During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ...ligand binding specificity and physiological relevance of two D7 long proteins from Culex quinquefasciatus mosquito, the vector of filaria parasites or West Nile viruses. CxD7L2 binds biogenic amines and eicosanoids. CxD7L1 exhibits high affinity for ADP and ATP, a binding capacity not reported in any D7. We solve the crystal structure of CxD7L1 in complex with ADP to 1.97 Å resolution. The binding pocket lies between the two protein domains, whereas all known D7s bind ligands either within the N- or the C-terminal domains. We demonstrate that these proteins inhibit hemostasis in ex vivo and in vivo experiments. Our results suggest that the ADP-binding function acquired by CxD7L1 evolved to enhance blood-feeding in mammals, where ADP plays a key role in platelet aggregation.
Transmission of pathogens by ticks is greatly supported by tick saliva released during feeding. Dendritic cells (DC) act as immunological sentinels and interconnect the innate and adaptive immune ...system. They control polarization of the immune response towards Th1 or Th2 phenotype. We investigated whether salivary cystatins from the hard tick Ixodes scapularis, sialostatin L (Sialo L) and sialostatin L2 (Sialo L2), influence mouse dendritic cells exposed to Borrelia burgdorferi and relevant Toll-like receptor ligands.
DCs derived from bone-marrow by GM-CSF or Flt-3 ligand, were activated with Borrelia spirochetes or TLR ligands in the presence of 3 μM Sialo L and 3 μM Sialo L2. Produced chemokines and IFN-β were measured by ELISA test. The activation of signalling pathways was tested by western blotting using specific antibodies. The maturation of DC was determined by measuring the surface expression of CD86 by flow cytometry.
We determined the effect of cystatins on the production of chemokines in Borrelia-infected bone-marrow derived DC. The production of MIP-1α was severely suppressed by both cystatins, while IP-10 was selectively inhibited only by Sialo L2. As TLR-2 is a major receptor activated by Borrelia spirochetes, we tested whether cystatins influence signalling pathways activated by TLR-2 ligand, lipoteichoic acid (LTA). Sialo L2 and weakly Sialo L attenuated the extracellular matrix-regulated kinase (Erk1/2) pathway. The activation of phosphatidylinositol-3 kinase (PI3K)/Akt pathway and nuclear factor-κB (NF-κB) was decreased only by Sialo L2. In response to Borrelia burgdorferi, the activation of Erk1/2 was impaired by Sialo L2. Production of IFN-β was analysed in plasmacytoid DC exposed to Borrelia, TLR-7, and TLR-9 ligands. Sialo L, in contrast to Sialo L2, decreased the production of IFN-β in pDC and also impaired the maturation of these cells.
This study shows that DC responses to Borrelia spirochetes are affected by tick cystatins. Sialo L influences the maturation of DC thus having impact on adaptive immune response. Sialo L2 affects the production of chemokines potentially engaged in the development of inflammatory response. The impact of cystatins on Borrelia growth in vivo is discussed.
Salivary glands from blood-feeding arthropods secrete several molecules that inhibit mammalian hemostasis and facilitate blood feeding and pathogen transmission. The salivary functions from
, the ...main vector of Onchocerciasis in South America, remain largely understudied. Here, we have characterized a salivary protease inhibitor (Guianensin) from the blackfly
.
A combination of bioinformatic and biophysical analyses, recombinant protein production, in vitro and in vivo experiments were utilized to characterize the molecula mechanism of action of Guianensin. Kinetics of Guianensin interaction with proteases involved in vertebrate inflammation and coagulation were carried out by surface plasmon resonance and isothermal titration calorimetry. Plasma recalcification and coagulometry and tail bleeding assays were performed to understand the role of Guianensin in coagulation.
Guianensin was identified in the sialotranscriptome of adult
flies and belongs to the Kunitz domain of protease inhibitors. It targets various serine proteases involved in hemostasis and inflammation. Binding to these enzymes is highly specific to the catalytic site and is not detectable for their zymogens, the catalytic site-blocked human coagulation factor Xa (FXa), or thrombin. Accordingly, Guianensin significantly increased both PT (Prothrombin time) and aPTT (Activated partial thromboplastin time) in human plasma and consequently increased blood clotting time
. Guianensin also inhibited prothrombinase activity on endothelial cells. We show that Guianensin acts as a potent anti-inflammatory molecule on FXa-induced paw edema formation in mice.
The information generated by this work highlights the biological functionality of Guianensin as an antithrombotic and anti-inflammatory protein that may play significant roles in blood feeding and pathogen transmission.
Mosquito salivary glands have important roles in blood feeding and pathogen transmission. However, the biological relevance of many salivary components has yet to be determined. Aegyptin, a secreted ...salivary protein from Aedes aegypti , binds collagen and inhibits platelet aggregation and adhesion. We used a transgenic approach to study the relevance of Aegyptin in mosquito blood feeding. Aedes aegypti manipulated genetically to express gene-specific inverted-repeat RNA sequences exhibited significant reductions in Aegyptin mRNA accumulation (85–87%) and protein levels (>80-fold) in female mosquito salivary glands. Transgenic mosquitoes had longer probing times (78–300 s, P < 0.0001) when feeding on mice compared with controls (15–56 s), feeding success was reduced, and those feeding took smaller blood meals. However, no differences in feeding success or blood meal size were found in membrane feeding experiments using defibrinated human blood. Salivary gland extracts from transgenic mosquitoes failed to inhibit collagen-induced platelet aggregation in vitro. Reductions of Aegyptin did not affect salivary ADP-induced platelet aggregation inhibition or disturb anticlotting activities. Our results demonstrate the relevance of Aegyptin for A. aegypti blood feeding, providing further support for the hypothesis that platelet aggregation inhibition is a vital salivary function in blood feeding arthropods. It has been suggested that the multiple mosquito salivary components mediating platelet aggregation (i.e., Aegyptin, apyrase, D7) represent functional redundancy. Our findings do not support this hypothesis; instead, they indicate that multiple salivary components work synergistically and are necessary to achieve maximum blood feeding efficiency.
Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from ...Lutzomyia longipalpis have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from Lu. longipalpis. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized in vitro. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of Leishmania infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the Lu. longipalpis-Leishmania major combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity in vitro. Moreover, in vivo experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-Leishmania vaccine.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the ...parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1β and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R-deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.
Mosquitoes inject saliva into the host skin to facilitate blood meal acquisition through active compounds that prevent hemostasis. D7 proteins are among the most abundant components of the mosquito ...saliva and act as scavengers of biogenic amines and eicosanoids. Several members of the D7 family have been characterized at the biochemical level; however, none have been studied thus far in
, a permissive vector for several arboviruses that causes extensive human morbidity and mortality. Here, we report the binding capabilities of a D7 long form protein from
(AlboD7L1) by isothermal titration calorimetry and compared its model structure with previously solved D7 structures. The physiological function of AlboD7L1 was demonstrated by ex vivo platelet aggregation and in vivo leukocyte recruitment experiments. AlboD7L1 binds host hemostasis agonists, including biogenic amines, leukotrienes, and the thromboxane A2 analog U-46619. AlboD7L1 protein model predicts binding of biolipids through its N-terminal domain, while the C-terminal domain binds biogenic amines. We demonstrated the biological function of AlboD7L1 as an inhibitor of both platelet aggregation and cell recruitment of neutrophils and eosinophils. Altogether, this study reinforces the physiological relevance of the D7 salivary proteins as anti-hemostatic and anti-inflammatory molecules that help blood feeding in mosquitoes.
Female mosquitoes require blood meals for egg development. The saliva of blood feeding arthropods contains biochemically active molecules, whose anti-hemostatic and anti-inflammatory properties ...facilitate blood feeding on vertebrate hosts. While transcriptomics has presented new opportunities to investigate the diversity of salivary proteins from hematophagous arthropods, many of these proteins remain functionally undescribed. Previous transcriptomic analysis of female salivary glands from Culex quinquefasciatus, an important vector of parasitic and viral infections, uncovered a 12-member family of putatively secreted proteins of unknown function, named the Cysteine and Tryptophan-Rich (CWRC) proteins. Here, we present advances in the characterization of two C. quinquefasciatus CWRC family members, CqDVP-2 and CqDVP-4, including their enrichment in female salivary glands, their specific localization within salivary gland tissues, evidence that these proteins are secreted into the saliva, and their native crystal structures, at 2.3 Å and 1.87 Å, respectively. The β-trefoil fold common to CqDVP-2 and CqDVP-4 is similar to carbohydrate-binding proteins, including the B subunit of the AB toxin, ricin, from the castor bean Ricinus communis. Further, we used a glycan array approach, which identifies carbohydrate ligands associated with inflammatory processes and signal transduction. Glycan array 300 testing identified 100 carbohydrate moieties with positive binding to CqDVP-2, and 77 glycans with positive binding to CqDVP-4. The glycan with the highest relative fluorescence intensities, which exhibited binding to both CqDVP-2 and CqDVP-4, was used for molecular docking experiments. We hypothesize that these proteins bind to carbohydrates on the surface of cells important to host immunology. Given that saliva is deposited into the skin during a mosquito bite, and acts as the vehicle for arbovirus inoculation, understanding the role of these proteins in pathogen transmission is of critical importance. This work presents the first solved crystal structures of C. quinquefasciatus salivary proteins with unknown function. These two molecules are the second and third structures reported from salivary proteins from C. quinquefasciatus, an important, yet understudied disease vector.
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•Saliva of blood feeding mosquitoes contains biochemically active molecules that facilitate blood feeding and pathogen transmission.•CqDVP-2 and CqDVP-4, two major proteins from Culex quinquefasciatus, are specific to adult female salivary glands and are secreted into the saliva.•Sex and tissue specificity of these proteins indicate their possible function(s) in blood feeding and pathogen transmission.•The native crystal structures of CqDVP-2 and CqDVP-4 have a ß-trefoil fold similar to carbohydrate-binding proteins.•CqDVP protein binding to carbohydrates is thought to play a role in inflammation and may be an evolutionary adaptation to blood feeding and help improve mosquito survival.
A Monoclonal Antibody for Malaria Prevention Gaudinski, Martin R; Berkowitz, Nina M; Idris, Azza H ...
The New England journal of medicine,
08/2021, Letnik:
385, Številka:
9
Journal Article
Recenzirano
Odprti dostop
Malaria remains a cause of substantial global morbidity and mortality. In this report, an engineered monoclonal antibody showed protection against malaria infection in a controlled human infection ...model.
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