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

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

Monoamine Neurotransmitters as Substrates for Novel Tick Sulfotransferases, Homology Modeling, Molecular Docking, and Enzyme Kinetics

Blacklegged ticks (Ixodes scapularis) transmit the causative agent of Lyme disease in the Northeastern United States. Current research focuses on elucidating biochemical pathways that may be disrupted to prevent pathogen transmission, thereby preventing disease. Genome screening reported transcripts coding for two putative sulfotransferases in whole tick extracts of the nymphal and larval stages. Sulfotransferases are known to sulfonate phenolic and alcoholic receptor agonists such as 17β-estradiol, thereby inactivating the receptor ligands. We used bioinformatic approaches to predict substrates for Ixosc Sult 1 and Ixosc Sult 2 and tested the predictions with biochemical assays. Homology models of 3D protein structure were prepared, and visualization of the electrostatic surface of the ligand binding cavities showed regions of negative electrostatic charge. Molecular docking identified potential substrates including dopamine, R-octopamine and S-octopamine, which docked into Ixosc Sult 1 with favorable binding affinity and correct conformation for sulfonation. Dopamine, but not R- or S-octopamine, also docked into Ixosc Sult 2 in catalytic binding mode. The predictions were confirmed using cytosolic fractions of whole tick extracts. Dopamine was a good substrate (Km = 0.1−0.4 μM) for the native Ixodes scapularis sulfotransferases from larval and nymphal stages regardless of their fed/unfed status. Octopamine sulfonation was detected only after feeding when gene expression data suggests that Ixosc Sult 1 is present. Because dopamine is known to stimulate salivation in ticks through receptor stimulation, these results imply that the function(s) of Ixosc Sult 1 or 2 may include inactivation of the salivation signal via sulfonation of dopamine and/or octopamine

Molecular characterization of novel sulfotransferases from the tick, Ixodes scapularis

<p>Abstract</p> <p>Background</p> <p><it>Ixodes scapularis</it>, commonly known as the blacklegged or deer tick, is the main vector of Lyme disease in the United States. Recent progress in transcriptome research has uncovered hundreds of different proteins expressed in the salivary glands of hard ticks, the majority of which have no known function, and include many novel protein families. We recently identified transcripts coding for two putative cytosolic sulfotransferases in these ticks which recognized phenolic monoamines as their substrates. In this current study, we characterize the genetic expression of these two cytosolic sulfotransferases throughout the tick life cycle as well as the enzymatic properties of the corresponding recombinant proteins. Interestingly, the resultant recombinant proteins showed sulfotransferase activity against both neurotransmitters dopamine and octopamine.</p> <p>Results</p> <p>The two sulfotransferase genes were coded as <it>Ixosc </it>SULT 1 & 2 and corresponding proteins were referred as <it>Ixosc </it>Sult 1 and 2. Using gene-specific primers, the sulfotransferase transcripts were detected throughout the blacklegged tick life cycle, including eggs, larvae, nymphs, adult salivary glands and adult midgut. Notably, the mRNA and protein levels were altered upon feeding during both the larval and nymphal life stages. Quantitative PCR results confirm that <it>Ixosc </it>SULT1 was statistically increased upon blood feeding while <it>Ixosc </it>SULT 2 was decreased. This altered expression led us to further characterize the function of these proteins in the Ixodid tick. The sulfotransferase genes were cloned and expressed in a bacterial expression system, and purified recombinant proteins <it>Ixosc </it>Sult 1(R) and 2(R) showed sulfotransferase activity against neurotransmitters dopamine and octopamine as well as the common sulfotransferase substrate <it>p-</it>nitrophenol. Thus, dopamine- or octopamine-sulfonation may be involved in altering the biological signal for salivary secretion in <it>I. scapularis.</it></p> <p>Conclusions</p> <p>Collectively, these results suggest that a function of <it>Ixosc </it>Sult 1 and Sult 2 in <it>Ixodid </it>tick salivary glands may include inactivation of the salivation signal via sulfonation of dopamine or octopamine.</p

Determination of Ki-67-7 and curcumin-induced apoptosis by flow cytometry.

<p>AY-27 cells were treated with anti-Ki-67 siRNA, curcumin or in the combined presence of both as described in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048567#pone-0048567-g003" target="_blank">Figures 3A and 3B</a>. At the end of the treatment period, the cells were harvested, stained with fluorescence-conjugated Annexin V and 7AAD as described in Methods. Following flow cytometric analysis, the percentage (number inserts in the figure) of normal cells or those undergoing apoptosis and necrosis was calculated using appropriate software (Flow Jo v.9). The rate of apoptosis was measured by adding the percentages in the right two quadrants in each of the figures. The data are from a representative (of two identical) experiment.</p

Molecular targets of CusiRNA.

<p>The scheme displays the possible molecular targets of Ki-67-7 and curcumin when used in combination (CusiRNA). Arrowheads indicate the activation of indicated proteins; hammer heads indicate inhibition and the broken lines represent the possible role for putative intermediates. Further details are provided in the text.</p

Effect of Ki-67-7 and curcumin on cell cycle phases.

<p>AY-27 cells were treated with Ki-67-7, curcumin or with both, as described earlier in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048567#pone-0048567-g003" target="_blank">Figures 3A and 3B</a>. At the end of the incubation period, the cells were treated with PI and the distribution of cells in various cell cycle phases were determined by flow cytometry as described in Methods. Cells were scored as percentage distribution of cells in each of the cell cycle phases (G_o/G_1, S and G2/M). Bars indicate values which are the means ± S.E of three determinations.</p

Dose-dependent effects of curcumin on bladder cancer cell proliferation.

<p>T-24 (2a) and AY-27 (2b) cells were plated in 12-well plates (0.8×10⁵ cells/well) and cultured for 48 hr (40–50% confluence) as described in Methods. Following exposure to various concentrations of curcumin for 24 h, cells were washed with PBS and incubated for another 24 h in curcumin-free medium. Cells incubated with DMSO (0.1%) alone were treated as controls. DMSO alone had little impact on tumor cell growth (data not shown). Cell proliferation was assessed on the basis of cell viability, measured by MTT assay. Cell viability was expressed as percent of viability observed in DMSO-treated control cells. Values are from a representative (of two) (2a) or 3–8 (mean ± S.E) determinations (2b). ^*P<0.05, ^**P<0.01,^***P<0.005.</p

Effect of Ki-67-7 and curcumin on the regulatory proteins of cell cycle phases and apoptosis: Western blot analysis.

<p>Bladder cancer cells (T-24 and AY-27) were cultured and exposed to Ki-67-7 and curcumin as described above in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048567#pone-0048567-g003" target="_blank">Figures 3A and 3B</a>. At the end of the treatment period, total protein was extracted and subjected to Western blotting using appropriate antibodies as described in Methods. For purposes of associating proteins with specific roles, they were grouped in separate categories, such as gene transcription (A), cell-cycle progression (B) and apoptosis (C). β-actin was used as the internal control. Since different proteins (e.g. Cyclin D1 and NF-κB) from the same membrane were identified in separate categories, the same β-actin blot is displayed on more than one occasion. Data presented are western blots from a single experiment, which is representative of 2–3 identical experiments.</p