Ticks salivate while feeding on their hosts. Saliva helps blood feeding through host anti-hemostatic and immunomodulatory components. Previous transcriptomic and proteomic studies revealed the ...complexity of tick saliva, comprising hundreds of polypeptides grouped in several multi-genic families such as lipocalins, Kunitz-domain containing peptides, metalloproteases, basic tail secreted proteins, and several other families uniquely found in ticks. These studies also revealed that the composition of saliva changes with time; expression of transcripts from the same family wax and wane as a function of feeding time. Here, we examined whether host immune factors could influence sialome switching by comparing sialomes of ticks fed naturally on a rabbit, to ticks artificially fed on defibrinated blood depleted of immune components. Previous studies were based on transcriptomes derived from pools of several individuals. To get an insight into the uniqueness of tick sialomes, we performed transcriptomic analyses of single salivary glands dissected from individual adult female I. ricinus ticks. Multivariate analysis identified 1,279 contigs differentially expressed as a function of time and/or feeding mode. Cluster analysis of these contigs revealed nine clusters of differentially expressed genes, four of which appeared consistently across several replicates, but five clusters were idiosyncratic, pointing to the uniqueness of sialomes in individual ticks. The disclosure of tick quantum sialomes reveals the unique salivary composition produced by individual ticks as they switch their sialomes throughout the blood meal, a possible mechanism of immune evasion.
Hard ticks feed for several days or weeks on their hosts. Blood feeding is assisted by tick saliva, which is injected in the host skin regularly, alternating with blood ingestion. Tick saliva ...contains hundreds or thousands of different peptides and other bioactive compounds that assist feeding by inhibiting their hosts' blood clotting, platelet aggregation, vasoconstriction, as well as pain and itching. Immunomodulatory and antimicrobial peptides are also found in tick saliva. Molecular characterization of tick salivary compounds, or its sialome (from the Greek sialos = saliva), helps identification of possible antigens that might confer anti-tick immunity, as well as identifying novel pharmacologically active compounds. Amblyomma americanum is a major nuisance tick in Eastern and Southern US, being a vector of Theileria and Ehrlichia bacteria to animals and humans. Presently we report an RNAseq study concerning the salivary glands of adult female A. americanum ticks, which involved sequencing of four libraries collected at different times of feeding. A total of 5,792 coding sequences were deduced from the transcriptome assembly, 3,139 of which were publicly deposited, expanding from the previously available 146 salivary sequences found in GenBank. A remarkable time-dependent transcript expression was found, mostly related to secretory products, supporting the idea that ticks may have several "sialomes" that are expressed at different times during feeding. The molecular nature of this sialome switching remains unknown. The hyperlinked spreadsheet containing the deduced coding sequences can be found at http://exon.niaid.nih.gov/transcriptome/Amb_americanum/Ambame-web.xlsx.
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.
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.
This data set of proteins constitutes a mining platform for novel pharmacologically active proteins and for uncovering vaccine targets against A. maculatum and the diseases they carry.
Loa loa, the African eyeworm, is a major filarial pathogen of humans. Unlike most filariae, L. loa does not contain the obligate intracellular Wolbachia endosymbiont. We describe the 91.4-Mb genome ...of L. loa and that of the related filarial parasite Wuchereria bancrofti and predict 14,907 L. loa genes on the basis of microfilarial RNA sequencing. By comparing these genomes to that of another filarial parasite, Brugia malayi, and to those of several other nematodes, we demonstrate synteny among filariae but not with nonparasitic nematodes. The L. loa genome encodes many immunologically relevant genes, as well as protein kinases targeted by drugs currently approved for use in humans. Despite lacking Wolbachia, L. loa shows no new metabolic synthesis or transport capabilities compared to other filariae. These results suggest that the role of Wolbachia in filarial biology is more subtle than previously thought and reveal marked differences between parasitic and nonparasitic nematodes.
The variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of P. falciparum-infected red blood cells, is a critical virulence factor for ...malaria. Each parasite has 60 antigenically distinct var genes that each code for a different PfEMP1 protein. During infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune-evasion mechanism to avoid the host antibody response. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown. Here we show that knocking out the P. falciparum variant-silencing SET gene (here termed PfSETvs), which encodes an orthologue of Drosophila melanogaster ASH1 and controls histone H3 lysine 36 trimethylation (H3K36me3) on var genes, results in the transcription of virtually all var genes in the single parasite nuclei and their expression as proteins on the surface of individual infected red blood cells. PfSETvs-dependent H3K36me3 is present along the entire gene body, including the transcription start site, to silence var genes. With low occupancy of PfSETvs at both the transcription start site of var genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long noncoding RNA. These results uncover a previously unknown role of PfSETvs-dependent H3K36me3 in silencing var genes in P. falciparum that might provide a general mechanism by which orthologues of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1 proteins may also be applied to the development of a malaria vaccine.
The habit of blood feeding evolved independently in many insect orders of families. Sand flies and mosquitoes belong to separate lineages of blood-feeding Diptera and are thus considered to have ...evolved the trait independently. Because of this, sand fly salivary proteins differ structurally from those of mosquitoes, and orthologous groups are nearly impossible to define. An exception is the long-form D7-like proteins that show conservation with their mosquito counterparts of numerous residues associated with the N-terminal domain binding pocket. In mosquitoes, this pocket is responsible for the scavenging of proinflammatory cysteinyl leukotrienes and thromboxanes at the feeding site. Here we show that long-form D7 proteins AGE83092 and ABI15936 from the sand fly species, Phlebotomus papatasi and P. duboscqi, respectively, inhibit the activation of platelets by collagen and the thromboxane A
analog U46619. Using isothermal titration calorimetry, we also demonstrate direct binding of U46619 and cysteinyl leukotrienes C
, D
and E
to the P. papatasi protein. The crystal structure of P. duboscqi ABI15936 was determined and found to contain two domains oriented similarly to those of the mosquito proteins. The N-terminal domain contains an apparent eicosanoid binding pocket. The C-terminal domain is smaller in overall size than in the mosquito D7s and is missing some helical elements. Consequently, it does not contain an obvious internal binding pocket for small-molecule ligands that bind to many mosquito D7s. Structural similarities indicate that mosquito and sand fly D7 proteins have evolved from similar progenitors, but phylogenetics and differences in intron/exon structure suggest that they may have acquired the ability to bind vertebrate eicosanoids independently, indicating a convergent evolution scenario.
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.
Only adult female mosquitoes feed on blood, while both genders take sugar meals. Accordingly, several compounds associated with blood feeding (i.e. vasodilators, anti-clotting, anti-platelets) are ...found only in female glands, while enzymes associated with sugar feeding or antimicrobials (such as lysozyme) are found in the glands of both sexes. We performed de novo assembly of reads from adult Aedes aegypti female and male salivary gland libraries (285 and 90 million reads, respectively). By mapping back the reads to the assembled contigs, plus mapping the reads from a publicly available Ae. aegypti library from adult whole bodies, we identified 360 transcripts (including splice variants and alleles) overexpressed tenfold or more in the glands when compared to whole bodies. Moreover, among these, 207 were overexpressed fivefold or more in female vs. male salivary glands, 85 were near equally expressed and 68 were overexpressed in male glands. We call in particular the attention to C-type lectins, angiopoietins, female-specific Antigen 5, the 9.7 kDa, 12-14 kDa, 23.5 kDa, 62/34 kDa, 4.2 kDa, proline-rich peptide, SG8, 8.7 kDa family and SGS fragments: these polypeptides are all of unknown function, but due to their overexpression in female salivary glands and putative secretory nature they are expected to affect host physiology. We have also found many transposons (some of which novel) and several endogenous viral transcripts (probably acquired by horizontal transfer) which are overexpressed in the salivary glands and may play some role in tissue-specific gene regulation or represent a mechanism of virus interference. This work contributes to a near definitive catalog of male and female salivary gland transcripts from Ae. aegypti, which will help to direct further studies aiming at the functional characterization of the many transcripts with unknown function and the understanding of their role in vector-host interaction and pathogen transmission.
The hematophagous behaviour emerged independently in several instances during arthropod evolution. Survey of salivary gland and saliva composition and its pharmacological activity led to the ...conclusion that blood-feeding arthropods evolved a distinct salivary mixture that can interfere with host defensive response, thus facilitating blood acquisition and pathogen transmission. The cat flea, Ctenocephalides felis, is the major vector of several pathogens, including Rickettsia typhi, Rickettsia felis and Bartonella spp. and therefore, represents an important insect species from the medical and veterinary perspectives. Previously, a Sanger-based sialome of adult C. felis female salivary glands was published and reported 1,840 expressing sequence tags (ESTs) which were assembled into 896 contigs. Here, we provide a deeper insight into C. felis salivary gland composition using an Illumina-based sequencing approach. In the current dataset, we report 8,892 coding sequences (CDS) classified into 27 functional classes, which were assembled from 42,754,615 reads. Moreover, we paired our RNAseq data with a mass spectrometry analysis using the translated transcripts as a reference, confirming the presence of several putative secreted protein families in the cat flea salivary gland homogenates. Both transcriptomic and proteomic approaches confirmed that FS-H-like proteins and acid phosphatases lacking their putative catalytic residues are the two most abundant salivary proteins families of C. felis and are potentially related to blood acquisition. We also report several novel sequences similar to apyrases, odorant binding proteins, antigen 5, cholinesterases, proteases, and proteases inhibitors, in addition to putative novel sequences that presented low or no sequence identity to previously deposited sequences. Together, the data represents an extended reference for the identification and characterization of the pharmacological activity present in C. felis salivary glands.
No commercially licensed vaccine or treatment is available for dengue fever, a potentially lethal infection that impacts millions of lives annually. New tools that target mosquito control may reduce ...vector populations and break the cycle of dengue transmission. Male mosquito seminal fluid proteins (Sfps) are one such target since these proteins, in aggregate, modulate the reproduction and feeding patterns of the dengue vector, Aedes aegypti. As an initial step in identifying new targets for dengue vector control, we sought to identify the suite of proteins that comprise the Ae. aegypti ejaculate and determine which are transferred to females during mating.
Using a stable-isotope labeling method coupled with proteomics to distinguish male- and female-derived proteins, we identified Sfps and sperm proteins transferred from males to females. Sfps were distinguished from sperm proteins by comparing the transferred proteins to sperm-enriched samples derived from testes and seminal vesicles. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera.
This is the first study to directly identify Sfps transferred from male Ae. aegypti to females. Our data lay the groundwork for future functional analyses to identify individual seminal proteins that may trigger female post-mating changes (e.g., in feeding patterns and egg production). Therefore, identification of these proteins may lead to new approaches for manipulating the reproductive output and vectorial capacity of Ae. aegypti.
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