Purification of a diagnostic, secreted cysteine protease-like protein from the hookworm Ancylostoma caninum

The enteric infection of humans with the canine hookworm Ancylostoma caninum varies in its clinical presentation, ranging from asymptomatic to eosinophilic gastroenteritis requiring surgical intervention. Infections are not patent, but can be diagnosed immunologically by detecting antibodies to an immunodominant secreted hookworm protein termed Ac68. To characterise Ac68, we purified the native protein from A. caninum excretory/secretory products using size exclusion followed by anion exchange chromatography. The epitopes in the purified protein recognised by human infection sera were shown to be proteins and not carbohydrates. The N-terminal amino acid sequence of the purified Ac68 was determined and six of the 11 residues obtained were shared with a previously characterised cysteine protease of A. caninum, AcCP1.     

Activation of Nippostrongylus brasiliensis infective larvae is regulated by a pathway distinct from the hookworm Ancylostoma caninum

Developmentally arrested infective larvae of strongylid nematodes are activated to resume growth by host-derived cues encountered during invasion of the mammalian host. Exposure of Nippostrongylus brasiliensis infective larvae to elevated temperature (37 °C) is sufficient to activate signalling pathways which result in resumption of feeding and protein secretion. This occurs independently of exposure to serum or glutathione, in contrast to the hookworm Ancylostoma caninum, and is not initiated by chemical exsheathment. No qualitative differences in protein secretion were induced by host serum as visualised by two-dimensional SDS–PAGE, although exposure of larvae to an aqueous extract of rat skin did stimulate secretion of a small pre-synthesised bolus of proteins. Infective larvae began feeding after a lag period of 3–4 h at 37 °C, reaching a maximum of 90% of the population feeding by 48 h. Neither a membrane permeant analogue of cyclic GMP nor muscarinic acetylcholine receptor agonists stimulated feeding at 20 °C, and high concentrations of both compounds inhibited temperature-induced activation. LY294002, an inhibitor of phosphatidylinositol 3-kinase, Akt inhibitor IV, an inhibitor of Akt protein kinase, and ketoconazole, an inhibitor of cytochrome P450, all blocked resumption of feeding and protein secretion at 37 °C. Serotonin increased the rate of feeding assessed by uptake of radiolabelled BSA, but could not initiate feeding independently of elevated temperature. Collectively, the data suggest that the early signalling events for larval activation in N. brasiliensis differ substantially from A. caninum, but that they may converge at pathways downstream of phosphatidylinositol 3-kinase involving steroid hormone synthesis.     

Sequence differences in the internal transcribed spacer ribosomal DNA among four species of hookworm (Ancylostomatoidea: Ancylostoma)

The two ribosomal DNA internal transcribed spacers (1 and 2) of the hookworms Ancylostoma caninum, A. tubaeforme, A. ceylanicum and A. duodenale were sequenced. The sequence lengths were similar among the four species, except that A. ceylanicum had slightly longer (by 5–7 bp) internal transcribed spacer 1 and 2 sequences. The predicted secondary structure of the internal transcribed spacer 2 precursor rRNA was similar for all species, despite interspecific differences in primary sequence ranging from 0.9% to 13.2%. Interspecific differences in internal transcribed spacer 1 sequence ranged from 0.9% to 7.5%. A cladistic analysis of the sequence data, using the human hookworm Necator americanus as the outgroup, provided little resolution of the phylogenetic relationships, except that A. ceylanicum occurred on a branch external to the other three species. Nonetheless, internal transcribed spacers 1 and 2 may provide useful phylogenetic information at higher taxonomic levels within the superfamily Ancylostomatoidea.     

Purification and Characterization of a DnaK-like and a GroEL-like Protein from Porphyromonas gingivalis

Heat-shock proteins of Porphyromonas gingivalis were demonstrated and two of them were purified and further characterized. The amplified de novo synthesis of two different proteins, with apparent molecular weights of 75 kDa and 68 kDa, was observed by autofluorography when a P. gingivalis culture incubated in a ¹⁴C-labeled amino acid mixture was shifted from 37°C to 44°C. Both proteins possessed ATP-binding abilities and were purified to almost homogeneity employing affinity chromatography on ATP-agarose followed by preparative SDS-PAGE. Purified 75 kDa and 68 kDa proteins had isoelectric points of 4.4 and 4.6, respectively. They were shown to be immunoreactive with commercial anti-DnaK and anti-GroEL polyclonal antibodies, respectively. Immunoblotting analysis of whole cells using antiserum raised against each purified protein from P. gingivalis, confirmed elevated synthesis of both proteins during thermal shock. A GroEL protein reacted strongly with antiserum against the 68 kDa protein. However, a DnaK protein reacted weakly with antiserum to the 75 kDa protein. Analysis of the N-terminal amino acid sequence of the DnaK-like protein (75 kDa) showed a high degree of homology with those of the HSP70 family including both prokaryotic and eukaryotic cells. The N-terminal amino acid analysis of the GroEL-like protein (68 kDa) indicated that it was identical to those of cloned GroEL homologues from P. gingivalis.     

Transcriptional changes in the hookworm, Ancylostoma caninum, during the transition from a free-living to a parasitic larva

Background

Third-stage larvae (L3) of the canine hookworm, Ancylostoma caninum, undergo arrested development preceding transmission to a host. Many of the mRNAs up-regulated at this stage are likely to encode proteins that facilitate the transition from a free-living to a parasitic larva. The initial phase of mammalian host invasion by A. caninum L3 (herein termed “activation”) can be mimicked in vitro by culturing L3 in serum-containing medium.

Methodology/Principal Findings

The mRNAs differentially transcribed between activated and non-activated L3 were identified by suppression subtractive hybridisation (SSH). The analysis of these mRNAs on a custom oligonucleotide microarray printed with the SSH expressed sequence tags (ESTs) and publicly available A. caninum ESTs (non-subtracted) yielded 602 differentially expressed mRNAs, of which the most highly represented sequences encoded members of the pathogenesis-related protein (PRP) superfamily and proteases. Comparison of these A. caninum mRNAs with those of Caenorhabditis elegans larvae exiting from developmental (dauer) arrest demonstrated unexpectedly large differences in gene ontology profiles. C. elegans dauer exiting L3 up-regulated expression of mostly intracellular molecules involved in growth and development. Such mRNAs are virtually absent from activated hookworm larvae, and instead are over-represented by mRNAs encoding extracellular proteins with putative roles in host-parasite interactions.

Conclusions/Significance

Although this should not invalidate C. elegans dauer exit as a model for hookworm activation, it highlights the limitations of this free-living nematode as a model organism for the transition of nematode larvae from a free-living to a parasitic state.     

Cloning and characterization of an extracellular serine protease from the nematode-trapping fungus <Emphasis Type="Italic">Arthrobotrys conoides</Emphasis>

An extracellular serine protease (Ac1) with a molecular mass of 35 kDa was purified from the nematode-trapping fungus Arthrobotrys conoides. The optimum activity of Ac1 is at pH 7.0 and 53.2°C (over 20 min). Ac1 can degrade a broad range of substrates including casein, gelatin, bovine serum albumin, collagen, and nematode cuticles. Moreover, the enzyme can immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, indicating Ac1 may be involved in infection against nematodes. The encoding gene of Ac1 contains one intron of 60-bp and two exons encoding a polypeptide of 411 amino acid residues. The deduced polypeptide sequence of Ac1 showed a high degree of similarity to two previously reported serine proteases PII and Mlx from other nematode-trapping fungi (81% aa sequence identity). However, three proteases Ac1, Aoz1 and Mlx showed optimum temperatures at 53.2, 45 and 65°C, respectively. Compared to PII, Ac1 appears to have a significantly higher activity against gelatin, bovine serum albumin, and non-denatured collagen. Moreover, our bioassay experiments showed that Ac1 is more effective at immobilizing P. redivivus than B. xylophilus.     

HSP68 — a DnaK-like heat-stress protein of plant mitochondria

A 68-kDa heat-stress protein (HSP68) has been purified from cell-suspension cultures of tomato (Lycopersicon peruvianum L.). Antibodies raised against HSP68 cross-react with the Escherichia coli heat-stress protein DnaK. HSP68 was found to be a hydrophilic, ATP-binding protein. Immunological analysis of subcellular fractions and immunogold-labelling of ultrathin sections showed consistently that HSP68 is localized in the mitochondrial matrix. In-vitro translation experiments indicated that HSP68 is synthesized as a precursor protein. Immunoscreening of cDNA libraries from tomato and potato (Solanum tuberosum L.) led to the isolation of corresponding cDNA clones. The deduced amino-acid sequences show strong relationships to the DnaK-like proteins from bacteria and organelles of eukaryotic cells. The protein HSP68 is constitutively expressed, but its synthesis is increased during heat stress in all cells of higher plantes investigated so far.Abbreviations HSP heat-stress protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by the Deutsche Forschungsgemeinschaft.     

Characterization of cathepsin B proteinase (AcCP-2) in eggs and larvae stages of hookworm Ancylostoma caninum

Cathepsin B proteinase constitutes a large multigenes family in parasitic and non-parasitic nematodes. The localization of cathepsin B proteinases (AcCP-1 and AcCP-2) in adult worm of Ancylostoma caninum has been characterized (Harrop et al., 1995), but the localization and function in eggs and larval stages remained undiscovered. Here we described the expressing of cathepsin B proteinase (AcCP-2) in Escherichia coli, and immuno-localization of cathepsin B proteinase in eggs and larvae stages of A. caninum. A cDNA fragment encoding a cathepsin B proteinase (AcCP-2) was cloned from A. caninum and expressed in E. coli. Gelatin digestion showed that recombinant cathepsin B proteinase (AcCP-2) has protease activity. The protein level of cathepsin B proteinase in larval and adult worm was detected by western blot. The immuno-localization of cathepsin B proteinase in eggs and larval stages was characterized. The expression of cathepsin B proteinase was more abundant in eggs and larvae stages of A. caninum. It implied that cathepsin B proteinase might play roles in the early development of A. caninum.     

Molecular Detection of Ancylostoma duodenale,Ancylostoma ceylanicum,and Necator americanus in Humans in Northeastern and Southern Thailand

The 2 principal species of hookworms infecting humans are Necator americanus and Ancylostoma duodenale. Case studies on zoonotic hookworm infections with Ancylostoma ceylanicum and/or Ancylostoma caninum are known mainly from Asian countries. Of these 2 zoonotic species, only A. ceylanicum can develop to adulthood in humans. In the present study, we report a molecular-based survey of human hookworm infections present in southern and northeastern Thailand. Thirty larval hookworm samples were obtained from fecal agar plate cultures of 10 patients in northeastren Thailand and 20 in southern Thailand. Partial ITS1, 5.8S, and ITS2 regions of the ribosomal DNA genes were amplified using PCR. The amplicons were sequenced, aligned, and compared with other hookworm sequences in GenBank database. The results showed that, in Thailand, N. americanus is more prevalent than Ancylostoma spp. and is found in both study areas. Sporadic cases of A. ceylanicum and A. duodenale infection were seen in northeastern Thailand.     

Early postabortion recovery of Neospora-infected lactating dairy cows

This study was designed to assess the reproductive performance of Neospora caninum–infected dairy cows (Bos taurus) after abortion. The population examined was composed of 92 aborting cows subsequently subjected to at least one artificial insemination (AI) attempt. Of these animals, 68 were N. caninum seropositive and 24 were seronegative. Only animals producing at least 25 kg milk at the estrus after abortion were inseminated. Normal uterine involution was verified at first AI after abortion in cows showing estrous signs within 30 d of abortion. Of the 92 aborting cows, 73 (79.3%) became pregnant after abortion: 57 (83.8%) and 16 (66.7%) of the N. caninum seropositive and seronegative cows, respectively. The factors days of gestation and lactation number at abortion failed to differ between the two Neospora serostatus groups, based on Student's t-test or Kruskal-Wallis tests, respectively. The interval from abortion to first AI, the number of AIs necessary for an animal to become pregnant, and the interval from abortion to next pregnancy in cows becoming pregnant were lower for the N. caninum–seropositive than for the N. caninum–seronegative cows (P < 0.0001). Logistic regression analysis indicated no significant effects of herd, lactation number, days of gestation at abortion, and the interval from abortion to AI on the likelihood of pregnancy at first AI after abortion. Based on the odds ratio, Neospora-seropositive cows were 6.22 times more likely to become pregnant at first AI after abortion compared with their seronegative partners. Fifty-three of the Neospora-seropositive cows (78%) were inseminated within 30 d after abortion and 26 (49%) became pregnant, whereas only one seronegative cow (4.2%) was ready for insemination within this 30-d period and remained open after first AI. These results indicate that Neospora-infected cows suffering abortion can be inseminated with a likelihood of high fertility within the first month of abortion. We recommend checking that uterine involution is normal early after abortion in N. caninum–seropositive cows.     

Electron and light microscopy of neutrophil responses in mice vaccinated and challenged with third-stage infective hookworm (Ancylostoma caninum) larvae

The role of neutrophils in mediating host inflammation was examined in mice vaccinated with living third-stage infective hookworm larvae (L₃). Mice were vaccinated by oral immunization with 500 L₃ (Ancylostoma caninum) once every 2 weeks for a total of three immunizations. The vaccinated mice were then challenged intraperitoneally with 2000 L_3, 1 week after the final immunization. To stimulate peritoneal production of neutrophils, 2 ml of 2% glycogen were injected intraperitoneally at 16 h prior to the challenge infection. Neutrophils were found to comprise 85% of the peritoneal cell population. L₃ from the challenge infection were collected and then examined at timed intervals by inverted light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Greater than a fivefold increase in the total numbers of peritoneal cells was noted in the vaccinated mice as compared to unvaccinated mice. In the peritoneal cavity of vaccinated mice, the neutrophils adhered to the L₃ within 2 h, and over 55% of the L₃ were surround by clusters of neutrophils to form a sausage-like sheath 4 h later. At 24–72 h after challenge, almost all of the L₃ recovered from the vaccinated mice were covered with thick clusters of cells. Both SEM and TEM demonstrated extensive ultrastructural damage to the L_3. In contrast, the L₃ recovered from the unvaccinated mice appeared to be unaffected by neutrophils. These studies suggest that neutrophils, like macrophages, can have an important role as effector cells in L₃-vaccinated mice.     

First epidemiological study on exposure to Neospora caninum in different canine populations in the Algiers District (Algeria)

Neospora caninum is an important cause of abortion in cattle worldwide. Dogs act as final hosts shedding oocysts in the environment. They can also harbour the extraintestinal stage of the parasite and this may be associated with a fairly rare neuromuscular condition. The sera of 781 dogs from the Algiers District were screened by IFAT for the presence of anti-N. caninum antibodies. These dogs were distributed into four populations: local stray dogs, police dogs, dogs from breeding kennels and farm dogs. The overall seroprevalence was 21.90%. Significant differences were observed between the different populations, the highest prevalence being observed in farm (44.44%) and stray dogs (22.55%). Additionally, the highest titres were observed in farm dogs. Among studied epidemiological parameters, breed, dog origin, season and vaccination status were significantly associated with IFAT results. Additionally, a recently described real time PCR was used on the blood of 100 pound dogs and the results were compared with the serological data. A higher proportion of dogs was found to be positive by PCR when compared to the IFAT results. There was only a fairly low agreement between PCR and IFAT results which suggests that these techniques measured different aspects of the host–parasite relationship. This study indicates that the level of exposure of the canine population of Algiers area to N. caninum is very high. This would indicate a potentially high risk for N. caninum induced abortion in cattle in this region and in Algeria.     

Ancylostoma caninum: calibration and comparison of diagnostic accuracy of flotation in tube, McMaster and FLOTAC in faecal samples of dogs

We performed a calibration of flotation in tube, McMaster and FLOTAC to determine the optimal flotation solution (FS) and the influence of faecal preservation for the diagnosis of Ancylostoma caninum in dogs, and compared the accuracy of the three copromicroscopic techniques. Among nine different FS, sodium chloride and sodium nitrate performed best for detection and quantification of A. caninum eggs. Faecal samples, either fresh or preserved in formalin 5%, resulted in higher A. caninum egg counts, compared to frozen samples or preserved in formalin 10% or sodium acetate–acetic acid–formalin. FLOTAC consistently resulted in higher A. caninum eggs per gram of faeces (EPG) and lower coefficient of variation (CV) than McMaster and flotation in tube. The best results in terms of mean faecal egg counts (highest value, i.e. 117.0 EPG) and CV (lowest value, i.e. 4.8%) were obtained with FLOTAC using sodium chloride and faecal samples preserved in formalin 5%. Our findings suggest that the FLOTAC technique should be considered for the diagnosis of A. caninum in dogs.     

The Lectin from the Crustacean Liocarcinus depurator Recognizes O-acetylsialic Acids

A lectin that recognized sialic acids and agglutinated mouse erythrocytes was purified from hemolymph of the crab Liocarcinus depurator. It consisted of 38-kDa subunits and had a pI about 6.0. The specificity of the lectin was assayed by hemagglutination inhibition. N-acetylneuraminic acid (Neu5Ac) was a good inhibitor and its N-acetyl group at C-5 was critical for lectin-ligand interaction. Substitution of the C-9 hydroxyl on Neu5Ac with an O-acetyl group (9-O-Ac-Neu5Ac) increased the inhibitory potency of this molecule. Furthermore, O-acetyl substitution of all the hydroxyl groups yielded even better inhibitors (2,4,7,8,9-O-Ac-Neu5Ac and its 1-O-methyl ester). Removal of the hydroxyl or O-acetyl group connected to C-2 reduced the potency of these inhibitors. The lectin agglutinated and stimulated human but not mouse lymphocytes. It was also inhibited by Escherichia coli (O111:B4) lipopolysaccharide and agglutinated specific gram-negative bacteria. In vitro labeling with [³⁵S]methionine indicated that the lectin was synthesized in hepatopangreas of L. depurator. Immunofluorescence showed that among hemocytes it localized mainly in the large-granule population.     

副溶血弧菌SH112株OmpA蛋白的高效表达及免疫学特性

【目的】我们前期研究表明副溶血弧菌SH112株的OmpA蛋白在该菌的致病过程中发挥重要作用,是亚单位疫苗研制的潜在靶标抗原。本研究进一步对ompA(VPA1186)基因进行克隆表达,并研究其免疫学特性。【方法】扩增去除信号肽序列的成熟外膜蛋白OmpA的基因片段,定向克隆至表达载体,基因测序后对其编码蛋白质进行生物信息学分析。重组蛋白His-OmpA经纯化后,免疫ICR小鼠制备鼠多抗血清。Western blotting检测该蛋白的免疫原性及鼠多抗血清的特异性。动物实验验证其免疫保护率。【结果】成功表达分子量约为40.0 kDa的重组蛋白His-OmpA。制备的鼠多抗血清ELISA效价可达1∶50000以上。Westernblotting检测结果显示,该血清可与His-OmpA蛋白、总外膜蛋白和全菌蛋白发生特异性反应,说明所表达的目的蛋白保持原蛋白的免疫原性。此外,该高免血清可与其他主要血清型的副溶血弧菌发生特异性交叉反应,而与其他非副溶血弧菌菌株无交叉反应,表明该血清特异性较高,且提示OmpA蛋白可能是副溶血弧菌属的共同保护性抗原。小鼠免疫保护实验结果表明,该蛋白可提供约35%的免疫保护率。【结论】OmpA蛋白可作为诊断副溶血弧菌感染和亚单位疫苗研制的靶蛋白,为进一步开展该蛋白的功能研究提供了参考。     

Neospora caninum: comparative gene expression profiling of Neospora caninum wild type and a temperature sensitive clone

To understand the genetic basis of virulence, gene expression profiles of a temperature-sensitive clone (NCts-8, relatively avirulent) and its wild type (NC-1) of Neospora caninum were characterized and compared using a high-density microarray with approximately 63,000 distinct oligonucleotides. This microarray consists of 5692 unique N. caninum sequences, including 1980 Tentative Consensus sequences and 3712 singleton ESTs from the TIGR N. caninum Gene Index (NCGI, release 5.0). Each sequence was represented by 11 distinct 60mer oligonucleotides synthesized in situ on the microarray. The results showed that 111 genes were significantly repressed and no up-regulated genes were identified in the NCts-8 clone. The level of 10 randomly selected genes from the repressed genes was confirmed using real-time RT-PCR. Of the 111 repressed genes, 58 were hypothetical protein products and 53 were annotated genes. Over 70% of the repressed genes identified in this study are clustered on five chromosomes (I, VII, VIII, X and XII). These results suggest that the down-regulated genes may be in part responsible for the reduced pathogenesis of NCts-8; further characterization of the regulated genes may aid in understanding of molecular basis of virulence and development of countermeasures against neosporosis.     

Purification and characterization of sialidase fromClostridium sordellii G12

Sialidase secreted by the urease-positiveClostridium sordellii strain G12 was isolated from culture medium and purified to apparent homogeneity as estimated by Fast Protein Liquid Chromatography (FPLC) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, ion-exchange chromatography, gel filtration, isoelectric focusing, and FPLC on ion-exchange resin and gel filtration materials were used. The sialidase was purified 159 300-fold from 5 l of culture medium, yielding 9 g of enzyme protein with a specific activity of 480 U/mg. For the denatured (SDS-PAGE) and native (FPLC) sialidase relative molecular masses of 40 000 and 38 500 Da, respectively, were estimated. The substrate specificity, kinetic data, and pH-optimum of the enzyme are similar to those of other bacterial sialidases. The influences of salt or serum proteins on enzyme activity are of interest.Abbreviations MU-Neu5Ac 4-methylumbelliferyl -d-N-acetylneuraminic acid - Ganglioside GD1a IV³NeuAc, ll³NeuAc-GgOse₄Cer - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid