of AMPs in the hemolymph of S. frugiperda. The same phenomenon was observed for another immune-related protein lacking antimicrobial activity. Altogether, our data suggest that Xenorhabdus strains have developed different strategies to supplant the humoral defense mechanisms of S. frugiperda, either by increasing their resistance to AMPs or by preventing their expression during such host-pathogen interaction.

[Cellular defence reactions and their depression in insects]. / Réactions de défense cellulaire et leur dépression chez les insectes.

In insects the main cellular defence reactions are phagocytosis and encapsulation of foreign bodies. Free cells of haemolymph called haemocytes are the effectors of these reactions. They are achieved under the control of humoral factors of the plasma or of the serum. Humoral factors are able to enhance or to decrease the cellular defence reactions. As in mammals, potential parasites or pathogens need to avoid or to inhibit the defence reactions before developing inside the insect body. As an example of the depression of immunity induced by a parasite we will study the relationships between an insect and a nematobacterial complex.

Insect immunity-effects of factors produced by a nematobacterial complex on immunocompetent cells(1).

During in vitro incubations, the nematobacterial complex Steinernema carpocapsae-Xenorhabdus nematophilus produces different factors having toxic activities in vitro towards haemocytes, the insect cells responsible for cellular immune defense reactions. Among others, two effects were evident on haemocyte monolayers; one of them was a cytotoxic activity while the other was an unsticking effect. The factors responsible for cytotoxic activity and unsticking effect, were separated from each other by a single chromatography on anion exchange column. These two effects on haemocytes were lost after heat treatment at 57 degrees C for 1 h and 45 degrees C for 30 min, respectively. Both factors were recovered after dialysis in a 10(4) Da cut off membrane. The cytotoxic activity was susceptible to proteases. Cytotoxic and unsticking factors did not show any lipase or lecithinase activity but the unsticking factor had protease activity. Lipopolysaccharides, purified from the bacteria harvested after incubation of the complex, did not have cytotoxic or unsticking effect on the insect cells in vitro.

Cooperation of dopachrome conversion factor with phenoloxidase in the eumelanin pathway in haemolymph of Locusta migratoria (Insecta).

Dopachrome Conversion Factor (DCF) was found in the plasma of the locust Locusta migratoria. It has an apparent molecular mass of 85,000. Its K(m) was 0.2 mM at 22 degrees C and pH 7 with L-dopachrome as substrate. It had a high substrate specificity for L-dopachrome, methyl-L-dopachrome and L-dopachrome methyl ester but no activity on the corresponding D-isomers or on dopaminechrome. DCF was devoid of any phenoloxidase activity. Under action of DCF, L-dopachrome was converted into dihydroxyindole, which showed that a decarboxylation occured in the course of reaction. Locust DCF was inhibited by indole-3-propionic acid but not by indole-3- or indole-2-carboxylic acid. It was also inhibited by L-tryptophan in a competitive manner. Inhibition and substrate specificity suggest that a carboxyl group, either free or as a methyl ester, is necessary but not sufficient for enzyme recognition. When purified prophenoloxidase was activated and then added to dihydroxyindole either prepared by chemical synthesis or obtained by action of purified DCF on dopachrome, black pigments with a maximum absorption at 540 nm were generated. Therefore in the eumelanin pathway of locust plasma, phenoloxidase can catalyze the reaction that converts the product generated by DCF.

Isolation and entomotoxic properties of the Xenorhabdus nematophilus F1 lecithinase.

Xenorhabdus spp. and Photorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the families Steinernematidae and Heterorhabditidae, respectively, were shown to produce different lipases when they were grown on suitable nutrient agar. Substrate specificity studies showed that Photorhabdus spp. exhibited a broad lipase activity, while most of the Xenorhabdus spp. secreted a specific lecithinase. Xenorhabdus spp. occur spontaneously in two variants, phase I and phase II. Only the phase I variants of Xenorhabdus nematophilus and Xenorhabdus bovienii strains produced lecithinase activity when the bacteria were grown on a solid lecithin medium (0.01% lecithin nutrient agar; 24 h of growth). Five enzymatic isomers responsible for this activity were separated from the supernatant of a X. nematophilus F1 culture in two chromatographic steps, cation-exchange chromatography and C18 reverse-phase chromatography. The substrate specificity of the X. nematophilus F1 lecithinase suggested that a phospholipase C preferentially active on phosphatidylcholine could be isolated. The entomotoxic properties of each isomer were tested by injection into the hemocoels of insect larvae. None of the isomers exhibited toxicity with the insects tested, Locusta migratoria, Galleria mellonella, Spodoptera littoralis, and Manduca sexta. The possible role of lecithinase as either a virulence factor or a symbiotic factor is discussed.

Two major proteins from locust plasma are involved in coagulation and are specifically precipitated by laminarin, a beta-1,3-glucan.

Incubation of plasma of the locust Locusta migratoria, with laminarin induced the precipitation of two major proteins with molecular masses of about 260,000 (P260) and 85,000 Da (P85). This precipitation was not observed when other polysaccharides, such as curdlan, dextran, chitin, cellulose or mannan were used. P260 and P85 were purified to homogeneity by a single step on heparin-sepharose chromatography. Since all attempts to separate P260 from P85, other than the use of sodium dodecyl sulfate, were unsuccessful, it is likely that these two molecules form a complex non-covalently associated. Treatment of P260-P85 complex with N-glycosidase F showed that P260 did not appear to be glycosylated whereas 6% of P85 molecular mass was due to N-linked carbohydrates. On the other hand, no change in molecular masses of P260 or P85 was observed once the complex had been treated with lipase. SDS-PAGE and Western blots of plasma and serum stained with blue Coomassie for proteins or with highly specific polysera to P260 or P85, respectively, showed that P260 was only present in plasma and P85 remained in both samples. This indicates that P260 is likely to be one of the most abundant plasma proteins directly involved in the coagulation process in Locusta migratoria. The addition of plasma or P260-P85 complex to a hemocyte lysate supernatant prior to its activation by laminarin induced a lower protease as well as phenoloxidase activity compared with the control. This reduction of activities was not observed in the presence of serum or when P260-P85 complex was added to a fully activated proPO system.

Structure and biological activity of a 1,3-beta-D-glucan-binding protein in crustacean blood.

The prophenoloxidase activating system, an enzyme cascade present in arthropod blood, has been shown to be involved in defense and recognition reactions. This system is converted to its active form by fungal 1,3-beta-D-glucans through binding to a plasma protein, a 1,3-beta-D-glucan-binding protein. Here the molecular cloning and carbohydrate composition of the 1,3-beta-D-glucan-binding protein from the freshwater crayfish Pacifastacus leniusculus are reported. It is also demonstrated that this protein can act as an opsonin, stimulating phagocytic uptake of yeast particles by isolated blood cells. The deduced amino acid sequence of 1,339 residues shows no significant similarity to proteins with similar functions in other animals such as the mannan-binding and lipopolysaccharide-binding proteins present in mammals. However, a short sequence motif with similarity to the active site of microbial 1,3-1,4-beta-D-glucan 4-glucanohydrolases was found to occur twice in the 1,3-beta-D-glucan-binding protein.

Purification and partial characterization of a beta-1,3-glucan-binding-protein membrane receptor from blood cells of the crayfish Pacifastacus leniusculus.

A receptor for the 100 kDa beta-1,3-glucan-binding protein [Duvic, B. and Söderhäll, K. (1990) J. Biol. Chem. 265, 9327-9332] has been purified from hemocyte membranes of the crayfish Pacifastacus leniusculus. The purification was achieved by DEAE-cellulose chromatography of detergent-solubilized membranes. The receptor had an apparent molecular mass of 350 kDa when subjected to native polyacrylamide-gel electrophoresis and was composed of two non-covalently associated subunits of about 230 kDa and 90 kDa, as judged by SDS/polyacrylamide-gel electrophoresis or two-dimensional electrophoresis. The receptor could only bind the beta-1,3-glucan-binding protein if this protein had previously reacted with a beta-1,3-glucan, laminarin, and the binding site was located on the 230 kDa subunit. The binding of laminarin-treated beta-1,3-glucan-binding protein to its receptor was a saturable process and binding data indicated a single high-affinity-binding site with a Kd of 0.35 +/- 0.15 microM as determined by Scatchard analysis. The receptor had a requirement for divalent cations and a pH optimum of 6.5 for binding the laminarin-treated beta-1,3-glucan-binding protein. Laminarin, as well as oligosaccharides such as D-glucose, sialic acid, N-acetyl glucosamine or methyl-alpha-D-mannoside, could not affect the binding of the beta-1,3-glucan-binding protein to its receptor.

Purification and characterization of a beta-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus.

The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two independent methods, affinity precipitation with a beta-1,3-glucan or immunoaffinity chromatography. The purified beta-1,3-glucan binding protein was homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a monomeric glycoprotein with a molecular mass of approximately 100,000 Da and an isoelectric point of approximately 5.0. Amino acid analysis showed a very high similarity with the amino acid composition of beta-1,3-glucan binding proteins recently purified from two insects, the cockroach Blaberus craniifer and the silkworm Bombyx mori. The N-terminal amino acid sequence was determined to be: H2N-Asp-Ala-Gly-X-Ala-Ser-Leu-Val-Thr-Asn-Phe-Asn-Ser-Ala-Lys-Leu-X-X-Ly s--- Using monospecific rabbit polyclonal antibodies, the presence of this protein has also been shown within the blood cells. The purified beta-1,3-glucan binding protein did not show any peptidase or phenoloxidase activity but was able to enhance the activation of hemocyte-derived peptidase and prophenoloxidase only in the presence of the beta-1,3-glucan, laminarin, whereas mannan, dextran (alpha-glucan), or cellulose (beta-1,4-glucan) incubated with the beta-1,3-glucan binding protein had no effect on these enzyme activities. The beta-1,3-glucan binding protein could only be affinity-precipitated from crayfish plasma by the beta-1,3-glucans laminarin or curdlan (an insoluble beta-1,3-glucan), while mannan or dextran did not bind to the beta-1,3-glucan binding protein. No hemagglutinating activity of the purified beta-1,3-glucan binding protein could be detected.