Hemelipoglycoprotein from the ornate sheep tick, dermacentor marginatus: structural and functional characterization

<p>Abstract</p> <p>Background</p> <p>Tick carrier proteins are able to bind, transport, and store host-blood heme, and thus they function also as antioxidants. Nevertheless, the role of carrier proteins in ticks is not fully understood. Some of them are found also in tick males which do not feed on hosts to such an extent such as females (there are differences in male feeding in different tick species) and thus they are not dealing with such an excess of heme; some of the carrier proteins were found in salivary glands where the processing of blood and thus release of heme does not occur. Besides, the carrier proteins bind relatively low amounts of heme (in one case only two molecules of heme per protein) compared to their sizes (above 200 kDa).</p> <p>The main aim of this study is the biochemical characterization of a carrier protein from the ornate sheep tick <it>Dermacentor marginatus</it>, hemelipoglycoprotein, with emphasis on its size in native conditions, its glycosylation and identification of its modifying glycans, and examining its carbohydrate-binding specificity.</p> <p>Results</p> <p>Hemelipoglycoprotein from <it>D. marginatus </it>plasma was purified in native state by immunoprecipitation and denatured using electroelution from SDS-PAGE separated plasma. The protein (290 kDa) contains two subunits with molecular weights 100 and 95 kDa. It is glycosylated by high-mannose and complex <it>N</it>-glycans HexNAc₂Hex₉, HexNAc₂Hex₁₀, HexNAc₄Hex₇, and HexNAc₄Hex₈. The purified protein is able to agglutinate red blood cells and has galactose- and mannose-binding specificity. The protein is recognized by antibodies directed against plasma proteins with hemagglutination activity and against fibrinogen-related lectin Dorin M from the tick <it>Ornithodoros moubata</it>.</p> <p>It forms high-molecular weight complexes with putative fibrinogen-related proteins and other unknown proteins under native conditions in tick plasma. Feeding does not increase its amounts in male plasma. The hemelipoglycoprotein was detected also in hemocytes, salivary glands, and gut. In salivary glands, the protein was present in both glycosylated and nonglycosylated forms.</p> <p>Conclusion</p> <p>A 290 kDa hemelipoglycoprotein from the tick <it>Dermacentor marginatus</it>, was characterized. The protein has two subunits with 95 and 100 kDa, and bears high-mannose and complex <it>N</it>-linked glycans. In hemolymph, it is present in complexes with putative fibrinogen-related proteins. This, together with its carbohydrate-binding activity, suggests its possible involvement in tick innate immunity. In fed female salivary glands, it was found also in a form corresponding to the deglycosylated protein.</p

Fibrinogen-related proteins in ixodid ticks

<p>Abstract</p> <p>Background</p> <p>Fibrinogen-related proteins with lectin activity are believed to be part of the tick innate immune system. Several fibrinogen-related proteins have been described and characterised mainly on the basis of their cDNA sequences while direct biochemical evidence is missing. One of them, the haemolymph lectin Dorin M from the tick <it>Ornithodoros moubata </it>was isolated and characterised in more depth.</p> <p>Results</p> <p>Several fibrinogen-related proteins were detected in the haemolymph of ixodid ticks <it>Dermacentor marginatus</it>, <it>Rhipicephalus appendiculatus</it>, <it>R. pulchellus</it>, and <it>R. sanguineus</it>. These proteins were recognised by sera directed against the tick lectin Dorin M and the haemagglutination activity of the ticks <it>R. appendiculatus </it>and <it>D. marginatus</it>. Cross-reactivity of the identified proteins with antibodies against the fibrinogen domain of the human ficolin was also shown. The carbohydrate-binding ability of tick haemolymph was confirmed by haemagglutination activity assays, and this activity was shown to be inhibited by neuraminic acid and sialylated glycoproteins as well as by N-acetylated hexosamines. The fibrinogen-related proteins were shown to be glycosylated and they were localised in salivary glands, midguts, and haemocytes of <it>D. marginatus</it>. Hemelipoglycoprotein was also recognised by sera directed against the fibrinogen-related proteins in all three <it>Rhipicephalus </it>species as well as in <it>D. marginatus</it>. However, this protein does not contain the fibrinogen domain and thus, the binding possibly results from the structure similarity between hemelipoglycoprotein and the fibrinogen domain.</p> <p>Conclusions</p> <p>The presence of fibrinogen-related proteins was shown in the haemolymph of four tick species in high abundance. Reactivity of antibodies directed against ficolin or fibrinogen-related proteins with proteins which do not contain the fibrinogen domain points out the importance of sequence analysis of the identified proteins in further studies. Previously observed expression of fibrinogen-related proteins in haemocytes together with the results of this study suggest involvement of fibrinogen-related proteins in tick immunity processes. Thus, they have potential as targets for anti-tick vaccines and as antimicrobial proteins in pharmacology. Research on fibrinogen-related proteins could reveal further details of tick innate immunity processes.</p

Neural and endocrine regulation of osmoregulatory organs in tick: recent discoveries and implications

International audienceTicks can survive in harsh and fluctuating vegetated environments for long durations between blood feedings with highly developed osmoregulatory mechanisms. Like the unique life history of hematophagous ticks, osmoregulatory organs and their regulatory mechanisms are significantly different from those in the closely related insect taxa. Over the last ten years, research has uncovered several neuropeptidergic innervations of the primary osmoregulatory organ, the salivary glands: myoinhibitory peptide (MIP), SIFamide, and elevenin. These neuropeptides are thought to be modulators of dopamine's autocrine or paracrine actions controlling the salivary glands, including the activation of fluid transport into the lumen of salivary acini and the pumping and gating action of salivary acini for expelling fluids out into salivary ducts. These actions are through two different dopamine receptors, D1 receptor and invertebrate D1-like dopamine receptor, respectively. Interestingly, MIP and SIFamide are also involved in the control of another important excretory/osmoregulatory organ, the hindgut, where SIFamide is myostimulatory, with MIP having antagonistic effects. FGLamide related allatostatin is also found to have axonal projections located on the surface of the rectum. Investigations of the osmoregulatory mechanisms of these critical vector species will potentially lead to the development of a measure to control tick species

Ultrastructural mapping of salivary gland innervation in the tick Ixodes ricinus

The salivary gland of hard ticks is a highly innervated tissue where multiple intertwined axonal projections enter each individual acini. In the present study, we investigated the ultrastructural architecture of axonal projections within granular salivary gland type II and III acini of Ixodes ricinus female. Using immunogold labeling, we specifically examined the associations of SIFamide neuropeptide, SIFamide receptor (SIFa_R), neuropeptide pigment dispersing factor (PDF), and the invertebrate-specific D1-like dopamine receptor (InvD1L), with acinar cells. In both acini types, SIFamide-positive axons were found to be in direct contact with either basal epithelial cells or a single adlumenal myoepithelial cell in close proximity to the either the acinar duct or its valve, respectively. Accordingly, SIFa_R staining correlated with SIFamide-positive axons in both basal epithelial and myoepithelial cells. Immunoreactivity for both InvD1L and PDF (type II acini exclusively) revealed positive axons radiating along the acinar lumen. These axons were primarily enclosed by the adlumenal myoepithelial cell plasma membrane and interstitial projections of ablumenal epithelial cells. Our study has revealed the detailed ultrastructure of I. ricinus salivary glands, and provides a solid baseline for a comprehensive understanding of the cell-axon interactions and their functions in this essential tick organ

Ultrastructure of <em>Ehrlichia mineirensis</em>, a new member of the <em>Ehrlichia</em> genus

Contribution: Study design, in vitro culture and drafting the mansucriptInternational audienceRecently, we reported the in vitro isolation and the molecular characterization of a new species of Ehrlichia (Ehrlichia mineirensis) from haemolymph of Brazilian Rhipicephalus (Boophilus) microplus ticks. This organism shows an ortholog of Ehrlichia canis major immunogenic protein gp36 with a new structure of tandem repeats. In the present study, we used electron microscopy (high pressure freezing and freeze substitution preparative techniques) to characterize morphologically this new agent growing in IDE8 tick cells. The results showed that E. mineirensis shares ultrastructural features with other members of the genus Ehrlichia (Ehrlichia muris, E. canis and Ehrlichia chaffeensis); typical parasitophorous vacuoles (morulae) contain electron-dense and reticulated Ehrlichiae embedded inside a fibrillar matrix. We observed the characteristic Gram-negative-type cell wall composed of both cytoplasmic and rippled outer membrane. We found organisms undergoing binary fission and rarely altered cells with unusual invagination of the cytoplasmic membrane

Characterisation of Zika virus infection in primary human astrocytes

Abstract Background The recent Zika virus (ZIKV) outbreak has linked ZIKV with microcephaly and other central nervous system pathologies in humans. Astrocytes are among the first cells to respond to ZIKV infection in the brain and are also targets for virus infection. In this study, we investigated the interaction between ZIKV and primary human brain cortical astrocytes (HBCA). Results HBCAs were highly sensitive to representatives of both Asian and African ZIKV lineages and produced high viral yields. The infection was associated with limited immune cytokine/chemokine response activation; the highest increase of expression, following infection, was seen in CXCL-10 (IP-10), interleukin-6, 8, 12, and CCL5 (RANTES). Ultrastructural changes in the ZIKV-infected HBCA were characterized by electron tomography (ET). ET reconstructions elucidated high-resolution 3D images of the proliferating and extensively rearranged endoplasmic reticulum (ER) containing viral particles and virus-induced vesicles, tightly juxtaposed to collapsed ER cisternae. Conclusions The results confirm that human astrocytes are sensitive to ZIKV infection and could be a source of proinflammatory cytokines in the ZIKV-infected brain tissue

Ehrlichia minasensis sp. nov., isolated from the tick Rhipicephalus microplus

Contribution: Conceived the idea and drafted the manuscriptInternational audienceRecently, we obtained a rickettsial isolate (Ehrlichia sp. UFMG-EVT ) from the haemolymph of engorged Rhipicephalus microplus tick females. On the basis of maximum-likelihood phylogenetic analysis using 16S rRNA gene, groEL, dsb, gltA and trp36 sequences we showed that Ehrlichia sp. UFMG-EVT belongs to the a-Proteobacteria, family Anaplasmataceae, genus Ehrlichia. Ehrlichia sp. UFMG-EVT is a sister taxon of Ehrlichia canis with 16S rRNA gene, groEL, dsb, gltA and trp36 sequence similarities of 98.3 %, 97.2 %, 94.7 %, 94.3 % and 49.1 %, respectively. Ehrlichia sp. UFMG-EVT has been maintained in the laboratory by continuous passage in the IDE8 tick cell line where the ultrastructure was characterized using electron microscopy and was found to resemble that of E. canis, Ehrlichia muris and Ehrlichia chaffeensis, but not Ehrlichia ruminantium and Ehrlichia ewingii. We propose the name Ehrlichia minasensis sp. nov. for this bacterium to acknowledge the place from where it was initially isolated, Minas Gerais, Brazil. The type strain is strain Ehrlichia sp. UFMG-EVT (5DSM 100393T 5TCB-TBB-0018T )