An Insight into the Sialome of the Lone Star Tick, \u3ci\u3eAmbylomma americanum\u3c/i\u3e, With a Glimpse On Its Time Dependent Gene Expression

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

A Divergent Strain of Culex pipiens-Associated Tunisia Virus in the Malaria Vector Anopheles epiroticus

Here, we report the draft genome sequence of a divergent strain ofCulex pipiens-associated Tunisia virus (CpATV) identified in the malaria vectorAnoph-eles epiroticus(CpATV-AnE). CpATV-AnE expands the reference virus sequence, intro-ducing an extended replicase with novel virga-like domains. Our results suggest thatthe host range of CpATV includesAnophelessp. mosquitoes.Instituto de Patología VegetalFil: Debat, Humberto J. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal (IPAVE). Córdoba; ArgentinaFil: Ribero, Jose M C. National Institute of Allergy and Infectious Diseases (NIAID-NIH). Laboratory of Malaria and Vector Research.Section of Vector Biology; Estados Unido

A Deep Insight into the Sialotranscriptome of the Gulf Coast Tick, \u3ci\u3eAmblyomma maculatum\u3c/i\u3e

Background Saliva of blood sucking arthropods contains compounds that antagonize their hosts\u27 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 \u3e75% 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 active proteins and for uncovering vaccine targets against A. maculatum and the diseases they carry

Purification and characterization of a novel salivary antimicrobial peptide from the tick, \u3cem\u3eIxodes scapularis\u3c/em\u3e

A novel antimicrobial peptide was isolated from the saliva of the Lyme disease tick vector, Ixodes scapularis, henceforth designated as ISAMP (I. scapularis Antimicrobial Peptide). ISAMP was purified using a sequential method including ultra filtration, gel filtration and reverse-phase high performance liquid chromatography. The purified peak had a molecular weight of 5.3 kDa by MALDI/TOF-MS and its amino acid sequence, determined by Edman degradation was PDxGxPxxVKAGRxPxxSI. A BLASTP search revealed that the protein is a putative 5.3 kDa secreted protein (AAM93656) from I. scapularis. The predicted protein is composed of 69 amino acids with no conserved domain motifs. Purified ISAMP was found to have antimicrobial activities against bacteria. Gene expression studies were carried out to observe ISAMP expression in different tick tissues. RT-PCR results indicated that the gene was expressed in hemocytes, fat body and salivary gland but virtually no expression was observed in the midgut. ISAMP is only similar to other Ixodid tick proteins, thus it is a member of a unique family

Tick saliva is a potent inhibitor of endothelial cell proliferation and angiogenesis

We report for the first time that saliva of the hard tick and Lyme disease vector,Ixodes scapularis,is a potent inhibitor of angiogenesis. Saliva (≤ 1:500 dilutions) or salivary gland (0.1–0.5 pairs/assay) dose-dependently inhibits microvascular endothelial cell (MVEC) proliferation. Inhibition was also detected with the saliva of the cattle tick Boophilus microplus but not with the salivary gland of Anopheles gambiae, An. stephensi, Lutzomyia longipalpis, Phlebotomus papatasi, Aedes aegypti, Culex quinquefasciatus, and Cimex lectularius. Inhibition of MVEC proliferation by I. scapularis saliva was accompanied by a change in cell shape (shrinkage of the cytoplasm with loss of cell-cell interactions) and apoptosis which was estimated by expression of phosphatidylserine using the Apopercentage dye, and by a typical pattern of chromatin margination, condensation, and fragmentation as revealed by nuclear staining with Hoechst 33258.The effect of saliva appears to be mediated by endothelial cell α5β1 integrin, because monoclonal antibodies against this but not αvβ3, αvβ5, α9β1, or α2β1 integrins remarkably block its effect. In addition, SDS/PAGE shows that saliva specifically degrades purified α5β1 but not αvβ5 or αvβ3 integrins. Incubation of saliva with EDTA and 1,10-phenanthroline, but not phenylmethylsulfonyl fluoride (PMSF), inhibits saliva-dependent degradation of purified α5β1 integrin, suggesting that a metalloprotease is responsible for the activity. Finally, saliva at ≤ 1:1,000 dilutions blocks sprouting formation from chick embryo aorta implanted in Matrigel, an in vitro model of angiogenesis. These findings introduce the concept that tick saliva is a negative modulator of angiogenesis-dependent wound healing and tissue repair, therefore allowing ticks to feed for days. Inhibition of angiogenesis was hitherto an unidentified biologic property of the saliva of any blood-sucking arthropod studied so far. Its presence in tick saliva may be regarded as an additional source of angiogenesis inhibitors with potential applications for the study of both vector and vascular biology

Cloning of a salivary gland metalloprotease and characterization of gelatinase and fibrin(ogen)lytic activities in the saliva of the Lyme disease tick vector Ixodes scapularis

The full-length sequence of tick salivary gland cDNA coding for a protein similar to metalloproteases (MP) of the reprolysin family is reported. The Ixodes scapularis MP is a 488 amino acid (aa) protein containing pre- and pro-enzyme domains, the zinc-binding motif HExxHxxGxxH common to metalloproteases, and a cysteine-rich region. In addition, the predicted amino-terminal sequences of I. scapularis MPs were found by Edman degradation of PVDF-transferred SDS/PAGE-separated tick saliva proteins, indicating that these putative enzymes are secreted. Furthermore, saliva has a metal-dependent proteolytic activity towards gelatin, fibrin(ogen), and fibronectin, but not collagen or laminin. Accordingly, I. scapularis saliva has a rather specific metalloprotease similar to the hemorrhagic proteases of snake venoms. This is the first description of such activity in tick saliva and its role in tick feeding and Borrelia transmission is discussed

Purification of a serine protease and evidence for a protein C activator from the saliva of the tick, \u3cem\u3eIxodes scapularis\u3c/em\u3e

The saliva of ticks is critical to their survival as parasites and hematophagous animals. In this study, we have purified an enzyme with trypsin-like activity from the saliva of the tick vector of Lyme Disease, Ixodes scapularis. This enzyme, named as IXOSP (I. scapularis salivary serine protease), is a 29.9 kDa molecule with N-terminus FPxMVxLRIKxR. A BLAST search identified IXOSP as a secreted serine protease (AAY66740) with a conserved catalytic triad His, Asp, and Ser. In vitro studies demonstrated that IXOSP cleaves chromogenic substrates with arginine in the P1 position, by a mechanism inhibited by PMSF or aprotinin. Gene expression studies revealed that IXOSP is expressed at different tick developmental stages, including eggs, and unfed or fed adult tick salivary glands, but not in nymphs or in the midgut. While the physiological substrate for IXOSP remains to be identified, we demonstrated that I. scapularis saliva activate protein C (PC) resulting in the production of activated PC, a potent anticoagulant that also regulates a myriad of inflammatory responses through protease activated receptors. In contrast, the salivary glands of Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Aedes aegypti, Lutzomyia longipalpis, and Phlebotomus ariasi did not activate protein C. These discoveries are discussed in the context of blood coagulation, inflammation and vector–host interactions

Knockdown of Selenocysteine-Specific Elongation Factor in \u3ci\u3eAmblyomma maculatum\u3c/i\u3e Alters the Pathogen Burden of \u3ci\u3eRickettsia parkeri\u3c/i\u3e with Epigenetic Control by the Sin3 Histone Deacetylase Corepressor Complex

Selenocysteine is the 21st naturally-occurring amino acid. Selenoproteins have diverse functions and many remain uncharacterized, but they are typically associated with antioxidant activity. The incorporation of selenocysteine into the nascent polypeptide chain recodes the TGA stop codon and this process depends upon a number of essential factors including the selenocysteine elongation factor (SEF). The transcriptional expression of SEF did not change significantly in tick midguts throughout the blood meal, but decreased in salivary glands to 20% at the end of the fast feeding phase. Since selenoprotein translation requires this specialized elongation factor, we targeted this gene for knockdown by RNAi to gain a global view of the role selenoproteins play in tick physiology. We found no significant differences in tick engorgement and embryogenesis but detected no antioxidant capacity in tick saliva. The transcriptional profile of selenoproteins in R. parkeri-infected Amblyomma maculatum revealed declined activity of selenoprotein M and catalase and increased activity of selenoprotein O, selenoprotein S, and selenoprotein T. Furthermore, the pathogen burden was significantly altered in SEF-knockdowns. We then determined the global impact of SEF-knockdown by RNA-seq, and mapped huge shifts in secretory gene expression that could be the result of downregulation of the Sin3 histone deacetylase corepressor complex

The transcriptome of the salivary glands of the female western black-legged tick \u3cem\u3eIxodes pacificus\u3c/em\u3e (Acari: Ixodidae)

Sequencing of an Ixodes pacificus salivary gland cDNA library yielded 1068 sequences with an average undetermined nucleotide of 1.9% and an average length of 487 base pairs. Assembly of the expressed sequence tags yielded 557 contigs, 138 of which appear to code for secreted peptides or proteins based on translation of a putative signal peptide. Based on the BLASTX similarity of these contigs to 66 matches of Ixodes scapularis peptide sequences, only 58% sequence identity was found, indicating a rapid divergence of salivary proteins as observed previously for mosquito and triatomine bug salivary proteins. Here we report 106 mostly full-length sequences that clustered in 16 different families: Basic-tail proteins rich in lysine in the carboxy-terminal, Kunitz-containing proteins (monolaris, ixolaris and penthalaris families), proline-rich peptides, 5-, 9.4- and 18.7-kDa proteins of unknown functions, in addition to metalloproteases (class PIII-like) similar to reprolysins. We also have found a family of disintegrins, named ixodegrins that display homology to variabilin, a GPIIb/IIIa antagonist from the tick Dermacentor variabilis. In addition, we describe peptides (here named ixostatins) that display remarkable similarities to the cysteine-rich domain of ADAMST-4 (aggrecanase). Many molecules were assigned in the lipocalin family (histamine-binding proteins); others appear to be involved in oxidant metabolism, and still others were similar to ixodid proteins such as the anticomplement ISAC. We also identified for the first time a neuropeptide-like protein (nlp-31) with GGY repeats that may have antimicrobial activity. In addition, 16 novel proteins without significant similarities to other tick proteins and 37 housekeeping proteins that may be useful for phylogenetic studies are described. Some of these proteins may be useful for studying vascular biology or the immune system, for vaccine development, or as immunoreagents to detect prior exposure to ticks