Anopheles gambiae immune responses to Sephadex beads: involvement of anti-Plasmodium factors in regulating melanization

We have performed a global genome expression analysis of mosquito responses to CM-25 Sephadex beads and identified 27 regulated immune genes, including several anti-Plasmodium factors and other components with likely roles in melanization. Silencing of two bead injection responsive genes, TEP1 and LRIM1, which encode proteins known to mediate Plasmodium killing, significantly compromised the ability to melanize the beads. In contrast, silencing of two Plasmodium protective c-type lectins, CTL4 and CTLMA2, did not affect bead melanization. This data suggest that the anti-Plasmodium factors have dual functions, as determinants of both Plasmodium killing and melanization of the parasite and other foreign bodies, while the Plasmodium protective factors are specifically utilized by the parasite for evasion of mosquito defense mechanisms.     

The cost of immunity in the yellow fever mosquito, Aedes aegypti depends on immune activation

Although host immunity offers the obvious benefit of reducing parasite infection, it is often traded-off with other fitness components. We investigated whether the cost of an immune response in the yellow fever mosquito, Aedes aegypti, is modulated by the antigen that activates the melanization immune response. Thus, one of three different novel antigens were injected into the mosquito's thorax--either a glass bead, a negatively charged (C-25) Sephadex bead, or a neutral (G-25) Sephadex bead--and fecundity and bead melanization were observed. Glass beads are immunologically inert and were therefore used as an inoculation control. The fecundity of mosquitoes inoculated with these beads did not differ from the fecundity of mosquitoes that did not melanize negatively charged or neutral beads. The ability of A. aegypti to melanize negatively charged Sephadex beads was associated with reduced fecundity, showing a clear cost of immunity. In contrast, melanization of the neutral beads was quite strong but had no effect on fecundity. Thus, the cost of what appeared to be the same immune response--melanization of a bead--depended on the type of bead that stimulated the immune system. Such differences might help to explain variation of immune efficacy against different parasites in natural populations.     

Effect of adult nutrition on the melanization immune response of the malaria vector Anopheles stephensi

Two dietary resources - blood and sugar - were assessed for effects on the melanization immune response of the mosquito Anopheles stephensi Liston (Diptera: Culicidae) towards inoculated Sephadex beads (negatively charged C-25). This melanization is conferred by genetic factors capable of making the mosquito refractory to malaria parasites. If An. stephensi females had obtained a bloodmeal one day before inoculation with a bead, the efficacy of their immune response increased with the concentration of sugar ingested. At the highest sugar concentration (6%) tested, 38% of the mosquitoes completely melanized their bead, whereas at the lowest sugar concentration (2%), none of the mosquitoes were able to melanize their bead completely. Among mosquitoes not having a bloodmeal, the immuno-competence was low (c. 9% of the mosquitoes completely melanized their bead) and independent of sugar concentration. The observed interaction between these two resources indicates that both resources are required for the Anopheles female to develop an effective melanization immune response.     

Silencing the genes for dopa decarboxylase or dopachrome conversion enzyme reduces melanization of foreign targets in Anopheles gambiae

The production of melanin is a complex biochemical process in which several enzymes may play a role. Although phenoloxidase and serine proteases are clearly key components, the activity of other enzymes, including dopa decarboxylase and dopachrome conversion enzyme may also be required. We tested the effect of knockdown of gene expression for these two enzymes on melanization of abiotic targets in the mosquito, Anopheles gambiae. Knockdown of dopa decarboxylase and dopachrome conversion enzyme resulted in a significant reduction of melanization of Sephadex beads at 24 h after injection. Knockdown of a third enzyme, phenylalanine hydroxylase, which is involved in endogenous production of tyrosine, had no effect on bead melanization. Quantitative analysis of gene expression demonstrated significant upregulation of phenylalanine hydroxylase, but not the other two genes, following injection.     

Surface characteristics of foreign targets that elicit an encapsulation response by the moth Pseudoplusia includens

Hemocytes from the moth Pseudoplusia includens encapsulate a variety of biotic and abiotic targets. Prior studies indicated that granular cells are usually the first hemocyte type to attach to foreign targets. Thereafter, large numbers of plasmatocytes attach to the target and form a capsule. To identify surface features that induce an encapsulation response, chromatography beads that differed in matrix composition, charge, and functional groups were tested using in vitro and in vivo bioassays. We first conducted in vitro assays using hemocytes with no plasma components present. These experiments indicated that bead types having sulfonic, diethylaminoethyl, and quaternary amine functional groups were encapsulated significantly more often than beads with other functional groups. Charge also significantly affected encapsulation with positively charged beads being encapsulated more often than negatively charged or neutral beads. In vitro assays using purified populations of hemocytes confirmed that these targets were recognized as foreign by granular cells, and that plasmatocytes only formed capsules after granular cells attached to the target. Bead types that were encapsulated under these in vitro conditions were always rapidly encapsulated when injected into P. includens larvae. However, some bead types, like CM-Sephadex, not encapsulated in vitro were encapsulated in vivo if left in the insect hemocoel for a longer period of time (ca. 24 h). Purified plasmatocytes encapsulated these beads in vitro if they were preincubated in plasma. Basic characterization studies suggest these humoral recognition molecules are proteins or small peptides. Comparative studies with other species of noctuid moths also indicated that encapsulation of some bead types differed significantly among species. Collectively, these results reveal that P. includens recognizes some targets as foreign by pattern recognition receptors on granular cells, whereas others are recognized by pattern recognition molecules in plasma. The binding affinities of these recognition molecules also appear to differ among closely related species of Lepidoptera.     

The role of surface characteristics in eliciting humoral encapsulation of foreign bodies in Plasmodium-refractory and -susceptible strains of Anopheles gambiae

A refractory strain of the mosquito, Anopheles gambiae, melanotically encapsulates and kills many species of malaria parasites, whereas susceptible strains allow the parasites to develop normally. To study the role of surface characteristics in eliciting this immune response, 27 types of chromatography beads that differed in matrix type, charge, functional group, and functional group density were assayed for degree of melanotic encapsulation in refractory and susceptible mosquitoes. Overall, two glucan-based matrices, Sephadex (dextran) and cellulose, stimulated the strongest responses, regardless of functional group. Substituting matrix hydroxyl groups with functional groups on Sephadex and cellulose beads decreased the level of encapsulation. These results demonstrate that glucans induce melanotic encapsulation in An. gambiae. Beads with agarose, polystyrene, and acrylic matrices, and most methacrylate-based beads elicited little or no melanization; however, epoxide-methacrylate beads were encapsulated, demonstrating that glucans are not essential for eliciting a response. Comparisons between the two strains demonstrated that refractory mosquitoes melanized many bead types to a greater degree than did susceptible mosquitoes. On this basis, we propose that an important difference between the two strains is that one of the enzymes involved in the melanization pathway functions at a higher level in the refractory strain. Finally, of all beads tested, only 85% substituted CM-Sephadex beads were virtually unmelanized in susceptible mosquitoes but highly melanized in the refractory strain; thus, a specific surface microenvironment is necessary to demonstrate this effect.     

Laminin and a Plasmodium ookinete surface protein inhibit melanotic encapsulation of Sephadex beads in the hemocoel of mosquitoes

In refractory mosquitoes, melanotic encapsulation of Plasmodium ookinetes and oocysts is a commonly observed immune response. However, in susceptible mosquitoes, Plasmodium oocysts develop extracellularly in the body cavity without being recognized by the immune system. Like Plasmodium gallinaceum oocysts, negatively charged carboxymethyl (CM)-Sephadex beads implanted in the hemocoel of Aedes aegypti female mosquitoes were not usually melanized, but were coated with mosquito-derived laminin. Conversely, electrically neutral G-Sephadex beads were routinely melanized. Since mosquito laminin coated both CM-Sephadex beads and P. gallinaceum oocysts, we hypothesized that laminin prevents melanization of both. To test this hypothesis, we coated cyanogen-bromide-activated G-Sephadex beads with laminin, recombinant P. gallinaceum ookinete surface protein (PgS28) or bovine serum albumin (BSA). Beads were implanted into the abdominal body cavity of female Aedes aegypti and retrieved 4 days later. Uncoated controls as well as BSA-coated G-Sephadex beads were melanized in a normal manner. However, melanization of beads coated with mouse laminin, Drosophila L2-secreted proteins or PgS28 was markedly reduced. Fluorescent antibody labeling showed that PgS28-coated beads had adsorbed mosquito laminin on their surface. Thus, mosquito laminin interacting with Plasmodium surface proteins probably masks oocysts from the mosquito's immune system, thereby facilitating their development in the body cavity.     

Mosquito phenoloxidase and defensin colocalize in melanization innate immune responses.

Mosquitoes mount strong humoral and cellular immune responses against foreign organisms. Two components of the mosquito immune response that have received much attention are the phenoloxidase cascade that leads to melanization and antimicrobial peptides. The purpose of the current study was to use immunocytochemistry and transmission electron microscopy to identify the location of the melanization rate-limiting enzyme phenoloxidase and the antimicrobial peptide defensin in innate immune reactions against Escherichia coli and Micrococcus luteus by the mosquito Aedes aegypti. Our results show that both phenoloxidase and defensin are present at the sites of melanin biosynthesis in immune reactions against bacteria. Furthermore, both proteins are often present inside the same melanotic capsules. When hemocytes were analyzed, phenoloxidase was present in the cytosol of oenocytoids, but no significant amounts of defensin were detected inside any hemocytes. In summary, these data show that phenoloxidase and defensin colocalize in melanization reactions against bacteria and argue for further studies into the potential role of defensin in phenoloxidase-based melanization innate immune responses in mosquitoes.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.     

Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti

Mosquitoes are vectors of many deadly and debilitating pathogens. In the current study, we used light and electron microscopies to study the immune response of Aedes aegypti hemocytes to bacterial inoculations, Plasmodium gallinaceum natural infections, and latex bead injections. After challenge, mosquitoes mounted strong phagocytic and melanization responses. Granulocytes phagocytosed bacteria singly or pooled them inside large membrane-delimited vesicles. Phagocytosis of bacteria, Plasmodium sporozoites, and latex beads was extensive; we estimated that individual granulocytes have the capacity to phagocytose hundreds of bacteria and thousands of latex particles. Oenocytoids were also seen to internalize bacteria and latex particles, although infrequently and with low capacity. Besides phagocytosis, mosquitoes cleared bacteria and sporozoites by melanization. Interestingly, the immune response toward 2 species of bacteria was different; most Escherichia coli were phagocytosed, but most Micrococcus luteus were melanized. Similar to E. coli, most Plasmodium sporozoites were phagocytosed. The immune response was rapid; phagocytosis and melanization of bacteria began as early as 5 min after inoculation. The magnitude and speed of the cellular response suggest that hemocytes, acting in concert with the humoral immune response, are the main force driving the battle against foreign invaders.     

Gene silencing of serine proteases affects melanization of Sephadex beads in Anopheles gambiae

Serine proteases play an important role in activation of prophenoloxidase (proPO), a critical enzyme in the production of melanin. We tested the effect of knockdown of gene expression for five clip domain serine proteases on melanization of abiotic targets in Anopheles gambiae. Knockdown of CLIPB4 resulted in a striking lack of melanization of Sephadex beads while knockdown of CLIPB8 caused a strong shift towards incompletely melanized beads. Knockdown of CLIPB1, B9 and B10 had lesser effects. CLIPB4 and CLIPB8 are strong candidates for activating enzymes in the proPO enzymatic cascade.     

The dual roles of Armigeres subalbatus prophenoloxidase V in parasite melanization and egg chorion melanization in the mosquito Ar. subalbatus

Phenoloxidases (POs) play key roles in various physiological functions in insects, e.g., cuticular sclerotization, wound healing, egg tanning, cuticle formation and melanotic encapsulaction of pathogens. Previously, we identified five POs, designated As-pro-PO I–V, from the mosquito Armigeres subalbatus and demonstrated that the functions of As-pro-PO I, II and III, were associated with filarial parasite melanization, blood feeding and cuticle formation, respectively. In the present study, we delineate the dual functions of As-pro-PO V. We found that the level of As-pro-PO V mRNA in mosquitoes was significantly increased after microfilaria challenge or blood feeding, and decreased to normal level after oviposition. Knockdown of As-pro-PO V by dsRNA resulted in significant decreases in the degree of microfilaria melanization, egg chronic melanization rates and egg hatching rates in Ar. subalbatus. Further transfection and electrophoretic mobility-shift assays verified the As-pro-PO V gene might regulated by both AP-1, a putative immune-related regulatory element and CdxA, a developmental regulatory element. The binding of AP-1 and CdxA motif with mosquito nuclear extracts was significantly enhanced after microfilaria challenge and blood-feeding in Ar. subalbatus, respectively. These results indicate that As-pro-PO V is a critical enzyme that is required for both an effective melanization immune response and egg chorion melanization in this mosquito.     

DNA probes on beads arrayed in a capillary, 'Bead-array', exhibited high hybridization performance

A DNA analysis platform called 'Bead-array' is presented and its features when used in hybridization detection are shown. In 'Bead-array', beads of 100- micro m diameter are lined in a determined order in a capillary. Each bead is conjugated with DNA probes, and can be identified by its order in the capillary. This probe array is easily produced by just arraying beads conjugated with probes into the capillary in a fixed order. The hybridization is also easily completed by introducing samples (1-300 micro l) into the capillary with reciprocal flow. For hybridization detection, as little as 1 amol of fluorescent-labeled oligo DNA was detected. The hybridization reaction was completed in 1 min irrespective of the amount of target DNA. When the number of target molecules was smaller than that of probe molecules on the bead, 10 fmol, almost all targets were captured on the bead. 'Bead-array' enables reliable and reproducible measurement of the target quantity. This rapid and sensitive platform seems very promising for various genetic testing tasks.     

Enhanced binding of fibronectin-coated latex beads to quiescent 3T3-L1 cells is correlated with escape from growth arrest

The possible involvement of fibronectin receptors in growth stimulation was investigated by an analysis of fibronectin-coated latex bead binding to 3T3-L1 cells under various conditions. 3T3-L1 cells, growth-arrested in a medium with a low concentration of calf serum, bound few fibronectin-coated beads. After addition of serum at concentrations of 1.0% or higher, there was a rapid and transient increase in the number of cells with bound beads and a subsequent increase in the incorporation of bromodeoxyuridine (BrdU) into cell nuclei. Incorporation of BrdU was observed in about 60% of the cells with bound beads. Fibroblast growth factor and platelet-derived growth factor at concentrations of 5 ng/ml or higher also enhanced binding of fibronectin-coated beads to cells. Stimulation of bead binding by epidermal growth factor and insulin was weak. Fibroblast growth factor, but not epidermal growth factor, increased the incorporation of BrdU into nuclei. These results indicate a relationship between stimulation of cell proliferation in quiescent cells and increased binding by cells of fibronectin-coated latex beads.     

The melanization reaction is not required for survival of Anopheles gambiae mosquitoes after bacterial infections

The melanization reaction of insects requires activation of pro-phenoloxidase by a proteolytic cascade leading to melanin production. Studies in adult mosquitoes have shown that bacteria are efficiently melanized in the hemocoel, but the contribution of melanization to survival after bacterial infections has not been established. Here we show that the Anopheles gambiae noncatalytic serine protease CLIPA8, an essential factor for Plasmodium ookinete melanization, is also required for melanization of bacteria in adult mosquitoes. CLIPA8 silencing by RNA interference inhibits pro-phenoloxidase activation and melanization of bacteria in the hemolymph following microbial challenge. However, CLIPA8 is not required for wound melanization nor for melanotic pseudotumor formation in serpin2 knockdown mosquitoes, suggesting a specific role for pathogen melanization. Surprisingly, CLIPA8 knockdown mosquitoes are as resistant to bacterial challenge as controls, indicating that melanization is not essential for defense against bacteria and questions its precise role in mosquito immunity.     

Regulation and function of the melanization reaction in Drosophila

The melanization reaction, involving the synthesis of melanin to encapsulate pathogens, is a prominent immune response in Drosophila, the mosquito, and other insects and arthropods. Biochemical studies with large insects have defined a basic model for how melanization is activated and regulated upon microbial infection. In this model, recognition of a microorganism triggers a serine protease cascade that activates phenol oxidase (PO), a key enzyme in the melanin biosynthetic pathway, and serpin-type protease inhibitors are involved in inhibiting the cascade. In the past few years, genetic studies in Drosophila have identified serine proteases and serpins that regulate activation of PO and melanization in vivo. These studies, along with molecular genetic analysis of melanization in the mosquito, have provided new insight into the role that melanization plays in fighting microbial infection.     

In vivo production of scFv-displaying biopolymer beads using a self-assembly-promoting fusion partner

Recombinant production and, in particular, immobilization of antibody fragments onto carrier materials are of high interest with regard to diagnostic and therapeutic applications. In this study, the recombinant production of scFv-displaying biopolymer beads intracellularly in Escherichia coli was investigated. An anti-beta-galactosidase scFv (single chain variable fragment of an antibody) was C-terminally tagged with the polymer-synthesizing enzyme PhaC from Cupriavidus necator by generating the respective hybrid gene. The functionality of the anti-beta-galactosidase scFv-PhaC fusion protein was assessed by producing the respective soluble fusion protein in an Escherichia coli AMEF mutant strain. AMEF (antibody-mediated enzyme formation) strains contain an inactive mutant beta-galactosidase, which can be activated by binding of an anti-beta-galactosidase antibody. In vivo activation of AMEF beta-galactosidase indicated that the scFv is functional with the C-terminal fusion partner PhaC. It was further demonstrated that polymer biosynthesis and bead formation were mediated by the scFv-PhaC fusion protein in the cytoplasm of recombinant E. coli when the polymer precursor was metabolically provided. This suggested that the C-terminal fusion partner PhaC acts as a functional insolubility partner, providing a natural cross-link to the bead and leading to in vivo immobilization of the scFv. Overproduction of the fusion protein at the polymer bead surface was confirmed by SDS-PAGE and MALDI-TOF/MS analysis of purified beads. Antigen binding functionality and specificity of the beads was assessed by analyzing the binding of beta-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of beta-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of beta-galactosidase to the scFv-displaying beads was quantitatively analyzed by enzyme-linked assays measuring beta-galactosidase activity. These indicated that the anti-beta-galactosidase scFv-displaying beads bound a maximum of 38 ng of beta-galactosidase per 1 microg of bead protein, showing an apparent equilibrium dissociation constant ( KD) of 12 x 10 (-7) M. This study clearly demonstrated that anti-beta-galactosidase scFv-displaying polymer beads can be produced in engineered E. coli in a one-step process by using PhaC as a self-assembly-promoting fusion partner.     

Ristocetin-dependent reconstitution of binding of von Willebrand factor to purified human platelet membrane glycoprotein Ib-IX complex

Whether the human platelet membrane glycoprotein (GP) Ib-IX complex is the receptor for ristocetin-dependent binding of von Willebrand factor (vWF) has been examined by reconstitution with the purified components using a solid-phase bead assay. Purified GP Ib-IX complex was bound and orientated on the beads via a monoclonal antibody, FMC 25, directed against the membrane-associated region of the complex. Specific binding of 125I-labeled vWF to the GP Ib-IX complex coated beads was strictly ristocetin dependent with maximal binding occurring at ristocetin concentrations greater than or equal to 1 mg/mL. Ristocetin-dependent specific binding of 125I-labeled vWF was saturable. The observed binding was specific to the interaction between vWF and the GP Ib-IX complex since there was no ristocetin-dependent specific binding of vWF if the physicochemically related platelet membrane glycoprotein, GP IIb, was substituted for the GP Ib-IX complex in a corresponding bead assay. Further, neither bovine serum albumin nor other adhesive glycoproteins, such as fibrinogen or fibronectin, specifically bound to the GP Ib-IX complex in the presence of ristocetin. Ristocetin-dependent binding of vWF to platelets and to GP Ib-IX complex coated beads was inhibited by monoclonal antibodies against a 45,000 molecular weight N-terminal region of GP Ib but not by monoclonal antibodies directed against other regions of the GP Ib-IX complex. Similar correspondence between platelets and purified GP Ib-IX complex with respect to the ristocetin-dependent binding of vWF was obtained with anti-vWF monoclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of an activator required for elevation of maturation- promoting factor (MPF) activity by gamma-S-ATP

Maturation-promoting factor (MPF) is a cell cycle control element able to cause cells to enter M-phase upon microinjection and will induce metaphase in nuclei incubated in cell extracts. Previous work has shown that MPF is composed of a complex between p34cdc 2 protein kinase and a B-type cyclin. In the present work gamma-S-ATP was found to cause activation of MPF activity in partially purified preparations, but this activation was lost upon chromatography on Matrex Green gel A. Readdition of other Matrex Green fractions to purified MPF restored the ability of gamma-S-ATP to activate MPF for nuclear breakdown as well as phosphorylation of histone H1. Use of the system described here will facilitate study of p34cdc 2 kinase activation and identification of elements involved in MPF regulation.