Periasamy Mullainadhan

Isolation, purification and characterization of β-1,3-glucan binding protein from the plasma of marine mussel Perna viridis

A beta-1,3-glucan binding protein (betaGBP) specific for laminarin (a beta-1,3-glucan) was detected for the first time in a mollusc, Perna viridis. betaGBP was isolated and purified from the plasma using laminarin precipitation and affinity chromatography on laminarin-Sepharose 6B, respectively. It agglutinated bakers yeast, bacteria, and erythrocytes and enhanced prophenoloxidase (proPO) activity of the plasma in a dose-dependent manner. The purified betaGBP appeared as a single band in native-PAGE and the purity was conformed by HPLC. The protein has a molecular weight estimate of 510kDa as determined by SDS-PAGE and in isoelectric focusing the purified betaGBP was focused as a single band at pI 5.3. betaGBP was found to possess inherent serine protease activity but lacked beta-1,3-glucanase activity and all these results suggest that plasma betaGBP of P. viridis functions as a recognition molecule for beta-1,3-glucan on the surface of microbial cell walls. This recognition and binding lead to the activation of the prophenoloxidase cascade mediated by the inherent serine protease activity of betaGBP. Presence of agglutinating activity and serine protease activity shows that betaGBP is a bifunctional protein. The findings are discussed in light of the importance of this protein in the innate immune response of P. viridis, and they implicate evolutionary link with similar proteins found in other invertebrates.

Agglutinin-mediated phagocytosis-associated generation of superoxide anion and nitric oxide by the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii

Hemocyte mediated phagocytosis is one of the vital components of innate defence mechanisms in crustaceans and this phagocytic process is aided by serum agglutinins. However, literature on agglutinin mediated opsono-phagocytosis is unclear in the case of Macrobrachium rosenbergii hemocytes. Further, very few studies in the case of superoxide anion generation and none with regard to nitric oxide generation during phagocytosis exist among crustaceans. We investigated the occurrence of agglutinins in the serum and the role of serum agglutinins in mediating phagocytosis by the hemocytes. We show that the prawn serum possesses agglutinins that function as opsonins during phagocytosis of HB RBC by the hemocytes. Hemagglutination-inhibition assays revealed the specificity of serum agglutinins for N-acetylated hexoses, namely GalNAc, GlcNAc and ManNAc, with a higher affinity for ManNAc. In addition, ManNAc was able to inhibit the phagocytic response (by about 60%) of the hemocytes against serum pretreated HB RBC, wherein the serum was previously treated with ManNAc. We next investigated the ability of the hemocytes to generate superoxide anion and nitric oxide during HB RBC phagocytosis and results show generation of both these free radicals. In addition, there was an enhancement in generation (75% increase) of these free radicals during agglutinin mediated opsonophagocytosis, when compared to buffer treated targets and interestingly this enhanced generation was inhibited by ManNAc (27% for superoxide anion and 36% for nitric oxide), an inhibitory sugar for phagocytosis. Inhibition of phagocytosis induced superoxide anion generation by DPI (53%), sodium azide (56%) and tropolone (61%), reveals the possible involvement of NADPH-oxidases, peroxidases and probably phenoloxidases, respectively, in the generation of superoxide anion. Similarly, decrease in nitric oxide generation in the presence of l-NIO (47%) during phagocytosis lends support to the role of nitric oxide generation during cellular immune processes. These findings thus suggest a role for superoxide anion and nitric oxide in the innate defense mechanism, namely phagocytosis, in Macrobrachium rosenbergii.

Effects of extract of soapnut Sapindus emarginatus on esterases and phosphatases of the vector mosquito, Aedes aegypti (Diptera: Culicidae)

Our earlier investigations with kernels from the soapnut Sapindus emarginatus revealed it as a new source of botanical biocide with potent antimosquito activity, as evident from the proven unique ability of the aqueous kernel extract to kill all the developmental stages of three important vector mosquito species, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. This extract was also found to be safe for two non-target aquatic insects. As a sequel to these findings, we have further examined quantitative and qualitative changes in total proteins, esterases, and phosphatases in whole body homogenates of fourth instar larvae and pupae of A. aegypti exposed to this extract at an appropriate threshold time for its lethal effect to gain insights into the impact of the botanical biocide on biochemical characteristics of the target vector mosquito at two distinct developmental stages. The profiles of proteins, esterases (acetylcholinesterse, α- and β-carboxylesterases), and phosphatases (acid and alkaline) exhibited distinct patterns of variation during normal development of fourth instar larvae and pupae, indicating intrinsic difference in biochemical features between these two developmental stages of A. aegypti. Upon exposure of the larvae to the extract, significant reduction in the activities of acetylcholinesterse, β-carboxylesterase, and acid phosphatases were recorded, whereas the total proteins, α-carboxylesterase and alkaline phosphatase activities were unaffected. By contrast, only alkaline phosphatase activity was significantly affected in pupae exposed to the extract. Analysis of these enzymes in native PAGE revealed that they exist in isoforms in both the larvae and pupae. The alterations in the levels of enzymatic activities observed from the quantitative assays of various enzymes were reflected by the respective zymograms with perceptible differences in the intensity and the number of bands detected especially with β-carboxylesterase, acid and alkaline phosphatase activity between the control and exposed test organisms. Despite the fact that the soapnut kernel extract causes mortality of both the larvae and pupae of A. aegypti, the findings of this study demonstrate that the impact of this extract is most pronounced in various enzyme profiles of the larvae rather than the pupae. Such discrepancy implicates the presence of unique biochemical mechanisms in the pupae of mosquito for detoxification of botanical biocides.

Functional analysis of plasma prophenoloxidase system in the marine mussel Perna viridis

Abstract The role of prophenoloxidase (proPO) system in recognition and phagocytosis of yeast cells by hemocytes was examined in vitro using whole plasma and proPO system isolated from the plasma of the marine mussel, Perna viridis . The proPO was isolated from the plasma by ammonium sulphate precipitation and gel filtration. The final proPO preparation was homogeneous in native PAGE, and could be activated by trypsin, α -chymotrypsin and pronase-E. Laminarin (a polymer of β -1, 3-glucan) and lipopolysaccharides (LPS) from diverse bacterial species effectively activated the isolated proPO, demonstrating the ability of this proenzyme to interact directly with microbial surface components. The susceptibility of proPO activation to inhibition by serine protease inhibitors such as soybean trypsin inhibitor (STI) or p -nitrophenyl- p ′-guanidinobenzoate ( p -NPGB), indicates that the isolated fraction may contain an integral serine protease domain in an inactive state. The presence of laminarin- or LPS-activated whole plasma of P. viridis facilitated adherence of yeast cells to hemocyte surface as well as eventually stimulated phagocytic uptake of the target cells by hemocytes, and no such hemocytic response was recorded with STI controls. This and other results strongly suggest that the intermediary factors generated during activation of plasma proPO system by non-self molecules play a key role in recognition and opsono-phagocytosis by hemocytes. However, the proPO system isolated from P. viridis plasma, after activation with microbial surface components, failed to show an opsonic effect.

Antimosquito activity of aqueous kernel extract of soapnut Sapindus emarginatus: impact on various developmental stages of three vector mosquito species and nontarget aquatic insects

Aqueous (physiological saline) extracts of seed kernel from seven indigenous plants were initially screened for their antimosquito activity against eggs, larvae of all instars, and pupae of Aedes aegypti. Among various seed kernels tested, the soapnut Sapindus emarginatus (Sapindaceae) extract was found to exhibit, for the first time, a strong antimosquito activity as evident from its ability to inflict 100% mortality of all the developmental stages of A. aegypti. Furthermore, the kernel extract of S. emarginatus also exerted ovicidal, larvicidal, and pupicidal activity against two other important vector mosquitoes, namely, Anopheles stephensi and Culex quinquefasciatus. Differential susceptibility of the various developmental stages of the three mosquito species exposed to soapnut extract was also noticed. The kernel extract was found to be safe for two nontarget aquatic insects tested: the larvae of Chironomus costatus and the nymphs of Diplonychus rusticus. Lethal concentration values of soapnut extract to these nontarget insects were always threefold or fivefold higher than those that produced 100% mortality of the larvae of the three mosquito species examined. The findings of this study clearly demonstrate that the aqueous kernel extract of S. emarginatus has potent antimosquito activity detectable against all the developmental stages of three important vector mosquito species as well as it is safe for nontarget aquatic organisms, and thus this new botanical resource could be used as an eco-friendly alternative biocidal agent in control of mosquitoes.

Isolation and characterization of an acetyl group-recognizing agglutinin from the serum of the Indian white shrimp Fenneropenaeus indicus

A natural agglutinin from the serum of the Indian white shrimp Fenneropenaeus (Penaeus) indicus was purified to electrophoretic homogeneity by a single-step affinity chromatography on N-acetylglucosamine-Sepharose 6B. The expression of hemagglutinating (HA) activity of F. indicus agglutinin (FIA) was independent of the presence of divalent cations and insensitive to their chelators. FIA gave a single symmetrical peak in its native form with a molecular mass estimate of 200 kDa on gel filtration in HPLC, and SDS-PAGE under reducing conditions revealed that it is a homo-oligomer of a 27-kDa subunit protein. The pattern of reactivity of FIA against anti-FIA rabbit serum in immunodiffusion and immunoelectrophoretic analysis provided additional evidence for its purity and homogeneity. HA-inhibition studies documented exclusive specificity of FIA for acetyl groups in carbohydrates independently of the presence of these groups at the C-2 or C-5 position and its stereochemical arrangement in the axial or equatorial orientation. The unique ability of FIA to recognize acetyl groups was also explicitly demonstrated with sialo- and asialo-glycoproteins. Strikingly, FIA also interacted equally with amino acids and chemicals containing acetyl groups, thereby unambiguously demonstrating the exquisite specificity of FIA for an acetyl group, irrespective of the presence of this group in carbohydrate or noncarbohydrate ligands. The susceptibility of HA activity of FIA to inhibition by lipopolysaccharides from diverse gram-negative bacteria as well as its ability to selectively agglutinate several bacterial species isolated from infected shrimps implicate a potential role of this humoral agglutinin of F. indicus in the host immunodefense reactions against microbial invaders.

Purification and characterization of a natural agglutinin from the serum of the hermit crab Diogenes affinis.

A natural agglutinin from the serum of the hermit crab Diogenes affinis was purified to homogeneity by a single-step affinity chromatography using N-acetylglucosamine-coupled Sepharose 6B. The purified serum agglutinin (PSA) showed a strong affinity for rat RBC, and its hemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA. PSA in active form has a molecular mass estimate of 185 kDa and is composed of four non-identical subunits (51, 49, 42 and 39 kDa) cross-linked by interchain disulfide bonds. The homogeneity of PSA was corroborated by immunodiffusion and immunoelectrophoretic analyses using rabbit antiserum raised against the agglutinin. The antibodies in this antiserum appear to be specific for RBC-binding sites of the agglutinin molecules as revealed by the ability of the antiserum to neutralize HA activities of both whole serum and PSA of D. affinis. In HA-inhibition assays performed with several carbohydrates and glycoproteins, PSA showed a distinct and unique specificity for acetyl group in carbohydrates independently of the presence of this group on C-2 or C-5 and its stereochemical arrangement in the axial or equatorial orientation. Besides, this agglutinin appears to recognize the terminal N- and O- acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of D. affinis agglutinin was also susceptible to inhibition by lipopolysaccharides from diverse gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections.

A lipopolysaccharide-binding hemagglutinin with specificity for acetylated aminosugars in the serum of the hermit crab Diogenes affinis (Henderson)

A naturally occurring hemagglutinin was detected in the serum of the hermit crab Diogenes affinis, and its erythrocyte (RBC) binding activities, physicochemical properties, and carbohydrate binding specificity were characterized. Both the hemagglutination profile and the pattern of cross-reactivity of the serum with different RBC types in cross-adsorption tests suggested a strong affinity of the serum agglutinin for rat RBC. Further analysis revealed that the agglutinin was specifically dependent on Ca2+ for its hemagglutinating activity and reversibly sensitive to EDTA. The activity was found to be stable between pH 6.0 and 7.5, heat-labile, and completely precipitable by ammonium sulphate or TCA, suggesting the proteinaceous nature of the serum agglutinin. In hemagglutination-inhibition assays, the serum agglutinin of D. affinis showed a distinct and unique specificity for acetyl group-containing carbohydrates and glycoprotein. Furthermore, the hemagglutinating activity of the serum agglutinin was also inhibited by lipopolysaccharide from Salmonella abortus equi, which might indicate a significant role of humoral agglutinin in the immune response of crustaceans against bacterial infection.

Crustacean defense strategies I. Molecular weight dependent clearance of dyes in the mud crab Scyllaserrata (Forskal) (Portunidae: Brachyura)

Clearance rates of dyes injected into the hemocoel of the mud crab, Scylla serrata, revealed several patterns of clearance and retention associated with physico-chemical and biological variables. Clearance rates remained constant: 1) when the molar concentrations of the injected dyes were equal, 2) after repeated injections of dyes and 3) after serum opsonization. Rates increased: 1) with higher molecular weight irrespective of charge, 2) at higher dye concentration and 3) at night concomitant with an increase in the hemocyte population. In contrast, clearance rates decreased with increasing crab size. Dye cleared from the hemolymph accumulated in the gills but not in the hepatopancreas, antennary glands nor gut. The retention of dyes in the gills increased with higher molecular weights. Granular hemocytes accumulated in the gill rachis and lamellae of dye treated but not in gills of normal and saline injected crabs. A role for hemocytes in molecular weight and concentration dependent clearance of dyes is suggested. Our findings are important for understanding the molecular basis of recognition in crustaceans.

Immuno-suppressive effects of aqueous extract of soapnut Sapindus emarginatus on the larvae and pupae of vector mosquito, Aedes aegypti

We recently reported the presence of potent anti-mosquito activity in aqueous kernel extract of the soapnut, Sapindus emarginatus, and demonstrated its impact on marker enzymes in larvae and pupae of the vector mosquito, Aedes aegypti. As a sequel to these findings, the present study elucidates immunotoxicity of this extract with respect to hemocyte-mediated cellular immune responses in fourth instar larvae and pupae as well as cuticular melanization reaction in the larvae of A. aegypti. The exposure of these two developmental stages of the mosquito to the soapnut extract at a lethal threshold concentration neither affected hemocyte viability tested up to 3h in vitro nor did it influence the hemocyte count. By contrast, exposure of the mosquito larvae and pupae to this extract significantly reduced the ability of their hemocytes to bind yeast cells, an important early event in the process of non-self recognition by immune cells. Consequently, the phagocytic activity of these hemocytes against yeast cells was also found to be adversely affected upon exposure of larvae and pupae to the extract. Besides, a perceptible initial delay in melanization reaction at the injured site of the cuticle in the extract-exposed larvae was observed. All these findings demonstrate, for the first time, the immuno-suppressive potential of a botanical biocide in the vector mosquito.