Black fly salivary gland extract inhibits proliferation and induces apoptosis in murine splenocytes.

Black flies are known to be vectors of pathogens including Onchocerca volvulus, which causes human onchocerciasis, and Vesicular Stomatitis Virus. Their salivary secretion has been shown to contain a complex cocktail of anti-haemostatic factors and immunomodulatory activities, which may contribute to efficient transmission of the pathogens. Black fly salivary gland extract (SGE) inhibits mitogen-stimulated mouse splenocyte proliferation, including proliferation of both CD4(+) and CD8(+) T cells. The factor responsible for the inhibition was determined to be a protein (or protein complex) of a size larger than 50 kDa. Moreover, exposure to SGE results in activation of caspase 3 and characteristic morphological changes in CD4(+) and CD8(+) T cells, suggesting that induction of apoptosis could, at least in part, be responsible for this inhibition.

Saliva of the Yellow Fever mosquito, Aedes aegypti, modulates murine lymphocyte function.

Saliva of many vector arthropods contains factors that inhibit haemostatic responses in their vertebrate hosts. Less is known about the effect of vector saliva on host immune responses. We investigated the effect of Aedes aegypti salivary gland extracts on antigen-stimulated responses of transgenic OVA-TCR DO11 mouse splenocytes in vitro. T-cell proliferation was inhibited in a dose-dependent manner, with greater than 50% inhibition at 0.3 salivary gland pair (SGP) equivalents/mL. LPS-stimulated B-cell proliferation was also inhibited. Secretion of the Th1 cytokines IL-2 and IFN-gamma was reduced by 50% or more with 0.45-0.6 SGP/mL, as was secretion of the pro-inflammatory cytokines GM-CSF and TNF-alpha, and the Th2 cytokine IL-5. The Th2 cytokines IL-4 and IL-10 were similarly reduced with 0.6-2 SGP/mL. Inhibition of lymphocyte function involved modulation of viable T-cells at low salivary gland extract (SGE) concentrations, and decreased viability at higher concentrations. Dendritic cells were not killed by salivary gland extracts at concentrations as high as 25 salivary gland pairs/mL, but secretion of IL-12 was inhibited by 87% following exposure to 0.6 SGP/mL. Activity is present in saliva and extracts of female but not male salivary glands, and it is depleted from salivary glands of blood-fed mosquitoes. The activity is denatured by boiling and by digestion with the protease papain, indicating a protein; gel filtration HPLC indicates a mass of about 387 kDa. These results suggest that A. aegypti saliva exerts a marked immunomodulatory influence on the environment at the bite site.

Differential modulation of murine host immune response by salivary gland extracts from the mosquitoes Aedes aegypti and Culex quinquefasciatus.

Mosquitoes (Diptera: Culicidae) are major vectors of numerous infectious agents. Compounds in mosquito saliva not only facilitate blood-feeding, but may also have an impact upon the immune system of vertebrate hosts. Consequently, the exposure to mosquito saliva may influence pathogen transmission, establishment and disease development. Using two medically important vector mosquitoes, Aedes aegypti (L.) and Culex quinquefasciatus Say, we examined the effects of mosquito saliva on immune cells of host mice. After antigen-specific or non-specific stimulation, murine splenocyte proliferation and production of both Th1 and Th2 cytokines were significantly reduced in the presence of salivary gland extract (SGE) from Ae. aegypti, but not SGE from Cx. quinquefasciatus. T cell populations were highly susceptible to this suppression, showing increased mortality and reduced division rates - judged by flow cytometric analyses. Evidently these two culicine mosquitoes differ in their host immunomodulatory activities.

Kinetics and equilibria in ligand binding by nitrophorins 1-4: evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap.

Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking insect Rhodnius prolixus that transport nitric oxide (NO) to the victim, sequester histamine, and inhibit blood coagulation. Here, we report kinetic and thermodynamic analyses for ligand binding by all four proteins and their reduction potentials. All four undergo biphasic association and dissociation reactions with NO. The initial association is fast (1.5-33 microM(-)(1) s(-)(1)) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows ( approximately 50 s(-)(1)), and the stabilized final complexes are resistant to autoreduction (E degrees = +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependent step (0.02-1.4 s(-)(1)), followed by a faster phase that is again similar to that of metmyoglobin (3-52 s(-)(1)). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate constants and smaller association rate constants than NP2 and NP3, leading to values for K(d) that are about 10-fold greater. The results are discussed in light of the recent crystal structures of NP1, NP2, and NP4, which display open, polar distal pockets, and of NP4-NO, which displays an NO-induced conformational change that leads to expulsion of solvent and complete burial of the NO ligand in a now nonpolar distal pocket. Taken together, the results suggest that tighter NO binding in the nitrophorins is due to the trapping of the molecule in a nonpolar distal pocket rather than through formation of particularly strong Fe-NO or hydrogen bonds.

Characterization of the Sialokinin I gene encoding the salivary vasodilator of the yellow fever mosquito, Aedes aegypti.

The gene encoding sialokinin I, the principal vasodilatory peptide of Aedes aegypti, has been isolated and characterized. Degenerate oligonucleotide primers based on peptide amino acid sequence were used to amplify a gene fragment from messenger RNA (mRNA) isolated from female salivary glands. The amplification product was used to probe a salivary gland complementary DNA (cDNA) library, and a number of corresponding cDNAs were isolated and their primary sequence determined. Analysis of the conceptual translation product of a 406-bp cDNA indicates that sialokinin I is expressed as a preprosialokinin and is subsequently post-translationally processed to the active peptide. Northern analysis revealed a 490-bp transcription product expressed exclusively in female salivary glands, and hybridization in situ of probes to RNA in whole tissues localized gene expression to the medial lobe of female salivary glands. Screening of an Ae. aegypti genomic library with the cDNA resulted in the isolation of a clone containing the gene, designated Sialokinin I (Sia I). Comparison of the cDNA with the genomic clone reveals two introns of 62 bp and 833 bp. Primer extension analysis showed that several transcription initiation sites are present. Southern analysis of genomic DNA shows that Sia I is most probably a single-copy gene. Similarities of the Sia I gene product with other genes are confined to the region encoding the active decapeptide.

The crystal structure of nitrophorin 4 at 1.5 A resolution: transport of nitric oxide by a lipocalin-based heme protein.

BACKGROUND: Nitrophorins are nitric oxide (NO) transport proteins from the saliva of blood-feeding insects, which act as vasodilators and anti-platelet agents. Rhodnius prolixus, an insect that carries the trypanosome that causes Chagas' disease, releases four NO-loaded nitrophorins during blood feeding, whereupon the ligand is released into the bloodstream or surrounding tissue of the host. Histamine, a signaling molecule released by the host upon tissue damage, is tightly bound by the nitrophorins; this may facilitate the release of NO and reduce inflammation in the host. RESULTS: Recombinant nitrophorin 4 (NP4) was expressed in Escherichia coli, reconstituted with heme, and found to bind NO and histamine in a manner similar to that of the natural protein. The crystal structure of NP4 revealed a lipocalin-like eight-stranded beta barrel, with heme inserted into one end of the barrel. His59 ligates the proximal site on the heme, a solvent molecule (NH3) ligates the distal site, and three additional solvent molecules occupy the distal pocket. Buried in the protein interior are Glu55 and three solvent molecules. A detailed comparison with other lipocalins suggests that NP4 is closely related to the biliverdin-binding proteins from insects. CONCLUSIONS: The nitrophorins have a unique hemoprotein structure and are completely unlike the globins, the only other hemoproteins designed to transport dissolved gases. Compared with the recently described structure of NP1, the NP4 structure is considerably higher resolution, confirms the unusual placement of ionizable groups in the protein interior, and clarifies the solvent arrangement in the distal pocket. It also provides a striking example of structural homology where sequence homology is minimal.

Analyses of cDNA and recombinant protein for a potent vasoactive protein in saliva of a blood-feeding black fly, Simulium vittatum.

A cDNA was cloned from the salivary glands of a blood-feeding black fly Simulium vittatum. The encoded protein has been given the name Simulium vittatum erythema protein or SVEP, because of its ability to increase blood perfusion in skin capillaries, resulting in the well-characterized erythema of black fly bites. The full-length cDNA contains 548 base pairs which encode 152 amino acid residues of the nascent protein. Post-translational processing produces a mature, secreted protein of 133 residues with a molecular mass of 15.4 kDa. Recombinant SVEP (rSVEP) was produced in a baculovirus expression system and purified by a one-step reversed-phase HPLC procedure. Analyses of physical properties and biological potency demonstrated fidelity of rSVEP to the native protein. Recombinant SVEP relaxed rabbit aorta preparations when preconstricted with 2 micromol l-1 phenylephrine or 25 mmol l-1 K+ but not with 60 mmol l-1 K+. Further, the rSVEP-induced relaxation response of phenylephrine-constricted aorta was inhibited by glibenclamide (10 micromol l-1), suggesting that at least part of its action to relax smooth muscle may result from the opening of ATP-dependent K+ channels. SVEP is a novel salivary-gland-derived vasoactive protein that may be essential for blood feeding by black flies and could potentially enhance transmission of filarial parasites.

Crystal structures of a nitric oxide transport protein from a blood-sucking insect.

The nitrophorins are heme-based proteins from the salivary glands of the blood-sucking insect Rhodnius prolixus that deliver nitric oxide gas (NO) to the victim while feeding, resulting in vasodilation and inhibition of platelet aggregation. The nitrophorins also bind tightly to histamine, which is released by the host to induce wound healing. Here we present three crystal structures of nitrophorin 1 (NP1): bound to cyanide, which binds in a manner similar to NO (2.3 A resolution); bound to histamine (2.0 A resolution); and bound to what appears to be NH3 from the crystallization solution (2.0 A resolution). The NP1 structures reveal heme to be sandwiched between strands of a lipocalin-like beta-barrel, and in an arrangement unlike any other gas-transport protein discovered to date. The heme is six-coordinate with a histidine (His 59) on the proximal side, and ligand in a spacious pocket on the distal side. The structures confirm that NO and histamine compete for the same binding pocket and become buried on binding. The dissociation constant for histamine binding was found to be 19 nM, approximately 100-fold lower than that for NO.

Identification of a steroidogenic neurohormone in female mosquitoes.

In the female mosquito, Aedes aegypti, neurohormones are released from the brain in response to a blood meal and stimulate the ovaries to secrete ecdysteroid hormones, which modulate yolk protein synthesis in the fat body. Neuropeptides with this bioactivity were isolated from head extracts, and partial sequences from these peptides when aligned gave a 31-residue sequence at the amino terminus. Oligonucleotide primers for this sequence were used to amplify with the polymerase chain reaction a genomic DNA product that hybridized to a clone from a head cDNA library. The cDNA encodes a 149-residue preprohormone that is processed into an 86-residue peptide, as indicated by the mass value obtained from the native peptide, with the expected amino-terminal sequence. After modification, the cDNA for the putative neurohormone was expressed in a bacterial system, and the purified peptide had high specific activity in bioassays, thus confirming that it is a steroidogenic gonadotropin, the first to be identified for invertebrates.

Nitric oxide binding and crystallization of recombinant nitrophorin I, a nitric oxide transport protein from the blood-sucking bug Rhodnius prolixus.

A nitric oxide transport protein (nitrophorin I) from the salivary glands of the blood-sucking bug Rhodnius prolixus has been expressed as an insoluble form in Escherichia coli, reconstituted with heme, and characterized with respect to NO binding kinetics and equilibria. NO binding and absorption spectra for recombinant nitrophorin I were indistinguishable from those of the insect-derived protein. The degree of NO binding, the rate of NO release, and the Soret absorption maxima for nitrophorin I were all pH dependent. The NO dissociation constant rose 9-fold over the pH range 5.0-8.3, from 0.19 x 10(-6) to 1.71 x 10(-6). The NO dissociation rate rose 2500-fold between pH 5.0 and pH 8.3, from 1.2 x 10(-3) to 3.0 s(-1). Thus, the NO association rate must also be pH dependent and reduced at pH 5.0 by approximately 280-fold. These factors are consistent with nitrophorin function: NO storage in the apparent low pH of insect salivary glands and NO release into the tissue of the insect's host, where vasodilation is induced. The reversible nature of NO binding, which does not occur with most other heme proteins, and the apparent kinetic control of NO release are discussed. We also report crystals of nitrophorin I that are suitable for structure determination by X-ray crystallography. The most promising crystal form contains two protein molecules in the asymmetric unit and diffracts beyond 2.0 A resolution.

Purification, partial characterization, and cloning of nitric oxide-carrying heme proteins (nitrophorins) from salivary glands of the blood-sucking insect Rhodnius prolixus.

Four nitric oxide (NO)-carrying proteins have been isolated from salivary glands of the blood-sucking insect Rhodnius prolixus. These have been given the collective name "nitrophorins," and individual proteins are designated NP1-NP4 in order of their relative abundance in the glands. All four reversibly bind NO; spectral shifts associated with NO binding indicate the interaction of NO with an Fe(III) heme. Physical properties, amino acid composition, and amino-terminal sequences of the nitrophorins are reported. The most abundant nitrophorin was cloned, and its sequence was determined.

The salivary gland-specific apyrase of the mosquito Aedes aegypti is a member of the 5'-nucleotidase family.

The saliva of hematophagous insects contains a variety of pharmacologically active substances that counteract the normal hemostatic response to injury in vertebrate hosts. The yellow-fever mosquito, Aedes aegypti, secretes an apyrase that inhibits ADP-dependent platelet aggregation. Apyrase was purified as an active enzyme from adult female salivary glands and subjected to tryptic digestion, and the resulting peptides were sequenced. The amino acid sequences obtained match the conceptual translation product of a cDNA clone isolated from an adult female salivary gland library. Sequence comparisons indicate similarities with a ubiquitous family of 5'-nucleotidases. The mosquito protein differs from other members of the family by lacking a carboxyl-terminal hydrophobic domain. The apparent conversion of a gene encoding an enzyme involved in a common metabolic event at the cellular level to a gene involved in the antihemostatic response of mosquitoes illustrates one way this particular insect has adapted to the challenges of bloodfeeding.

Sialokinin I and II: vasodilatory tachykinins from the yellow fever mosquito Aedes aegypti.

The saliva of the mosquito Aedes aegypti has previously been reported to contain a 1400-Da peptide with pharmacological properties typical of a tachykinin. In the present study this vasodilator has been purified to homogeneity and found to consist of two peptides: sialokinin I, with the sequence Asn-Thr-Gly-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH2, and sialokinin II, identical to sialokinin I except for an Asp in position 1. These peptides are present in amounts of 0.62 and 0.16 pmol (711 and 178 ng), respectively, per salivary gland pair. When assayed on the guinea pig ileum, both peptides are as active as the mammalian tachykinin substance P, with K0.5 values of 5.07, 6.58, and 4.94 nM for sialokinin I, sialokinin II, and substance P, respectively.

The role of salivary vasodilators in bloodfeeding and parasite transmission.

In this paper, Donald Champagne reviews the salivary vasodilators, points to effects of similar compounds that may be shared by the insect substances, and discusses the potential significance of these effects with regard to parasite transmission.

Reversible binding of nitric oxide by a salivary heme protein from a bloodsucking insect.

The bloodsucking bug Rhodnius prolixus has a salivary vasodilator, previously characterized as a nitrovasodilator, with salivary smooth muscle-relaxing and antiplatelet activity. Rhodnius salivary glands are bright red owing to the abundance of heme proteins. Electron paramagnetic resonance and optical spectroscopic experiments indicated that the salivary vasodilator is a nitrosylheme protein with an Fe(III) heme that binds nitric oxide (NO) reversibly. Dilution of the protein in neutral pH promoted NO release. This protein thus appears to be the NO carrier that helps R. prolixus to feed on blood.

Light-mediated allelochemical effects of naturally occurring polyacetylenes and thiophenes from asteraceae on herbivorous insects.

Polyacetylenes and their thiophene derivatives, characteristic secondary metabolites of the Asteraceae, were examined for their effects on herbivorous insects. Three thiophenes (a monothiophene, a bithiophene, and α-terthienyl) and four polyacetylenes (phenylheptatriyne, phenylheptadiynene, phenylheptadiyene acetate, and matricaria lactone) were studied for their phototoxicity and light-independent toxicity to (1) a polyphagous lepidopteran,Ostrinia nubilalis, whose host range includes a number of phototoxic Asteraceae, (2) a polyphagous lepidoteran,Euxoa messoria, whose host range includes very few species of Asteraceae, and (3) an oligophagous lepidopteran,Manduca sexta, which is a specialist on Solanaceae. Several compounds were phototoxic toM. sexta andE. messoria even at very low irradiance levels, but behavioral adaptations, including spinning silk and boring into diet, allowedO. nubilalis to avoid photosensitization. Light-independent activity of the compounds to all three species involved feeding deterrence increasing in the orderO. nubilalis, E. messoria, andM. sexta, and longterm metabolic toxicity in the form of impaired nutrient utilization. The biosynthetically derived thiophenes were more toxic than their acetylenic precursors, and toxicity increased with increasing number of thiophene rings. The results are discussed in terms of plant-insect coevolution.