Andrezza C. Chagas

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.—Schwarz, A., von Reumont, B.M., Erhart, J., Chagas, AC., Ribeiro, J.M.C., Kotsyfakis, M. De novo Ixodes ricinus salivary gland transcriptome analysis using two next‐generation sequencing methodologies. FASEB J. 27, 4745–4756 (2013). www.fasebj.org

Lundep, a Sand Fly Salivary Endonuclease Increases Leishmania Parasite Survival in Neutrophils and Inhibits XIIa Contact Activation in Human Plasma

Neutrophils are the hosts 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.

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.

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

BackgroundPsorophora 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.ResultsIllumina 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.ConclusionsThis 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.

Tick sialostatins L and L2 differentially influence dendritic cell responses to Borrelia spirochetes

BackgroundTransmission 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.MethodsDCs 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.ResultsWe 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.ConclusionsThis 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.

Tick Salivary Sialostatin L Represses the Initiation of Immune Responses by Targeting IRF4-Dependent Transcription in Murine Mast Cells

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.

Leishmaniose Tegumentar Americana (LTA) em uma vila de exploração de minérios - Pitinga, município de Presidente Figueiredo, Amazonas, Brasil

A Vila de Pitinga, Presidente Figueiredo/AM, e uma area de exploracao de minerios e endemica para leishmaniose tegumentar americana (LTA). A vila e a sede administrativa e local de moradia de seus funcionarios. O comportamento epidemiologico da endemia, avaliado para o periodo de 2000 a 2004, foi relacionado com as medidas de controle adotadas para reduzir a incidencia da doenca na area e comparado com o registrado para o municipio e o Estado do Amazonas. A maior proporcao dos casos detectados no periodo ocorreu no genero masculino, com atividades laborais de contato com a floresta. O declinio na incidencia de casos de LTA na area do estudo, nao foi observado como ocorrido no Municipio e Estado e foi considerado resultante das medidas de controle para a doenca, aplicadas na area.

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

BackgroundLittle 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 hosts hemostasis, inflammation, and immunity.ResultsTranscriptome 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.ConclusionsOur 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.

Collagen-binding protein, Aegyptin, regulates probing time and blood feeding success in the dengue vector mosquito, Aedes aegypti

Significance Hematophagy is key to mosquito reproductive success and an important link in pathogen transmission cycles. Vertebrate hemostasis, specifically platelet aggregation, vasoconstriction, and coagulation, presents a significant challenge to successful blood feeding. Mosquitoes have been selected to secrete salivary proteins with powerful antihemostatic activities. Aegyptin, a salivary protein from the dengue, chikungunya, and yellow fever vector, Aedes aegypti, binds collagen and inhibits platelet aggregation and adhesion. A transgene-based, gene-silencing approach elicited a specific and significant reduction in Aegyptin mRNA and protein levels in female salivary glands. The resulting prolonged probing times and reductions in feeding quality and quantity support the conclusion that platelet aggregation inhibition is a vital salivary function and that Aegyptin plays a significant and nonredundant role in successful feeding. 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.