Exploring the sialome of the tick Ixodes scapularis

To attempt description of the set of mRNA and protein (sialome) expressed in the salivary glands of the tick Ixodes scapularis, we randomly sequenced 735 clones of a full-length salivary gland cDNA library of this arthropod and performed Edman degradation of protein bands from salivary gland homogenates (SGH) and saliva separated by SDS-PAGE. The sequences were grouped into 410 clusters, of which 383 are not associated with known I. scapularis sequences. 15- and 17-protein bands from PAGE yielded amino-terminal information on the saliva and salivary gland gels, respectively. We attributed 19 of these sequences to translation products of the cDNA library. Full-length sequences were obtained for 87 clones. Among these protein sequences are several protease inhibitors of distinct classes, metalloproteases, novel proteins with histamine-binding domains, and several peptide families of unknown function displaying different conserved cysteine residues, many of which contain single Kunitz domains. This work provides information into the diversity of messages expressed in the salivary glands of I. scapularis, describes novel sequences that may be responsible for known biological activites, indicates further biological activities that may be present in I. scapularis saliva and identifies novel vaccine targets that may be used in Lyme disease prevention

Ixonnexin from Tick Saliva Promotes Fibrinolysis by Interacting with Plasminogen and Tissue-Type Plasminogen Activator, and Prevents Arterial Thrombosis

Tick saliva is a rich source of modulators of vascular biology. We have characterized Ixonnexin, a member of the “Basic-tail” family of salivary proteins from the tick Ixodes scapularis. Ixonnexin is a 104 residues (11.8 KDa), non-enzymatic basic protein which contains 3 disulfide bonds and a C-terminal rich in lysine. It is homologous to SALP14, a tick salivary FXa anticoagulant. Ixonnexin was produced by ligation of synthesized fragments (51–104) and (1–50) followed by folding. Ixonnexin, like SALP14, interacts with FXa. Notably, Ixonnexin also modulates fibrinolysis in vitro by a unique salivary mechanism. Accordingly, it accelerates plasminogen activation by tissue-type plasminogen activator (t-PA) with Km 100 nM; however, it does not affect urokinase-mediated fibrinolysis. Additionally, lysine analogue ε-aminocaproic acid inhibits Ixonnexin-mediated plasmin generation implying that lysine-binding sites of Kringle domain(s) of plasminogen or t-PA are involved in this process. Moreover, surface plasmon resonance experiments shows that Ixonnexin binds t-PA, and plasminogen (KD 10 nM), but not urokinase. These results imply that Ixonnexin promotes fibrinolysis by supporting the interaction of plasminogen with t-PA through formation of an enzymatically productive ternary complex. Finally, in vivo experiments demonstrates that Ixonnexin inhibits FeCl3-induced thrombosis in mice. Ixonnexin emerges as novel modulator of fibrinolysis which may also affect parasite-vector-host interactions

Purification and functional characterisation of rhiminopeptidase A, a novel aminopeptidase from the venom of Bitis gabonica rhinoceros

This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites

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

<p>Abstract</p> <p>Background</p> <p>Little is known about the composition and function of the saliva in black flies such as <it>Simulium guianense</it>, the main vector of river blindness disease in Brazil. The complex salivary potion of hematophagous arthropods counteracts their host's hemostasis, inflammation, and immunity.</p> <p>Results</p> <p>Transcriptome analysis revealed ubiquitous salivary protein families--such as the Antigen-5, Yellow, Kunitz domain, and serine proteases--in the <it>S. guianense </it>sialotranscriptome. Insect-specific families were also found. About 63.4% of all secreted products revealed protein families found only in <it>Simulium</it>. 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 <it>Simulium vittatum </it>and <it>S. nigrimanum </it>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.</p> <p>Conclusions</p> <p>Our results contribute to understanding the role of <it>Simulium </it>saliva in transmission of <it>Onchocerca volvulus </it>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.</p

Modulation of the immune response to Mycobacterium tuberculosis during malaria/M. tuberculosis co-infection

Tuberculosis (TB) causes significant morbidity and mortality on a global scale. The African region has 24% of the world's TB cases. TB overlaps with other infectious diseases such as malaria and HIV, which are also highly prevalent in the African region. TB is a leading cause of death among HIV-positive patients and co-infection with HIV and TB has been described as a syndemic. In view of the overlapping epidemiology of these diseases, it is important to understand the dynamics of the immune response to TB in the context of co-infection. We investigated the cytokine response to purified protein derivative (PPD) in peripheral blood mononuclear cells from TB patients co-infected with HIV or malaria and compared it to that of malaria- and HIV-free TB patients. A total of 231 subjects were recruited for this study and classified into six groups; untreated TB-positive, TB positive subjects on TB drugs, TB- and HIV-positive, TB- and malaria-positive, latent TB and apparently healthy control subjects. Our results demonstrate maintenance of interferon (IFN)-γ production in HIV and malaria co-infected TB patients in spite of lower CD4 counts in the HIV-infected cohort. Malaria co-infection caused an increase in the production of the T helper type 2 (Th2)-associated cytokine interleukin (IL)-4 and the anti-inflammatory cytokine IL-10 in PPD-stimulated cultures. These results suggest that malaria co-infection diverts immune response against M. tuberculosis towards a Th-2/anti-inflammatory response which might have important consequences for disease progression

Maternal Malaria Induces a Procoagulant and Antifibrinolytic State That Is Embryotoxic but Responsive to Anticoagulant Therapy

Low birth weight and fetal loss are commonly attributed to malaria in endemic areas, but the cellular and molecular mechanisms that underlie these poor birth outcomes are incompletely understood. Increasing evidence suggests that dysregulated hemostasis is important in malaria pathogenesis, but its role in placental malaria (PM), characterized by intervillous sequestration of Plasmodium falciparum, proinflammatory responses, and excessive fibrin deposition is not known. To address this question, markers of coagulation and fibrinolysis were assessed in placentae from malaria-exposed primigravid women. PM was associated with significantly elevated placental monocyte and proinflammatory marker levels, enhanced perivillous fibrin deposition, and increased markers of activated coagulation and suppressed fibrinolysis in placental plasma. Submicroscopic PM was not proinflammatory but tended to be procoagulant and antifibrinolytic. Birth weight trended downward in association with placental parasitemia and high fibrin score. To directly assess the importance of coagulation in malaria-induced compromise of pregnancy, Plasmodium chabaudi AS-infected pregnant C57BL/6 mice were treated with the anticoagulant, low molecular weight heparin. Treatment rescued pregnancy at midgestation, with substantially decreased rates of active abortion and reduced placental and embryonic hemorrhage and necrosis relative to untreated animals. Together, the results suggest that dysregulated hemostasis may represent a novel therapeutic target in malaria-compromised pregnancies

A Deep Insight into the Sialotranscriptome of the Gulf Coast Tick, Amblyomma maculatum

Background: Saliva of blood sucking arthropods contains compounds that antagonize their hosts ’ hemostasis, which include platelet aggregation, vasoconstriction and blood clotting; saliva of these organisms also has anti-inflammatory and immunomodullatory properties. Perhaps because hosts mount an active immune response against these compounds, the diversity of these compounds is large even among related blood sucking species. Because of these properties, saliva helps blood feeding as well as help the establishment of pathogens that can be transmitted during blood feeding. Methodology/Principal Findings: We have obtained 1,626,969 reads by pyrosequencing a salivary gland cDNA library from adult females Amblyomma maculatum ticks at different times of feeding. Assembly of this data produced 72,441 sequences larger than 149 nucleotides from which 15,914 coding sequences were extracted. Of these, 5,353 had.75 % coverage to their best match in the non-redundant database from the National Center for Biotechnology information, allowing for the deposition of 4,850 sequences to GenBank. The annotated data sets are available as hyperlinked spreadsheets. Putative secreted proteins were classified in 133 families, most of which have no known function. Conclusions/Significance: This data set of proteins constitutes a mining platform for novel pharmacologically activ

Deep Sequencing of Pyrethroid-Resistant Bed Bugs Reveals Multiple Mechanisms of Resistance within a Single Population

A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD50 bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance

Novel immunomodulators from hard ticks selectively reprogramme human dendritic cell responses

Hard ticks subvert the immune responses of their vertebrate hosts in order to feed for much longer periods than other blood-feeding ectoparasites; this may be one reason why they transmit perhaps the greatest diversity of pathogens of any arthropod vector. Tick-induced immunomodulation is mediated by salivary components, some of which neutralise elements of innate immunity or inhibit the development of adaptive immunity. As dendritic cells (DC) trigger and help to regulate adaptive immunity, they are an ideal target for immunomodulation. However, previously described immunoactive components of tick saliva are either highly promiscuous in their cellular and molecular targets or have limited effects on DC. Here we address the question of whether the largest and globally most important group of ticks (the ixodid metastriates) produce salivary molecules that specifically modulate DC activity. We used chromatography to isolate a salivary gland protein (Japanin) from Rhipicephalus appendiculatus ticks. Japanin was cloned, and recombinant protein was produced in a baculoviral expression system. We found that Japanin specifically reprogrammes DC responses to a wide variety of stimuli in vitro, radically altering their expression of co-stimulatory and co-inhibitory transmembrane molecules (measured by flow cytometry) and their secretion of pro-inflammatory, anti-inflammatory and T cell polarising cytokines (assessed by Luminex multiplex assays); it also inhibits the differentiation of DC from monocytes. Sequence alignments and enzymatic deglycosylation revealed Japanin to be a 17.7 kDa, N-glycosylated lipocalin. Using molecular cloning and database searches, we have identified a group of homologous proteins in R. appendiculatus and related species, three of which we have expressed and shown to possess DC-modulatory activity. All data were obtained using DC generated from at least four human blood donors, with rigorous statistical analysis. Our results suggest a previously unknown mechanism for parasite-induced subversion of adaptive immunity, one which may also facilitate pathogen transmission