The Tick Protein Sialostatin L2 Binds to Annexin A2 and Inhibits NLRC4-Mediated Inflammasome Activation.

Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1ß (IL-1ß) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.

Simplagrin, a platelet aggregation inhibitor from Simulium nigrimanum salivary glands specifically binds to the Von Willebrand factor receptor in collagen and inhibits carotid thrombus formation in vivo.

BACKGROUND: Among the several challenges faced by bloodsucking arthropods, the vertebrate hemostatic response against blood loss represents an important barrier to efficient blood feeding. Here we report the first inhibitor of collagen-induced platelet aggregation derived from the salivary glands of a black fly (Simulium nigrimanum), named Simplagrin. METHODS AND FINDINGS: Simplagrin was expressed in mammalian cells and purified by affinity-and size-exclusion chromatography. Light-scattering studies showed that Simplagrin has an elongated monomeric form with a hydrodynamic radius of 5.6 nm. Simplagrin binds to collagen (type I-VI) with high affinity (2-15 nM), and this interaction does not involve any significant conformational change as determined by circular dichroism spectroscopy. Simplagrin-collagen interaction is both entropically and enthalpically driven with a large negative ΔG, indicating that this interaction is favorable and occurs spontaneously. Simplagrin specifically inhibits von Willebrand factor interaction with collagen type III and completely blocks platelet adhesion to collagen under flow conditions at high shear rates; however, Simplagrin failed to block glycoprotein VI and Iα2ß1 interaction to collagen. Simplagrin binds to RGQOGVMGF peptide with an affinity (K(D) 11 nM) similar to that of Simplagrin for collagen. Furthermore, Simplagrin prevents laser-induced carotid thrombus formation in vivo without significant bleeding in mice and could be useful as an antithrombotic agent in thrombosis related disease. CONCLUSION: Our results support the orthology of the Aegyptin clade in bloodsucking Nematocera and the hypothesis of a faster evolutionary rate of salivary function of proteins from blood feeding arthropods.

Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma.

Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET) were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep) from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep) shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate)- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized) Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.

An insight into the sialome of the frog biting fly, Corethrella appendiculata.

The Nematocera infraorder Culicomorpha is believed to have descended from bloodfeeding ancestors over 200 million years ago, generating bloodfeeding and non-bloodfeeding flies in two superfamilies, the Culicoidea-containing the mosquitoes, the frog-feeding midges, the Chaoboridae, and the Dixidae-and the Chironomoidea-containing the black flies, the ceratopogonids, the Chironomidae, and the Thaumaleidae. Blood feeding requires many adaptations, including development of a sophisticated salivary potion that disarms host hemostasis, the physiologic mechanism comprising platelet aggregation, vasoconstriction, and blood clotting. The composition of the sialome (from the Greek sialo = saliva) from bloodfeeding animals can be inferred from analysis of their salivary gland transcriptome. While members of the mosquitoes, black flies, and biting midges have provided sialotranscriptome descriptions, no species of the frog-biting midges has been thus analyzed. We describe in this work the sialotranscriptome of Corethrella appendiculata, revealing a complex potion of enzymes, classical nematoceran protein families involved in blood feeding, and novel protein families unique to this species of frog-feeding fly. Bacterial (Wolbachia) and novel viral sequences were also discovered.

A deep insight into the sialotranscriptome of the mosquito, Psorophora albipes.

BACKGROUND: Psorophora mosquitoes are exclusively found in the Americas and have been associated with transmission of encephalitis and West Nile fever viruses, among other arboviruses. Mosquito salivary glands represent the final route of differentiation and transmission of many parasites. They also secrete molecules with powerful pharmacologic actions that modulate host hemostasis, inflammation, and immune response. Here, we employed next generation sequencing and proteome approaches to investigate for the first time the salivary composition of a mosquito member of the Psorophora genus. We additionally discuss the evolutionary position of this mosquito genus into the Culicidae family by comparing the identity of its secreted salivary compounds to other mosquito salivary proteins identified so far. RESULTS: Illumina sequencing resulted in 13,535,229 sequence reads, which were assembled into 3,247 contigs. All families were classified according to their in silico-predicted function/ activity. Annotation of these sequences allowed classification of their products into 83 salivary protein families, twenty (24.39%) of which were confirmed by our subsequent proteome analysis. Two protein families were deorphanized from Aedes and one from Ochlerotatus, while four protein families were described as novel to Psorophora genus because they had no match with any other known mosquito salivary sequence. Several protein families described as exclusive to Culicines were present in Psorophora mosquitoes, while we did not identify any member of the protein families already known as unique to Anophelines. Also, the Psorophora salivary proteins had better identity to homologs in Aedes (69.23%), followed by Ochlerotatus (8.15%), Culex (6.52%), and Anopheles (4.66%), respectively. CONCLUSIONS: This is the first sialome (from the Greek sialo = saliva) catalog of salivary proteins from a Psorophora mosquito, which may be useful for better understanding the lifecycle of this mosquito and the role of its salivary secretion in arboviral transmission.

Novel family of insect salivary inhibitors blocks contact pathway activation by binding to polyphosphate, heparin, and dextran sulfate.

OBJECTIVE: Polyphosphate and heparin are anionic polymers released by activated mast cells and platelets that are known to stimulate the contact pathway of coagulation. These polymers promote both the autoactivation of factor XII and the assembly of complexes containing factor XI, prekallikrein, and high-molecular-weight kininogen. We are searching for salivary proteins from blood-feeding insects that counteract the effect of procoagulant and proinflammatory factors in the host, including elements of the contact pathway. APPROACH AND RESULTS: Here, we evaluate the ability of the sand fly salivary proteins, PdSP15a and PdSP15b, to inhibit the contact pathway by disrupting binding of its components to anionic polymers. We attempt to demonstrate binding of the proteins to polyphosphate, heparin, and dextran sulfate. We also evaluate the effect of this binding on contact pathway reactions. We also set out to determine the x-ray crystal structure of PdSP15b and examine the determinants of relevant molecular interactions. Both proteins bind polyphosphate, heparin, and dextran sulfate with high affinity. Through this mechanism they inhibit the autoactivation of factor XII and factor XI, the reciprocal activation of factor XII and prekallikrein, the activation of factor XI by thrombin and factor XIIa, the cleavage of high-molecular-weight kininogen in plasma, and plasma extravasation induced by polyphosphate. The crystal structure of PdSP15b contains an amphipathic helix studded with basic side chains that forms the likely interaction surface. CONCLUSIONS: The results of these studies indicate that the binding of anionic polymers by salivary proteins is used by blood feeders as an antihemostatic/anti-inflammatory mechanism.

De novo Ixodes ricinus salivary gland transcriptome analysis using two next-generation sequencing methodologies.

Tick salivary gland (SG) proteins possess powerful pharmacologic properties that facilitate tick feeding and pathogen transmission. For the first time, SG transcriptomes of Ixodes ricinus, an important disease vector for humans and animals, were analyzed using next-generation sequencing. SGs were collected from different tick life stages fed on various animal species, including cofeeding of nymphs and adults on the same host. Four cDNA samples were sequenced, discriminating tick SG transcriptomes of early- and late-feeding nymphs or adults. In total, 441,381,454 pyrosequencing reads and 67,703,183 Illumina reads were assembled into 272,220 contigs, of which 34,560 extensively annotated coding sequences are disclosed; 8686 coding sequences were submitted to GenBank. Overall, 13% of contigs were classified as secreted proteins that showed significant differences in the transcript representation among the 4 SG samples, including high numbers of sample-specific transcripts. Detailed phylogenetic reconstructions of two relatively abundant SG-secreted protein families demonstrated how this study improves our understanding of the molecular evolution of hematophagy in arthropods. Our data significantly increase the available genomic information for I. ricinus and form a solid basis for future tick genome/transcriptome assemblies and the functional analysis of effectors that mediate the feeding physiology and parasite-vector interaction of I. ricinus.

An insight into the sialome of Simulium guianense (DIPTERA:SIMulIIDAE), the main vector of River Blindness Disease in Brazil.

BACKGROUND: Little is known about the composition and function of the saliva in black flies such as Simulium guianense, the main vector of river blindness disease in Brazil. The complex salivary potion of hematophagous arthropods counteracts their host's hemostasis, inflammation, and immunity. RESULTS: Transcriptome analysis revealed ubiquitous salivary protein families--such as the Antigen-5, Yellow, Kunitz domain, and serine proteases--in the S. guianense sialotranscriptome. Insect-specific families were also found. About 63.4% of all secreted products revealed protein families found only in Simulium. Additionally, we found a novel peptide similar to kunitoxin with a structure distantly related to serine protease inhibitors. This study revealed a relative increase of transcripts of the SVEP protein family when compared with Simulium vittatum and S. nigrimanum sialotranscriptomes. We were able to extract coding sequences from 164 proteins associated with blood and sugar feeding, the majority of which were confirmed by proteome analysis. CONCLUSIONS: Our results contribute to understanding the role of Simulium saliva in transmission of Onchocerca volvulus and evolution of salivary proteins in black flies. It also consists of a platform for mining novel anti-hemostatic compounds, vaccine candidates against filariasis, and immuno-epidemiologic markers of vector exposure.