nan Anisuzzaman

Longistatin is an unconventional serine protease and induces protective immunity against tick infestation

Classical serine proteases use the conserved Ser/His/Asp catalytic triad to hydrolyze substrates. Here, we show that longistatin, a salivary gland protein with two EF-hand domains from the vector tick Haemaphysalis longicornis, does not have the conserved catalytic triad, but still functions as a serine protease. Longistatin was synthesized in and secreted from the salivary glands of ticks, and is injected into host tissues during the acquisition of blood-meals. Longistatin hydrolyzed fibrinogen, an essential plasma protein in the coagulation cascade, and activated plasminogen, into its active form plasmin, a serine protease that dissolves fibrin clots. Longistatin efficiently hydrolyzed several serine protease-specific substrates showing its specificity to the amide bond of Arg. Longistatin did not hydrolyze synthetic substrates specific for other groups of proteases. The enzyme was active at a wide range of temperatures and pHs, with the optimum at 37°C and pH 7. Its activity was efficiently inhibited by various serine protease inhibitors such as phenylmethanesulfonyl fluoride (PMSF), aprotinin, antipain, and leupeptin with the estimated IC(50) of 278.57 μM, 0.35 μM, 41.56 μM and 198.86 μM, respectively. In addition, longistatin was also potently inhibited by Zinc (Zn(2+)) in a concentration-dependent manner with an IC(50) value of 275 μM, and the inhibitory effect of Zn(2+) was revived by ethylenediaminetetra acetic acid (EDTA). Immunization studies revealed that longistatin sharply induced high levels of protective IgG antibodies against ticks. Immunization with longistatin reduced repletion of ticks by about 54%, post engorgement body weight by >11% and molting of nymphs by approximately 34%; thus, the vaccination trial was approximately 73% effective against tick infestation. Taken together, our results suggest that longistatin is a new potent atypical serine protease, and may be an interesting candidate for the development of anti-tick vaccines.

Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis

We report here the molecular characterization and possible function of a cysteine protease (termed HlCPL-A) identified in the midgut of the hard tick Haemaphysalis longicornis. HlCPL-A is a 333 amino acid protein belonging to the papain family of the cysteine protease. A construct encoding proHlCPL-A was expressed in Escherichia coli and purified as both procathepsin L and active processed cathepsin L forms. The HlCPL-A gene expression was up-regulated by blood-feeding process. HlCPL-A exhibited substrate specificity against synthetic peptidyl substrates (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA; k(cat)/K(m)=0.19 and 0.0023 M(-1) S(-1), respectively). The proteolytic activity of HlCPL-A was inhibited by leupeptin, antipain and E-64 but was unaffected by pepstatin. HlCPL-A was capable of degrading bovine hemoglobin at pH 3.2 to 5.6. These results suggest that HlCPL-A may play important roles in the digestion of host hemoglobin in ticks.

Longistatin, a novel EF-hand protein from the ixodid tick Haemaphysalis longicornis, is required for acquisition of host blood-meals☆

Calcium and the EF-hand Ca(++)-binding proteins have been undisputedly recognised as the key players in almost all aspect of cell functions, starting from the cells birth, during mitosis to its end with apoptosis. But in a few exceptional cases the EF-hand proteins are secreted from the cells and play their crucial roles extracellularly. Here, to our knowledge for the first time, we have identified and characterised an EF-hand Ca(++)-binding protein from the salivary glands of the ixodid tick, Haemaphysalis longicornis, herein called longistatin. Longistatin possesses two EF-hand domains which conserve canonical structure and bind with Ca(++). Both the recombinant and endogenous proteins were stained with Rutheninum red. Reverse-transcription PCR data showed that longistatin-specific transcript was expressed in all life-cycle stages of H. longicornis and was up-regulated only in blood-fed ticks. Organ-specific transcription analysis revealed a salivary gland-specific expression of the gene which peaked at 96-120 h of feeding when ticks acquired full blood-meals and become engorged but its expression declined sharply as they detached and dropped off the host. Consistently, endogenous protein was localised in the salivary glands of adult ticks and in the lumen of the functional acini of the salivary glands. Furthermore, longistatin was detected in feeding lesions at the site of attachment of ticks on the host. These results suggest that longistatin is synthesised in, and is secreted from, the salivary glands and may have functional roles in the feeding process of ixodid ticks.

Longistatin in tick saliva blocks advanced glycation end-product receptor activation

Ticks are notorious hematophagous ectoparasites and vectors of many deadly pathogens. As an effective vector, ticks must break the strong barrier provided by the skin of their host during feeding, and their saliva contains a complex mixture of bioactive molecules that paralyze host defenses. The receptor for advanced glycation end products (RAGE) mediates immune cell activation at inflammatory sites and is constitutively and highly expressed in skin. Here, we demonstrate that longistatin secreted with saliva of the tick Haemaphysalis longicornis binds RAGE and modulates the host immune response. Similar to other RAGE ligands, longistatin specifically bound the RAGE V domain, and stimulated cultured HUVECs adhered to a longistatin-coated surface; this binding was dramatically inhibited by soluble RAGE or RAGE siRNA. Treatment of HUVECs with longistatin prior to stimulation substantially attenuated cellular oxidative stress and prevented NF-κB translocation, thereby reducing adhesion molecule and cytokine production. Recombinant longistatin inhibited RAGE-mediated migration of mouse peritoneal resident cells (mPRCs) and ameliorated inflammation in mouse footpad edema and pneumonia models. Importantly, tick bite upregulated RAGE ligands in skin, and endogenous longistatin attenuated RAGE-mediated inflammation during tick feeding. Our results suggest that longistatin is a RAGE antagonist that suppresses tick bite-associated inflammation, allowing successful blood-meal acquisition from hosts.

High-throughput RNA sequencing profiles and transcriptional evidence of aerobic respiratory enzymes in sporulating oocysts and sporozoites of Eimeria tenella.

Seven species of Eimeria are responsible for coccidiosis in chickens. Eimeria tenella is one of the most pathogenic parasites since it is associated with high mortality and great economic impact. The life cycle of the parasite includes development in the environment and in the intestinal tract. We conducted RNA sequencing using a next generation sequencer to obtain transcriptome information from the sporulating oocysts, and sporozoites. We collected 2.8 million 75 bp reads of a short-tag sequence, and 25,880 contigs were generated by the Oases assembler. A Blastx search of GenBank databases revealed that 7780 contigs (30.1%) had significant homology with deposited sequence data (E-value <1e-6); among these contigs, 6051 contigs were similar to those of Toxoplasma gondii while only 513 contigs (6.6%) were similar to those of E. tenella. After an orthological analysis conducted with the UniProt database of T. gondii, 6661 contigs were distributed within the categories of cellular components (1528 gene categories), biological processes (861 gene categories), and molecular functions (241 gene categories). The significantly matched contigs contained high numbers of enzymes associated with glycolysis, TCA, and the pentose-phosphate pathway. Most of the enzymes, measured by quantitative reverse transcription-PCR, were up-regulated in sporulating stage. These results suggest that the intracellular carbohydrate amylopectin could be used as an energy source for ATP production including glycolysis and the pentose-phosphate pathway, which generates NADPH and pentoses. Our data also suggest that Eimeria might possess a partial or similar pathway to the TCA cycle essential for aerobic respiration. Furthermore, the newly annotated and non-annotated contigs might contain E. tenella-specific or novel sequences.

A Kunitz-type proteinase inhibitor from the midgut of the ixodid tick, Haemaphysalis longicornis, and its endogenous target serine proteinase.

Although previous studies strongly suggested the involvement of serine proteases in blood digestion in the midgut of ticks, the regulating molecules of these proteinases are still unidentified. A novel Haemaphysalis longicornis Kunitz-type serine proteinase inhibitor with a single Kunitz-domain (HlMKI) has been identified and its co-localization with a midgut-derived serine proteinase (HlSP) within the epithelial cells has been demonstrated. Recombinant HlMKI inhibited the hydrolytic activity of HlSP, suggesting that HlMKI is a possible inhibitor of HlSP and may be part of a regulatory system of midgut serine proteinases.

A hemocyte-derived Kunitz-BPTI-type chymotrypsin inhibitor, HlChI, from the ixodid tick Haemaphysalis longicornis, plays regulatory functions in tick blood-feeding processes.

Inhibitors of proteases play key roles in the biological processes of vertebrate and invertebrate animals, including arthropod parasites. Here, we describe a cDNA that encodes a functionally active chymotrypsin inhibitor of the BPTI/Kunitz family of serine protease inhibitors from the hemocytes of the ixodid tick, Haemaphysalis longicornis, herein called HlChI. HlChI sequence is evolutionarily conserved and contains six cysteine residues and three disulfide bonds with a calculated molecular weight of 9.1 kDa. HlChI-specific mRNA was expressed in all developmental stages of ticks and the expression was up-regulated by hosts blood-feeding processes. Endogenous HlChI was localized mainly in the hemocytes. HlChI potently inhibited bovine pancreatic α-chymotrypsin for hydrolyzing the fluorogenic substrate (IC(50) 8.32 nM, K(d) 5.35 ± 1.01 nM) and bovine casein digestion. However, HlChI weakly inhibited bovine pancreatic trypsin and could not affect the porcine elastase activity, suggesting its narrow specificity to chymotrypsin. HlChI was stable over the pH range 2-11 and heating up to 70 °C at pH 8. HlChI was highly stable to 8 M urea and 2% SDS at pH 8.0, when treated for 24 h at 37 °C. However, 0.2 M 2-mercaptoethanol caused complete but reversible inactivation of HlChI. Knockdown of HlChI gene by RNA interference (RNAi) caused death of the feeding ticks, failure of ticks to engorge and significantly reduced body weight gain. RNAi also resulted in significantly decreased egg conversion ratio and fecundity. These results suggest that HlChI is a chymotrypsin-specific inhibitor with high stability and may play regulatory functions in hosts blood-feeding processes and tick reproduction.

Semi-artificial mouse skin membrane feeding technique for adult tick, Haemaphysalis longicornis

BackgroundAn in vitro artificial feeding technique for hard ticks is quite useful for studying the tick-pathogen interactions. Here, we report a novel semi-artificial feeding technique for the adult parthenogenetic tick, Haemaphysalis longicornis, using mouse skin membrane.FindingsSkin with attached adult ticks was removed from the mouse body at 4 to 5 days post-infestation for the construction of the feeding system. This system supplied with rabbit blood was kept in >95% relative humidity at 30°C during the feeding, and ticks were fully engorged (artificially engorged, AE) within 12 to 48 h. For comparison, ticks were fed to engorgement solely on rabbit or mouse for 5 days as controls (naturally engorged on rabbit, NEr, or mouse, NEm). Blood digestion-related gene expression in the midgut and reproductive fitness were compared. Body weight, egg mass weight, egg conversion ratio, and hatchability of eggs did not show any significant differences. We analyzed transcription profiles of selected genes assayed by quantitative RT-PCR and revealed similar patterns of expression between NEr and AE but some differences between NEm and AE or NEm and NEr.ConclusionsOur results demonstrate that this semi-artificial feeding technique mimics natural feeding processes of ticks and can be utilized as a standardized method to inoculate pathogens, especially Babesia protozoa, into H. longicornis and possibly other tick species as well.

Longistatin, an EF-Hand Ca 2+ -Binding Protein from Vector Tick: Identification, Purification, and Characterization

EF-hand Ca(2+)-binding motif, a structural component of the EF-hand protein, functions as a calcium sensor and/or buffer in the cytosol of the cell. However, in a few exceptional cases, the EF-hand proteins are secreted from cells and play crucial roles extracellularly. We have identified longistatin, an EF-hand Ca(2+)-binding protein, from the salivary glands of the tick, Haemaphysalis longicornis. Longistatin possesses an N-terminal sequence of unknown structure and two EF-hand motifs in the C-terminus, which conserve a calmodulin-like canonical structure. Longistatin shows distinct changes in its migration during electrophoresis through SDS-PAGE gel containing calcium or ethylenediaminetetraacetic acid (EDTA). Both recombinant and endogenous forms of longistatin can be stained with rutheninum red, demonstrating that longistatin is a Ca(2+)-binding protein.

Transcriptional profiles of virulent and precocious strains of Eimeria tenella at sporozoite stage; novel biological insight into attenuated asexual development.

Chicken coccidiosis is caused by Eimeria spp., particularly Eimeria tenella, and is characterized by watery or hemorrhagic diarrhea, resulting in death in severe cases. Precociously attenuated live vaccines are widely used to control the disease, and these are produced by serially passaging virulent strains through chickens, and the collection of oocysts from feces at progressively earlier time points during oocyst shedding. Sporozoites of the precocious strain rapidly enter the intestinal mucosa, and their subsequent asexual development reduces their growth. However, there have been few detailed genetic or transcriptional analyses of the strains. Here, we used RNA sequencing to gain novel biological insight into the pathogenicity and precocity of E. tenella. We compared the differential transcription in the sporozoites (the initial stage of endogenous development) of virulent and precocious strains by mapping the sequence reads onto the draft genome of E. tenella. About 90% of the reads from both strains were mapped to the genome, and 16,630 estimated transcript regions were identified. Using Gene Ontology slim and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and the annotation of the estimated transcripts with Blastx, we found that the expression of some genes involved in carbohydrate metabolism were expressed two-fold more strongly in the virulent strain than in the precocious strain. Characteristically, genes related to proteins secreted from the apical complex, proteases, cell attachment proteins, mitochondrial proteins, and transporters were most strongly upregulated in the virulent strain. Interestingly, the expression of genes associated with cell survival, development, or proliferation was strongly upregulated in the precocious strain. These findings suggest that virulent strains survive long before invasion and invade actively/successfully into host cells, whereas proliferative processes appear to affect precocity.