A proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva.

BACKGROUND: The argasid tick Ornithodoros erraticus is the main vector of tick-borne human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin. The prevention and control of these diseases would greatly benefit from the elimination of O. erraticus populations, and anti-tick vaccines are envisaged as an effective and sustainable alternative to chemical acaricide usage for tick control. Ornithodoros erraticus saliva contains bioactive proteins that play essential functions in tick feeding and host defence modulation, which may contribute to host infection by tick-borne pathogens. Hence, these proteins could be candidate antigen targets for the development of vaccines aimed at the control and prevention of O. erraticus infestations and the diseases this tick transmits. The objective of the present work was to obtain and characterise the proteome of the saliva of O. erraticus adult ticks as a means to identify and select novel salivary antigen targets. METHODS: A proteomics informed by transcriptomics (PIT) approach was applied to analyse samples of female and male saliva separately using the previously obtained O. erraticus sialotranscriptome as a reference database and two different mass spectrometry techniques, namely liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-dependent acquisition mode and sequential window acquisition of all theoretical fragment ion spectra MS (SWATH-MS). RESULTS: Up to 264 and 263 proteins were identified by LC-MS/MS in the saliva of O. erraticus female and male ticks, respectively, totalling 387 non-redundant proteins. Of these, 224 were further quantified by SWATH-MS in the saliva of both male and female ticks. Quantified proteins were classified into 23 functional categories and their abundance compared between sexes. Heme/iron-binding proteins, protease inhibitors, proteases, lipocalins and immune-related proteins were the categories most abundantly expressed in females, while glycolytic enzymes, protease inhibitors and lipocalins were the most abundantly expressed in males. Ninety-seven proteins were differentially expressed between the sexes, of which 37 and 60 were overexpressed in females and males, respectively. CONCLUSIONS: The PIT approach demonstrated its usefulness for proteomics studies of O. erraticus, a non-model organism without genomic sequences available, allowing the publication of the first comprehensive proteome of the saliva of O. erraticus reported to date. These findings confirm important quantitative differences between sexes in the O. erraticus saliva proteome, unveil novel salivary proteins and functions at the tick-host feeding interface and improve our understanding of the physiology of feeding in O. erraticus ticks. The integration of O. erraticus sialoproteomic and sialotranscriptomic data will drive a more rational selection of salivary candidates as antigen targets for the development of vaccines aimed at the control of O. erraticus infestations and the diseases it transmits.

Characterization of a single-domain von Willebrand factor type C protein (HaSVC) from the salivary gland of the tick Hyalomma asiaticum.

As a widely distributed arthropod and vector for various pathogens, Hyalomma asiaticum presents great risk and potential losses in animal husbandry. Effective measures, including the use of vaccines, are necessary for controlling ticks and tick-borne diseases. A concise understanding of the tick-host interaction associated molecules and pathways is required for vaccine development. In the present study, a protein containing a single-domain von Willebrand factor type C (HaSVC) was isolated from H. asiaticum and was subjected to functional identification. As a result, the full-length sequence of the HaSVC (506 bp) gene was obtained, which putatively encodes 100 amino acids with a predicted molecular mass of 11 kDa, excluding the 23-amino acid signal peptide. HaSVC contains 8 cysteines to form 4 disulfide bonds. The native HaSVC protein was detected in multiple tick organs. HaSVC neither attenuated the anti-coagulation process nor directly affected the blood feeding of adult ticks. However, the purified recombinant protein HaSVC (rHaSVC/GST) significantly increased the proliferation of mice spleen cells. This might suggest a regulatory function for HaSVC on inflammation, thus providing new information that may explain the "crosstalk" between ticks and hosts.

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.

Rhipicephalus appendiculatus: characterization of a testis-associated protein.

A novel gene coding for Rhipicephalus appendiculatus Male-specific Protein (RAMP) was identified in a cDNA library constructed from the testis/vas deferens of R. appendiculatus ticks. This gene encodes a secreted protein exclusively expressed in the testis/vas deferens. The putative RAMP amino acid sequence contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMP mRNA was up-regulated in male ticks during blood feeding. RAMP was detected not only in the testis/vas deferens of males but also in postcoitum female ticks based on Western blotting, indicating that this protein is transferred to the female tick during copulation. Virgin female ticks, microinjected with recombinant RAMP, had significantly prolonged attachment duration during feeding, but there was no effect on fed weight. These results suggest that RAMP is a male-specific molecule in the spermatophore, and is related to female attachment behavior in R. appendiculatus ticks.

Ixodes ricinus: the potential of two-dimensional gel electrophoresis as a tool for studying host-vector-pathogen interactions.

Ixodes ricinus is a three-host tick, with three active instars. For moulting to occur the tick has to find a host where it can take a blood meal. Throughout feeding I. ricinus can be infected or infect the host with different pathogens, e.g., Tick-Borne Encephalitis virus or Borrelia burgdorferi. The host-vector-pathogen interaction is very complex, making a detailed study difficult. Here we analyse the potential of two-dimensional gel electrophoresis (2DE) to study the host-vector-pathogen interaction. We examined 20 nymphs, which as larvae parasitised either mouse or hen. After moulting, they were kept alive for up to 30 weeks, to analyse whether tick ageing influenced host determination, and for comparison of the 2D-gels. Even though the number of proteins in the gel decreased during ageing, some proteins of the host determination persisted for all 30 weeks. We also discovered persisting proteins in relation to nymphs. These findings showed that 2DE is suitable as a tool for studying host-vector-pathogen interactions.

Borreliacidal activity of saliva of the tick Amblyomma americanum.

Amblyomma americanum (Linneaus) (Acari: Ixodidae), an important tick vector of human and animal disease, is not a competent vector of the bacterial agent of Lyme disease, Borrelia burgdorferi, although its range overlaps the geographical distribution of Lyme disease within the United States. A possible mechanism that could prevent acquisition of B. burgdorferi spirochetes from infected hosts is the toxic effect of A. americanum saliva on B. burgdorferi. The data presented here indicate that after 24 and 48 h of exposure to A. americanum saliva, significantly fewer B. burgdorferi were alive compared to treatment controls as assessed by spirochete motility under dark-field microscopy and resistance to the dead stain, propidium iodide. After 48 h, fewer than 13% of saliva-exposed B. burgdorferi were alive. In contrast, significantly more B. burgdorferi exposed to Ixodes scapularis (Acari: Ixodidae) saliva survived after 24 or 48 h compared to A. americanum saliva or treatment controls.

Exploring tick saliva: from biochemistry to 'sialomes' and functional genomics.

Tick saliva, a fluid once believed to be only relevant for lubrication of mouthparts and water balance, is now well known to be a cocktail of potent anti-haemostatic, anti-inflammatory and immunomodulatory molecules that helps these arthropods obtain a blood meal from their vertebrate hosts. The repertoire of pharmacologically active components in this cocktail is impressive as well as the number of targets they specifically affect. These salivary components change the physiology of the host at the bite site and, consequently, some pathogens transmitted by ticks take advantage of this change and become more infective. Tick salivary proteins have therefore become an attractive target to control tick-borne diseases. Recent advances in molecular biology, protein chemistry and computational biology are accelerating the isolation, sequencing and analysis of a large number of transcripts and proteins from the saliva of different ticks. Many of these newly isolated genes code for proteins with homologies to known proteins allowing identification or prediction of their function. However, most of these genes code for proteins with unknown functions therefore opening the road to functional genomic approaches to identify their biological activities and roles in blood feeding and hence, vaccine development to control tick-borne diseases.

Characteristics of scrapie isolates derived from hay mites.

Previous epidemiological evidence suggested that in some instances a vector and/or reservoir is involved in the occurrence and spread of transmissible spongiform encephalopathies (TSEs). In a preliminary study, hay mite preparations from five Icelandic farms with a history of scrapie were injected into mice, and some of these mice became sick after long incubation periods. To confirm that the disease was scrapie, subsequent passages in mice were performed. In addition, the characteristics of the disease process in these passages were assessed and the results compared to those findings with standard scrapie strains. As expected for scrapie, subsequent passages in the same host led to shortened incubation periods compared to those in primary isolate mice, and all mice had spongiform changes in brain. Results were similar for three of four isolates with regard to clinical manifestations, the incubation periods in mice of the three scrapie incubation-period genotypes (s7s7, s7p7, p7p7), and the PrPSc Western blot (WB) pattern. The characteristics of the fourth isolate were markedly different from the other three isolates with regard to these parameters. Comparison of the characteristics of standard mouse-adapted scrapie strains and the four isolates revealed differences; these differences were particularly pronounced for the fourth isolate.

Comparison of surface proteins of Anaplasma marginale grown in tick cell culture, tick salivary glands, and cattle.

Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, infects bovine erythrocytes, resulting in mild to severe hemolytic disease that causes economic losses in domestic livestock worldwide. Recently, the Virginia isolate of A. marginale was propagated in a continuous tick cell line, IDE8, derived from embryonic Ixodes scapularis. Development of A. marginale in cell culture was morphologically similar to that described previously in ticks. In order to evaluate the potential of the cell culture-derived organisms for use in future research or as an antigen for serologic tests and vaccines, the extent of structural conservation of the major surface proteins (MSPs) between the cell culture-derived A. marginale and the bovine erythrocytic stage, currently the source of A. marginale antigen, was determined. Structural conservation on the tick salivary-gland stage was also examined. Monoclonal and monospecific antisera against MSPs 1 through 5, initially characterized against erythrocyte stages, also reacted with A. marginale from cell culture and tick salivary glands. MSP1a among geographic A. marginale isolates is variable in size because of different numbers of a tandemly repeated 28- or 29-amino-acid peptide. The cell culture-derived A. marginale maintained the same-size MSP1a as that found on the Virginia isolate of A. marginale in bovine erythrocytes and tick salivary glands. Although differences were observed in the polymorphic MSP2 antigen between culture and salivary-gland stages, MSP2 did not appear to vary, by two-dimensional gel electrophoresis, during continuous passage in culture. These data show that MSPs of erythrocyte-stage A. marginale are present on culture stages and may be structurally conserved during continuous culture. The presence of all current candidate diagnostic and vaccine antigens suggests that in vitro cultures are a valuable source of rickettsiae for basic research and for the development of improved diagnostic reagents and vaccines against anaplasmosis.

Displaced tick-parasite interactions at the host interface.

Reciprocal interactions of parasites transmitted by blood-sucking arthropod vectors have been studied primarily at the parasite-host and parasite-vector interface. The third component of this parasite triangle, the vector-host interface, has been largely ignored. Now there is growing realization that reciprocal interactions between arthropod vectors and their vertebrate hosts play a pivotal role in the survival of arthropod-borne viruses, bacteria, and protozoa. The vector-host interface is the site where the haematophagous arthropods feeds. To obtain a blood meal, the vector must overcome the host's inflammatory, haemostatic, and immune responses. This problem is greatest for ixodid ticks which may imbibe as much as 15 ml blood whilst continuously attached to their host for 10 days or more. To feed successfully, the interface between tick and host becomes a battle between the host's mechanisms for combating the tick and the tick's armoury of bioactive proteins and other chemicals which it secrets, via saliva, into the feeding lesion formed in the host's skin. Parasites entering this battlefield encounter a privileged site in their vertebrate host that has been profoundly modified by the pharmacological activities of their vector's saliva. For example, ticks suppress natural killer cells and interferons, both of which have potent antiviral activities. Not surprisingly, vector-bone parasites exploit the immunomodulated feeding site to promote their transmission and infection. Certain tick-bone viruses are so successful at this that they are transmitted from one infected tick, through the vertebrate host to a co-feeding uninfected tick, without a detectable viraemia (virus circulating in the host's blood), and with no untoward effect on the host. When such viruses do have an adverse effect on the host, they may impede their vectors' feeding. Thus important interactions between ticks and tick-borne parasites are displaced to the interface with their vertebrate host-the skin site of blood-feeding and infection.

Isolation and characterization of americanin, a specific inhibitor of thrombin, from the salivary glands of the lone star tick Amblyomma americanum (L.).

A thrombin (EC 3.4.21.5) inhibitor (americanin) was isolated from the salivary glands of the lone star tick Amblyomma americanum (L.) using reversed-phase chromatography and anion-exchange chromatography. Americanin did not inhibit any other protease tested, including factor Xa, plasmin, trypsin, chymotrypsin, elastase, papain, pepsin, and carboxypeptidase. The inhibition of thrombin by americanin decreased dramatically with dilution of the reaction mixture including thrombin, its substrate, and americanin. When thrombin assays were performed in the presence of americanin, the reaction curve showed a time-dependent inhibition. Significant inhibition was observed when americanin concentration was approximately equal to that of thrombin, with a Ki of 0.073 nM. The results suggest that americanin is a specific, reversible, competitive, slow, tight-binding inhibitor of thrombin.

Differentiation of Rhipicephalus ticks (Acari: Ixodidae) by gas chromatography of cuticular hydrocarbons.

Gas chromatography of the cuticular hydrocarbons of 4 species of ticks belonging to the genus Rhipicephalus (R. sanguineus, R. turanicus, R. pusillus, and R. bursa) showed a unique pattern for each taxon. The hydrocarbon fractions were composed of a mixture of straight-chain, terminally methylated, and internally branched alkanes; however, only a small quantity of alkenes was detected. Freshly collected, dried, and alcohol-stored specimens of R. sanguineus were analyzed and their patterns found to be nearly identical. Collection of specimens from separate localities demonstrated a species-specific pattern for Spanish material.

Comparison of known and suspected pheromonal constituents in males of African ticks, Amblyomma hebraeum Koch and Amblyomma variegatum (Fabricius).

Three low molecular weight compounds were found in hexane:diethyl ether extracts of fed males of the African ticks, Amblyomma variegatum (tropical bont tick) and A. hebraeum (bont tick), namely, o-nitrophenol, methyl salicylate and 2,6-dichlorophenol. These same compounds were also fond in a rinse of fed A. variegatum males, but were absent or present in only trace amounts in a rinse of fed A. hebraeum males, o-Nitrophenol and methyl salicylate were present in much higher concentrations (i.e., amounts/tick) in A. variegatum than in A. hebraeum. 2,6-Dichlorophenol was also more abundant in A. variegatum than in A. hebraeum, but the differences were not as great as with the former two compounds. Extraction in hexane over a 3-week period revealed four additional compounds, benzaldehyde, benzyl alcohol, benzothiazole and nonanoic acid. The first three compounds were found in males of both species; nonanoic acid was found only in A. hebraeum males. Published reports consistently show strong attraction by o-nitrophenol and methyl salicylate for both sexes of the two bont tick species; 2,6-dichlorophenol and benzaldehyde have been reported to be attractive to both sexes of A. hebraeum. The possible roles of these compounds, as well as others occasionally reported from A. hebraeum and A. variegatum, as components of the aggregation/attachment pheromone or other pheromones is discussed.