Molecular cloning and characterization of Ac-MTP-2, an astacin-like metalloprotease released by adult Ancylostoma caninum

Ac-MTP-2 is an astacin-like metalloprotease secreted by adult Ancylostoma caninum hookworms. Ac-mtp-2 cDNA was cloned by immunoscreening a cDNA library with antisera prepared against adult A. caninum excretory/secretory (ES) products. The full-length Ac-mtp-2 contains 850 bp cDNA encoding a 233 amino acid open reading frame (ORF) with 32% amino acid identity to Ce-NSP-4, a pharyngeal cell-derived secreted metalloprotease of the nematode Caenorhabditis elegans. The predicted ORF contained a conserved Met-turn sequence (SXMHY), but only a partial zinc-binding signature sequence (GXXXEHXRXER instead of HEXXHXXGXXHEXXRXDR) found in other astacins. However, by both gelatin gel electrophoresis and azocasein digestion, the recombinant Ac-MTP-2 exhibited proteolytic activity that was inhibited by the zinc chelator 1,10-phenanthroline and Ac-TMP, a putative tissue inhibitor of metalloprotease that was previously shown to be a highly abundant component of adult A. caninum ES products. By RT-PCR, Western blot Ac-MTP-2 was found only expressed in adult hookworms and secreted in the adult ES products. Immunolocalization with antisera shows that Ac-MTP-2 is located to the esophageal glands (confirming its role as a secretory protein), as well as to the parasite uterus. It is hypothesized that Ac-MTP-2 functions in the extracorporeal digestion of the intestinal mucosal plug lodged in the buccal capsule of the adult parasite.     

Ancylostoma caninum anticoagulant peptide-5: immunolocalization and in vitro neutralization of a major hookworm anti-thrombotic

Hookworm infection is a major cause of gastrointestinal blood loss and iron deficiency anemia in the developing world. Recently two major anticoagulant serine protease inhibitors have been identified and cloned from adult Ancylostoma caninum hookworms. One of these, A. caninum anticoagulant peptide 5 (AcAP5), is a potent and specific inhibitor of human coagulation factor Xa. A polyclonal IgG has been purified from rabbits immunized with recombinant AcAP5 using affinity chromatography. Using immunohistochemistry, the polyclonal alpha-rAcAP5 IgG localized to the cephalic or amphidial glands, confirming previous biochemical studies that had identified this secretory gland as the primary source of anticoagulant activity in the adult worm. This polyclonal IgG also neutralized the inhibitory activity of recombinant and native AcAP using a single stage chromogenic assay of coagulation factor Xa activity. In addition, the polyclonal IgG also neutralized the anticoagulant activity of native and recombinant AcAP5 as measured by the activated partial thromboplastin time clotting assay. Importantly, this neutralizing activity is species specific, as the polyclonal IgG failed to neutralize the anticoagulant activity of A. ceylanicum. Taken together, these data suggest that the hookworm anticoagulant AcAP5 represents a viable target for future immunization strategies aimed at inhibiting the ability of the adult hookworm to feed on blood in vivo.     

Isolation and molecular cloning of a secreted hookworm platelet inhibitor from adult Ancylostoma caninum

Hookworms, bloodfeeding intestinal nematodes, are a leading cause of iron deficiency anemia in the developing world. These parasites have evolved potent mechanisms of interfering with mammalian hemostasis, presumably for the purpose of facilitating bloodfeeding. Adult Ancylostoma caninum worm extracts contain an activity that inhibits platelet aggregation and adhesion by blocking the function of two cell surface integrin receptors, Glycoprotein IIb/IIIa and GPIa/IIa. Using rpHPLC, the hookworm platelet inhibitor activities have been purified from protein extracts of A. caninum. Because the two inhibitory activities co-purified through multiple chromatographic steps, have similar molecular masses and share identical N-terminal as well as internal amino acid sequence homology, it is likely that they represent a single gene product. A cDNA corresponding to the purified hookworm platelet inhibitor (HPI) protein has been cloned from adult A. caninum RNA, and the translated amino acid sequence shows significant homology to Neutrophil Inhibitory Factor and Ancylostoma Secreted Proteins, suggesting that these related hookworm proteins represent a novel class of integrin receptor antagonists. Polyclonal antibodies raised against the recombinant HPI protein recognize corresponding native proteins in A. caninum extracts and excretory/secretory products, and immunohistochemistry data have identified the cephalic glands as the major source of the inhibitor within the adult hookworm. These data suggest that HPI is secreted by the adult stage of the parasite at the site of intestinal attachment. As such, it may represent a viable target for a vaccine-based strategy aimed at interfering with hookworm-induced gastrointestinal hemorrhage and iron deficiency anemia.     

Molecular cloning and characterization of Ac-TMP-2, a tissue inhibitor of metalloproteinase secreted by adult Ancylostoma caninum

Ac-TMP-2, an immunodominant hookworm antigen encoding a tissue inhibitor of metalloproteinase (TIMP) was cloned by immunoscreening an Ancylostoma caninum larval cDNA library with sera pooled from dogs immunized with irradiated A. caninum third stage larvae (ir-L3). The open reading frame of Ac-tmp-2 cDNA encoded a 244 amino acids (predicted molecular weight of 27.7 kDa), which shared a common N-terminus with other vertebrate and invertebrate TIMPs, including Ac-TMP-1, the most abundant adult hookworm secreted protein. However Ac-TMP-2 also contains an unusual multicopy (ten) repeat of the amino acid sequence, KTVEENDE. By immunoblotting, Ac-TMP-2 was detected only in adult hookworms and their excretory secretory products although the corresponding mRNA was also detected in L3. Immunolocalization with specific antiserum showed that native Ac-TMP-2 was located in adult worm's esophagus and cephalic glands. Recombinant Ac-TMP-2 expressed in bacteria was highly immunogenic and recognized by ir-L3 immunized dog immune sera. The recombinant Ac-TMP-2 protein inhibited the human matrix metalloproteinases, MMP-2, MMP-7 and MMP-13. As an immunodominant protein having a possible role in the parasite-host relationship of canine hookworm infection, recombinant Ac-TMP-2 represents a plausible target for vaccine development.     

Ancylostoma ceylanicum anticoagulant peptide-1: role of the predicted reactive site amino acid in mediating inhibition of coagulation factors Xa and VIIa

Hookworm infection is a leading cause of gastrointestinal blood loss and iron deficiency anemia in developing countries. Ancylostoma hookworms secrete potent anticoagulants, which have been shown to target coagulation factors Xa and the factor VIIa/Tissue Factor complex, respectively. The goal of these experiments was to determine the mechanism of action of three recombinant hookworm anticoagulants using in vitro assays. Three hookworm coagulation inhibitors were expressed and purified, along with site directed mutants targeting each of the predicted P1 inhibitory reactive site amino acid residues. Using chromogenic assays, it has been confirmed that Ancylostoma caninum Anticoagulant Peptide 5 (AcAP5) inhibits coagulation factor Xa (fXa) by a canonical, substrate-like mechanism. In contrast, Ancylostoma ceylanicum Anticoagulant Peptide-1 (AceAP1) binds to and inhibits fXa by both active site and non-active site mediated interactions. Data from in vitro studies also demonstrates that AceAP1 inhibits the factor VIIa/Tissue complex (fVIIa/TF) and displays a distinct pattern of fXa binding. Together, these data suggest that the human hookworm A. ceylanicum has evolved a single anticoagulant that targets multiple components of the mammalian coagulation response, effectively mimicking the concerted action of the two related inhibitors from A. caninum. Despite the amino acid sequence similarity, AceAP1 appears to interact with coagulation proteases fXa and fVIIa by a novel mechanism, perhaps explaining its spectrum of inhibitory activity.     

Metalloproteases of infective Ancylostoma hookworm larvae and their possible functions in tissue invasion and ecdysis.

To infect their hosts, hookworm larvae must exsheath and migrate through connective tissue. A modified in vitro skin chamber was used to show that the human hookworm Ancylostoma duodenale and the zoonotic canine hookworm Ancylostoma caninum penetrate epidermis, basement membrane, and dermis in similar ways. These similarities in tissue invasion properties reflect the observed biochemical similarities in parasite protease composition. The larvae of both species contain protease activity that is inhibited by o-phenanthroline; this identifies the proteases as metalloproteases. The enzyme activities exhibit an alkaline pH optimum between pH 9 and 10. During modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which a protein substrate (either casein or gelatin) was used, the protease activities resolved into a major band at an Mr of 68,000 and a minor band at an Mr of 38,000. Proteases were released by living A. caninum larvae in vitro and degraded purified and radiolabeled casein to smaller peptides. Motile hookworm larvae were also incubated with purified and radiolabeled connective tissue macromolecules in vitro. Both Ancylostoma species degraded human fibronectin to a 60,000-Mr polypeptide intermediate, but could not degrade solubilized bovine elastin or human laminin. In contrast, the obligate skin-penetrating nematode Strongyloides stercoralis degraded all three substrates. This biochemical difference may explain some observed differences in invasiveness.     

Ancylostoma secreted protein 2: cloning and characterization of a second member of a family of nematode secreted proteins from Ancylostoma caninum.

Invading infective third-stage larvae (L3) of parasitic nematodes execute a series of programmed developmental events in response to a host-specific signal encountered during infection. One of these early events is the release of excretory/secretory products. Using an in vitro feeding assay that mimics these early events of infection, a protein released by in vitro activated larvae of the hookworm Ancylostoma caninum was identified. This protein, Ac-ASP-2, was partially sequenced, and the cDNA encoding it isolated by PCR and screening of an A. caninum L3 cDNA library. The Ac-asp-2 cDNA encodes a protein of 219 amino acids that is related to a previously identified protein, Ac-ASP-1, from hookworms. Both molecules are members of an evolutionarily diverse family of molecules that include the venom allergens of the Hymenoptera, and the testes specific proteins/sperm-coating glycoproteins of mammals. Homologues are present in nearly all nematodes tested, as demonstrated by PCR-hybridization and database searching. The Ac-asp-2 mRNA is synthesized in all life history stages, but the gene product is released only by L3 activated to feed in vitro. The wide distribution of the Ac-asp-2 in nematodes and its release in response to host specific signals suggests that Ac-ASP-2 serves an important function in nematode physiology and development, and possibly in the infective process of parasitic species.     

Extent and course of excretion of larva in the milk of female dogs after infections of different severity with Ancylostoma caninum (Ancylostomatidae)]

It was investigated whether there is a linear correlation between the number of applied infective larvae of Ancylostoma caninum and the number of larvae excreted by bitches with the milk. The investigations were carried out with nine bitches that were infected percutaneously each with 5,000, 10,000 or 20,000 third stage larvae of Ancylostoma caninum at the day of conception. A clear linear correlation between the number of applied infective larvae of Ancylostoma caninum and the number of larvae excreted with the milk could be demonstrated only for the first week of lactation. Regarding the total investigated period of 28 days only a tendency towards such a correlation between infective dosage and excretion of larvae with the milk could be found.     

AcAPc2自剪切融合蛋白表达载体pTWIN1-AcAPc2的构建和表达

为表达和获取具有抗凝血功能的犬钩虫抗凝血肽（AcAPc2），采用搭桥PCR方法合成犬钩虫抗凝血肽全长双链cDNA序列，AcAPc2cDNA全序列连入表达载体pTWIN1上构建成具有蛋白自剪切功能的表达载体pTWIN1-AcAPc2，将阳性重组子转入表达型大肠杆菌E．coli ER2566进行表达。表达的融合蛋白AcAPc2-intein2-CBD为可溶性蛋白，且融合蛋白约占菌体总蛋白的30．1％，经蛋白质印迹分析确定表达产物是具有CBD蛋白的特异性融合蛋白。融合蛋白AcAPc2-intein2-CBD经几丁质柱高效亲和纯化，并经β-巯基乙醇诱导的独特的在柱自剪切后，得到目的蛋白AcAPc2，经SDS—PAGE分析在21KD处呈现目的条带，所得的可溶性AcAPc2分子量符合其天然活性的二聚体形式。对AcAPc2表达和纯化工艺上的改进，方便了AcAPc2的获取，AcAPc2氨基酸序列的生物信息学分析初步阐明了AcAPc2的结构和功能的关系，为进一步研究AcAPc2的抗凝血机制及其作为抗凝血药的临床应用奠定了基础。     

Cloning and characterization of a novel gene encoding 16 kDa protein (Ac16) from Angiostrongylus cantonensis

Angiostrongylus cantonensis is the most common infectious cause of eosinophilic meningitis or meningoencephalitis. A novel gene (AC16) was isolated from a cDNA library of A. cantonensis fourth-stage larvae. The putative 16-kDa protein has 149 amino acids and is homologous to an immunodominant hypodermal antigen (IHA16) from Ancylostoma caninum (identities?=?57%). In this paper, we cloned the gene and purified the recombinant Ac16 (rAC16) protein. Real-time quantitative PCR revealed that Ac16 was expressed significantly higher in the fourth-stage larvae and adult worms derived from rats than that in the fourth-stage larvae derived from mice. Moreover, sera from rat (permissive host) infected with A. cantonensis detected Ac16 by Western blot, while sera from infected mouse (non-permissive host) could not. The results implied that Ac16 was related to the parasitic adaptation of A. cantonensis in different hosts and non-permissive host mouse had no circulating antibody to the antigen Ac16 from A. cantonensis and thus might contribute to understanding the mechanism of parasite immune evasion. Furthermore, we evaluated the ability of Ac16 antibody diagnosing A. cantonensis infection by an indirect enzyme-linked immunosorbent assay. The results showed that the Ac16 antibody had a 79.17% sensitivity to rAC16 and 83.33% to crude adult worm antigens (CA) (P?>?0.05), while the specificity to rAC16 and to CA were 95.89% and 86.30% respectively (P?相似文献   

Efficacy of emodepside plus toltrazuril (Procox(®) oral suspension for dogs) against Toxocara canis, Uncinaria stenocephala and Ancylostoma caninum in dogs

The efficacy of emodepside plus toltrazuril (Procox? oral suspension for dogs) against different species of gastrointestinal nematodes (Toxocara canis, Ancylostoma caninum, Uncinaria stenocephala) was evaluated in nine randomised,blinded and placebo-controlled laboratory studies in naturally or experimentally infected dogs. The product was used at the proposed minimum dose of 0.45 mg emodepside and 9 mg toltrazuril per kg body weight. Efficacy was calculated based on worm counts after necropsy. Worm burdens in the control dogs ranged between 0 and 409 worms of the respective stage for T. canis and between 4 and 655 worms for hookworms. The studies demonstrated 100 % efficacy of emodepside/toltrazuril suspension against mature adult, ≥ 94.7 %efficacy against immature adult and 99.3 % efficacy against the L4 larval stage of T. canis. The efficacy against mature adult A. caninum was ≥ 99.5 % and the efficacy against mature adult U. stenocephala was 100 %. All differences between treatment and control groups were statistically significant and no gender effect was found. It can be concluded that the emodepside/toltrazuril suspension represents a safe and highly effective product in dogs with nematode (T. canis, hookworms) infection.     

The canine hookworm genome: analysis and classification of Ancylostoma caninum survey sequences

Hookworms infect nearly a billion people. The Ancylostoma caninum hookworm of canids is a model for studying human infections and information from its genome coupled with functional genomics and proteomics can accelerate progress towards hookworm control. As a step towards a full-scale A. caninum genome project, we generated 104,000 genome survey sequences (GSSs) and determined the genome size of the canine hookworm. GSSs assembled into 57.6 Mb of unique sequence from a genome that we estimate by flow cytometry of isolated nuclei to be 347 +/- 1.2 Mb, substantially larger than other Rhabditina species. Gene finding identified 5538 genes in the GSS assembly, for a total of 9113 non-redundant A. caninum genes when EST sequences are also considered. Functional classifications of many of the 70% of genes with homology to genes in other species are provided based on gene ontology and KEGG associations and secreted and membrane-bound proteins are also identified.     

Biochemical characterization and vaccine potential of a heme-binding glutathione transferase from the adult hookworm Ancylostoma caninum

We report the cloning and expression of Ac-GST-1, a novel glutathione S-transferase from the adult hookworm Ancylostoma caninum, and its possible role in parasite blood feeding and as a vaccine target. The predicted Ac-GST-1 open reading frame contains 207 amino acids (mass, 24 kDa) and exhibited up to 65% amino acid identity with other nematode GSTs. mRNA encoding Ac-GST-1 was detected in adults, eggs, and larval stages, but the protein was detected only in adult hookworm somatic extracts and excretory/secretory products. Using antiserum to the recombinant protein, Ac-GST-1 was immunolocalized to the parasite hypodermis and muscle tissue and weakly to the intestine. Recombinant Ac-GST-1 was enzymatically active, as determined by conjugation of glutathione to a model substrate, and exhibited a novel high-affinity binding site for hematin. The possible role of Ac-GST-1 in parasite heme detoxification during hemoglobin digestion or heme uptake prompted interest in evaluating it as a potential vaccine antigen. Vaccination of dogs with Ac-GST-1 resulted in a 39.4% reduction in the mean worm burden and 32.3% reduction in egg counts compared to control dogs following larval challenge, although the reductions were not statistically significant. However, hamsters vaccinated with Ac-GST-1 exhibited statistically significant worm reduction (53.7%) following challenge with heterologous Necator americanus larvae. These studies suggest that Ac-GST-1 is a possible drug and vaccine target for hookworm infection.     

Characterization of a recombinant immunodiagnostic antigen (NIE) from Strongyloides stercoralis L3-stage larvae

Due to the process of internal autoinfection, even chronic asymptomatic infections with Strongyloides stercoralis have the potential to become severe disseminated disease with fatal outcome. Intermittent and scanty larval excretion makes parasitologic diagnosis difficult. Serodiagnosis is helpful, but antigen preparation from infective larvae requires access to patients or immunosuppressed experimental animals. For these reasons, attention has turned to recombinant antigens for immunodiagnosis. A 31-kDa candidate antigen (NIE) derived from an L3 cDNA library is described in this report. Multiple alignment of the deduced amino acid sequence of NIE showed approximately 12-18% identity with various other organisms, including 17.9% of Asp1 of Ancylostoma caninum, 12.6% of Hemonchus contortus, and 17.6% of insect venom allergen 5 of yellow jacket. By ELISA, antibodies to the purified recombinant NIE antigen were demonstrated in 87.5% of 48 sera from strongyloides-infected patients and in only 6.5% of sera from presumed normal controls. Immunoreactivity of purified NIE antigen with parasite-specific IgE from sera of strongyloides-infected patients indicated its potential use as an immediate sensitivity skin test antigen. This application of the NIE antigen was supported by its capacity to trigger release of histamine upon in vitro exposure to blood from strongyloides-infected patients and its failure to produce histamine release from blood of normal controls.     

Identification of ribosomal phosphoprotein P0 of Neospora caninum as a potential common vaccine candidate for the control of both neosporosis and toxoplasmosis

The characterization of the cross-reactive antigens of two closely related apicomplexan parasites, Neospora caninum and Toxoplasma gondii, is important to elucidate the common mechanisms of parasite-host interactions. In this context, a gene encoding N. caninum ribosomal phosphoprotein P0 (NcP0) was identified by immunoscreening of a N. caninum tachyzoite cDNA expression library with antisera from mice immunized with T. gondii tachyzoites. The NcP0 was encoded by a gene with open reading frame of 936 bp, which encoded a protein of 311 amino acids. The NcP0 gene existed as a single copy in the genome and was interrupted by a 432 bp intron. The NcP0 showed 94.5% amino acid identity to T. gondii P0 (TgP0). Anti-recombinant NcP0 (rNcP0) sera recognized a native parasite protein with a molecular mass of 34 kDa in Western blot analysis. Immunofluorescence analysis showed that the NcP0 was localized to the surface of N. caninum tachyzoites. A purified anti-rNcP0 IgG antibody inhibited the growth of N. caninum and T. gondii in vitro in a concentration-dependent manner. These results indicate that P0 is a cross-reactive antigen between N. caninum and T. gondii and a potential common vaccine candidate to control both parasites.