Longistatin, a Plasminogen Activator, Is Key to the Availability of Blood-Meals for Ixodid Ticks

Ixodid ticks are notorious blood-sucking ectoparasites and are completely dependent on blood-meals from hosts. In addition to the direct severe effects on health and productivity, ixodid ticks transmit various deadly diseases to humans and animals. Unlike rapidly feeding vessel-feeder hematophagous insects, the hard ticks feed on hosts for a long time (5−10 days or more), making a large blood pool beneath the skin. Tick's salivary glands produce a vast array of bio-molecules that modulate their complex and persistent feeding processes. However, the specific molecule that functions in the development and maintenance of a blood pool is yet to be identified. Recently, we have reported on longistatin, a 17.8-kDa protein with two functional EF-hand Ca++-binding domains, from the salivary glands of the disease vector, Haemaphysalis longicornis, that has been shown to be linked to blood-feeding processes. Here, we show that longistatin plays vital roles in the formation of a blood pool and in the acquisition of blood-meals. Data clearly revealed that post-transcriptional silencing of the longistatin-specific gene disrupted ticks' unique ability to create a blood pool, and they consequently failed to feed and replete on blood-meals from hosts. Longistatin completely hydrolyzed α, β and γ chains of fibrinogen and delayed fibrin clot formation. Longistatin was able to bind with fibrin meshwork, and activated fibrin clot-bound plasminogen into its active form plasmin, as comparable to that of tissue-type plasminogen activator (t-PA), and induced lysis of fibrin clot and platelet-rich thrombi. Plasminogen activation potentiality of longistatin was increased up to 4 times by soluble fibrin. Taken together, our results suggest that longistatin may exert potent functions both as a plasminogen activator and as an anticoagulant in the complex scenario of blood pool formation; the latter is critical to the feeding success and survival of ixodid ticks

The Kunitz-Like Modulatory Protein Haemangin Is Vital for Hard Tick Blood-Feeding Success

Ticks are serious haematophagus arthropod pests and are only second to mosquitoes as vectors of diseases of humans and animals. The salivary glands of the slower feeding hard ticks such as Haemaphysalis longicornis are a rich source of bioactive molecules and are critical to their biologic success, yet distinct molecules that help prolong parasitism on robust mammalian hosts and achieve blood-meals remain unidentified. Here, we report on the molecular and biochemical features and precise functions of a novel Kunitz inhibitor from H. longicornis salivary glands, termed Haemangin, in the modulation of angiogenesis and in persistent blood-feeding. Haemangin was shown to disrupt angiogenesis and wound healing via inhibition of vascular endothelial cell proliferation and induction of apoptosis. Further, this compound potently inactivated trypsin, chymotrypsin, and plasmin, indicating its antiproteolytic potential on angiogenic cascades. Analysis of Haemangin-specific gene expression kinetics at different blood-feeding stages of adult ticks revealed a dramatic up-regulation prior to complete feeding, which appears to be functionally linked to the acquisition of blood-meals. Notably, disruption of Haemangin-specific mRNA by a reverse genetic tool significantly diminished engorgement of adult H. longicornis, while the knock-down ticks failed to impair angiogenesis in vivo. To our knowledge, we have provided the first insights into transcriptional responses of human microvascular endothelial cells to Haemangin. DNA microarray data revealed that Haemangin altered the expression of 3,267 genes, including those of angiogenic significance, further substantiating the antiangiogenic function of Haemangin. We establish the vital roles of Haemangin in the hard tick blood-feeding process. Moreover, our results provide novel insights into the blood-feeding strategies that enable hard ticks to persistently feed and ensure full blood-meals through the modulation of angiogenesis and wound healing processes

Bovine theileriosis: an emerging problem in south-eastern Australia?

Bovine theileriosis is an arthropod-borne disease caused by one or more haemoprotozoan parasites of the genus Theileria. Traditionally, Theileria infection in cattle in Australia was largely asymptomatic and recognized to be associated with Theileria buffeli, now assigned to the Theileria orientalis-group. There have been some recent outbreaks of theileriosis in dairy and beef cattle, mainly in subtropical climatic zone (New South Wales) of Australia. Here, we provide the first published evidence of an outbreak of bovine theileriosis in the south-eastern Australia (state of Victoria) linked to the ikeda and chitose genotypes of T. orientalis. Future investigations should focus sharply on the elucidating the epidemiology and ecology of Theileria in this region to subvert the possible impact on the cattle industry

Persistent infection with Strongyloides venezuelensis in the Mongolian gerbil (Meriones unguiculatus)

To examine the fate of Strongyloides venezuelensis, Mongolian gerbils (Meriones unguicalatus) were orally infected with 1,000 L3 larvae per animal. Altogether, 50 gerbils divided into 5 groups of 10 each were monitored for a period of 570 days to document the kinetics of faecal egg output, adults worm population, morphological development, fecundity, and hematological changes including peripheral blood eosinophilia. This study chronicled a life long parasitism of S. venezuelensis in the gerbil host, and showed that S. venezuelensis infection was quite stable throughout the course of infection and the worms maintained their normal development as evidenced by their body dimension. A progressive loss of body condition of the infected gerbils was observed as the level of infection advanced. However, no detectable pathological changes were observed in the gastrointestinal tract. The present findings indicate that an immunocompetent host, such as the Mongolian gerbil, can serve as a life long carrier model of S. venezuelensis if the worms are not expelled within 570 days after infection

Examination of gastrointestinal helminth in livestock grazing in grassland of Bangladesh

To determine association of grassland with parasitic diseases of livestock in Bangladesh, the 'Tracer' animals (two cow calves and two goats) were released for a month in a grassland used for communal grazing of livestock near school premise in Kanthal, Trishal, Mymensingh, Bangladesh. After slaughtering of the tracer animals, their gastrointestinal tract examination revealed six species of nematode and one cestode. The nematode species were Haemonchus contortus, Trichostrongylus axei, Mecistocirrus digitatus, Oesophagostomum spp., Trichuris spp. and Bunostomum sp. The cestode was one of the genus Moniezia. With this preliminary study, grasslands are thought to be one of the main sources of gastrointestinal parasitic diseases of livestock in Bangladesh

Binding of longistatin with fibrin clot.

<p>(A) Detection of longistatin bound on fibrin meshwork. Fibrinogen at different concentrations (3.75, 7.5, 15, 30 and 60 mM; final concentration) was mixed in the absence or presence of longistatin (10 µg) or an equal amount of t-PA or u-PA in a buffer (50 mM Tris–HCl, pH 7.5; 100 mM NaCl and 5 mM CaCl₂) and thrombin (0.10 NIH unit/µl) was added immediately and was incubated at 25°C for 1 h. The clot was treated with anti-longistatin (1∶100), anti-t-PA (1∶100), anti-u-PA (1∶20) or pre-immune sera (1∶100). Bound antibodies were detected using green fluorescent-labeled secondary antibody (Alexa Flour 488 goat anti-mouse IgG). (B) Supernatant was analyzed by 12.5% SDS–PAGE under reducing conditions. (C) The target protein was extracted from the supernatant and its concentration was determined using micro-BCA reagent as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001312#s4" target="_blank">Materials and Methods</a>. The results are expressed as percentage of longistatin/t-PA/u-PA bound to the fibrin clot. Data represent mean ± SD, n = 3.</p

Plasminogen activation by longistatin.

<p>(A) Longistatin (40, 80, 160, 320 and 640 nM) was incubated without or with plasminogen (0.24 units) adding fibrin CNBr fragments (4 µg) in a total volume of 200 µl of buffer (50 mM Tris–HCl, pH 7.5; 100 mM NaCl and 5 mM CaCl₂) at 25°C for 2 h. Then, plasmin-specific fluorogenic substrate (100 µM, final concentration) was added and substrate hydrolysis was monitored by measuring excitation and emission wavelengths of 360 nm and 460 nm, respectively, at 15 min intervals. <i>Inset</i>, initial rate of plasminogen activation at different concentrations of longistatin. (B) Effects of fibrin CNBr fragments on the activation of plasminogen by longistatin. Plasminogen (0.24 units) was incubated with longistatin (640 nM) in the absence or presence of fibrin CNBr fragments (0.25, 1 and 4 µg) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001312#s4" target="_blank">Materials and Methods</a>. All assays were performed in triplicate.</p

Effects of post-transcriptional silencing of <i>longistatin</i> gene on blood pool formation and blood feeding.

<p>(A) Impact of longistatin-specific mRNA silencing on blood-meal feeding from hosts. Adult ticks were injected with <i>longistatin</i> dsRNA or <i>mal</i>E dsRNA (1 µg/tick) and were allowed to feed on a tick-naïve rabbit. RNAi-treated ticks failed to replete. (B) Postengorgement weight was significantly (p<0.01) lower in the RNAi-treated group than that of the control group. Dotted lines indicate mean±SD of body weight of ticks. (C) Effects of post-transcriptional silencing of <i>longistatin</i> gene on blood pool formation. RNAi was performed and ticks were fed in the same manner as in A. RNAi-treated ticks failed to establish a prominent blood pool. Arrows indicate site of tick attachment. (D) Blood pools were significantly (p<0.01) smaller in the RNAi-treated group. (E) Histopathological changes were studied using EVGS. Longistatin was detected in the feeding lesions of ticks on the host's tissues using mouse anti-longistatin sera (1∶100).</p

A schematic diagram showing roles of longistatin in blood coagulation and fibrinolysis events.

<p>In the initial phase, the tick bites and lacerates tissues at the site of attachment and damages vascular beds, which results in hemorrhage leading to the development of a blood pool. Longistatin is synthesized in and secreted from the salivary glands and injected into the blood pool during feeding process <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001312#ppat.1001312-Anisuzzaman1" target="_blank">[27]</a>. Longistatin degrades fibrinogen and activates plasminogen to its active form, plasmin. HMWK, high-molecular-weight kininogen; PKK, prekallikrein; TF, tissue factor. Yellow arrows, contact activation (intrinsic) pathway; olive-green arrows, tissue factor (extrinsic) pathway; green arrows, common pathway of coagulation cascade and white arrows, fibrinolytic pathway. Figure adapted from ref. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001312#ppat.1001312-Stark1" target="_blank">[1]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001312#ppat.1001312-CesarmanMaus1" target="_blank">[6]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001312#ppat.1001312-Murray2" target="_blank">[32]</a>.</p

Anti-coagulation and fibrinogenolytic activity of longistatin.

<p>(A) Effects of longistatin on the formation of fibrin clot. Fibrinogen (7.5 mM) was pre-incubated in a buffer in the absence or presence of longistatin (0.1, 0.2, 0.4, 0.8 and 1.6 µM) or plasmin (1.6 µM) and then thrombin was added (0.10 NIH unit/µl) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001312#s4" target="_blank">Materials and Methods</a>. Clot formation was detected visually and also by determining changes in turbidity at OD₄₅₀ using a spectrophotometer after 15 min. (B) Longistatin (1.6 µM) delayed fibrin clot formation up to 90 min. Fibrinogen was incubated in the absence or presence of longistatin (1.6 µM) following the same procedures as mentioned in A and then thrombin was added. OD₄₅₀ was measured at 15 min intervals. (C) Fibrinogenolytic effect of longistatin. Fibrinogen (7.5 mM) was incubated in the absence or presence of longistatin (0.4, 0.8 and 1.6 µM) or plasmin (1.6 µM). Samples were collected at the indicated time period and were subjected to 12.5% SDS–PAGE analysis under reducing conditions. A gradual degradation of the α, β and γ chains of fibrinogen was detectable with the concomitant deposition of degraded products. Asterisks (^*) indicate that the difference compared with the negative control group (buffer only) is significant as determined by Student's <i>t</i>-test with unequal variance (^*p<0.05, ^**p<0.01).</p