A Helminth-Derived Chitinase Structurally Similar to Mammalian Chitinase Displays Immunomodulatory Properties in Inflammatory Lung Disease.

Immunomodulation of airway hyperreactivity by excretory-secretory (ES) products of the first larval stage (L1) of the gastrointestinal nematode Trichuris suis is reported by us and others. Here, we aimed to identify the proteins accounting for the modulatory effects of the T. suis L1 ES proteins and studied six selected T. suis L1 proteins for their immunomodulatory efficacy in a murine OVA-induced allergic airway disease model. In particular, an enzymatically active T. suis chitinase mediated amelioration of clinical signs of airway hyperreactivity, primarily associated with suppression of eosinophil recruitment into the lung, the associated chemokines, and increased numbers of RELMα + interstitial lung macrophages. While there is no indication of T. suis chitinase directly interfering with dendritic cell activation or antigen presentation to CD4 T cells, treatment of allergic mice with the worm chitinase influenced the hosts' own chitinase activity in the inflamed lung. The three-dimensional structure of the T. suis chitinase as determined by high-resolution X-ray crystallography revealed high similarities to mouse acidic mammalian chitinase (AMCase) but a unique ability of T. suis chitinase to form dimers. Our data indicate that the structural similarities between the parasite and host chitinase contribute to the disease-ameliorating effect of the helminth-derived chitinase on allergic lung inflammation.

Improved strategy for the curation and classification of kinases, with broad applicability to other eukaryotic protein groups.

Despite the substantial amount of genomic and transcriptomic data available for a wide range of eukaryotic organisms, most genomes are still in a draft state and can have inaccurate gene predictions. To gain a sound understanding of the biology of an organism, it is crucial that inferred protein sequences are accurately identified and annotated. However, this can be challenging to achieve, particularly for organisms such as parasitic worms (helminths), as most gene prediction approaches do not account for substantial phylogenetic divergence from model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, whose genomes are well-curated. In this paper, we describe a bioinformatic strategy for the curation of gene families and subsequent annotation of encoded proteins. This strategy relies on pairwise gene curation between at least two closely related species using genomic and transcriptomic data sets, and is built on recent work on kinase complements of parasitic worms. Here, we discuss salient technical aspects of this strategy and its implications for the curation of protein families more generally.

Polypyridylruthenium(II) complexes exert in vitro and in vivo nematocidal activity and show significant inhibition of parasite acetylcholinesterases.

Over 4.5 billion people are at risk of infection with soil transmitted helminths and there are concerns about the development of resistance to the handful of frontline nematocides in endemic populations. We investigated the anti-nematode efficacy of a series of polypyridylruthenium(II) complexes and showed they were active against L3 and adult stages of Trichuris muris, the rodent homologue of the causative agent of human trichuriasis, T. trichiura. One of the compounds, Rubb12-mono, which was among the most potent in its ability to kill L3 (IC50 = 3.1 ± 0.4 µM) and adult (IC50 = 5.2 ± 0.3 µM) stage worms was assessed for efficacy in a mouse model of trichuriasis by administering 3 consecutive daily oral doses of the drug 3 weeks post infection with the murine whipworm Trichuris muris. Mice treated with Rubb12-mono showed an average 66% reduction (P = 0.015) in faecal egg count over two independent trials. The drugs partially exerted their activity through inhibition of acetylcholinesterases, as worms treated in vitro and in vivo showed significant decreases in the activity of this class of enzymes. Our data show that ruthenium complexes are effective against T. muris, a model gastro-intestinal nematode and soil-transmitted helminth. Further, knowledge of the target of ruthenium drugs can facilitate modification of current compounds to identify analogues which are even more effective and selective against Trichuris and other helminths of human and veterinary importance.

Whipworm kinomes reflect a unique biology and adaptation to the host animal.

Roundworms belong to a diverse phylum (Nematoda) which is comprised of many parasitic species including whipworms (genus Trichuris). These worms have adapted to a biological niche within the host and exhibit unique morphological characteristics compared with other nematodes. Although these adaptations are known, the underlying molecular mechanisms remain elusive. The availability of genomes and transcriptomes of some whipworms now enables detailed studies of their molecular biology. Here, we defined and curated the full complement of an important class of enzymes, the protein kinases (kinomes) of two species of Trichuris, using an advanced and integrated bioinformatic pipeline. We investigated the transcription of Trichuris suis kinase genes across developmental stages, sexes and tissues, and reveal that selectively transcribed genes can be linked to central roles in developmental and reproductive processes. We also classified and functionally annotated the curated kinomes by integrating evidence from structural modelling and pathway analyses, and compared them with other curated kinomes of phylogenetically diverse nematode species. Our findings suggest unique adaptations in signalling processes governing worm morphology and biology, and provide an important resource that should facilitate experimental investigations of kinases and the biology of signalling pathways in nematodes.

Serine protease(s) secreted by the nematode Trichuris muris degrade the mucus barrier.

The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a T(H)2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s).

Cytochrome oxidase subunit 1 and mitochondrial 16S rDNA sequences of Trichuris skrjabini (Tricocephalida: Trichuridae).

The partial mitochondrial cytochrome c-oxidase subunit 1 gene (cox 1) and partial mitochondrial 16S ribosomal DNA of Trichuris skrjabini (Baskakov 1924) isolated from Capra hircus have been amplified and sequenced. The analyses of multiple sequence alignments of mitochondrial 16S rDNA and cox 1 of T. skrjabini revealed high homology with those of Trichinella species. For the first time, the mitochondrial DNA gene sequences of one species of trichurid nematode have been cited.

Genetic and morphological heterogeneity in small rodent whipworms in southwestern Europe: characterization of Trichuris muris and description of Trichuris arvicolae n. sp. (Nematoda: Trichuridae).

Genetic and morphological variability of whipworms Trichuris Roederer, 1761 (Nematoda: Trichuridae), parasites of small rodents in southwestern Europe, was studied. Isozyme patterns of natural populations of nematodes parasitizing rodent species of the Muridae (Apodemus sylvaticus, Apodemus flavicollis, Mus musculus) and Arvicolidae (Clethrionomys glareolus, Microtus agrestis, Microtus arvalis) were analyzed at 6 putative loci. Two diagnostic loci were found in T. muris from Muridae and from Arvicolidae. Thus, the existence of 2 species of Trichuris restricted to different host families was indicated. They included Trichuris muris Schrank, 1788, originally described as being from mice, and Trichuris arvicolae n. sp., parasitizing the above species of Arvicolidae. The morphological variability of both species was compared. Although ranges of all morphological characters of the new species overlapped with those of T. muris, stepwise discriminant analysis yielded a 100% accurate classification of females when using vagina length and egg size. Males of T. muris and T. arvicolae cannot be separated entirely. A set of 6 variables yielded 95.7% discrimination; the most discriminating variables were spicule size and body width.

Rapid purification and characterization of L-dopachrome-methyl ester tautomerase (macrophage-migration-inhibitory factor) from Trichinella spiralis, Trichuris muris and Brugia pahangi.

Macrophage-migration-inhibition factor (MIF) is an essential stimulator of mammalian T-lymphocyte-dependent adaptive immunity, hence MIF orthologues might be expressed by infectious organisms as an immunosubversive stratagem. Since MIF actively catalyses the tautomerization of the methyl ester of l-dopachrome (using dopachrome tautomerase), the occurrence of MIF orthologues in several parasitic helminths was investigated by assaying and characterizing such activity. Evidence of MIF orthologues (dopachrome tautomerase) was found in the soluble fraction of the nematodes Trichinella spiralis (stage 4 larvae) and Trichuris muris (adults), and the filarial nematode Brugia pahangi (adults). The MIF orthologues of Tr. muris (TmMIF) and B. pahangi (BpMIF) were purified to homogeneity using phenyl-agarose chromatography, that of T. spiralis (TsMIF) required a further step: cation-exchange FPLC. Retention time on reverse-phase HPLC and Mr on SDS/PAGE of the nematode MIFs were similar to those of human MIF. N-terminal sequences (19 residues) of TsMIF and TmMIF showed 47 and 36% identity, respectively, with human MIF. The N-terminal sequence of BpMIF (14 residues) was identical to that of an MIF orthologue in the genome of B. malayi (Swiss-Prot, P91850) and showed 43% identity to either human or TsMIF. TsMIF had 10-fold higher dopachrome tautomerase activity than MIF from the other sources. The enzyme activities of TsMIF, BpMIF and TmMIF were less sensitive to inhibition by haematin (I50: >15 microM, >15 microM and 2.6 microM, respectively) than that of human MIF (I50 0.2 microM). Significant dopachrome tautomerase or phenyl-agarose-purifiable MIF-like protein was not detected in the soluble fraction of the nematodes Heligmosomoides polygyrus and Nippostrongylus brasiliensis, the cestode Hymenolepis diminuta, or the trematodes Schistosoma mansoni, S. japonicum and S. haematobium, or the free-living nematode, Caenorhabditis elegans, which does contain an MIF-related gene.

Morphologic, biometric, and isoenzyme characterization of Trichuris suis.

Trichuris suis isolates were collected from the cecum of Sus scrofa domestica (pig) and S. s. scrofa (wild boar). Morphology and biometry studies were carried out. Morphology studies showed the existence of typical caudal papillae in males of T. suis from wild boars, but no other difference was observed in the biometric parameters (total length, esophageal length, posterior-portion body length, and spicular length) of T. suis isolated from either host. Individual extracts were subjected to malate dehydrogenase (MDH), malic enzyme (ME), glucose 6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDH), and superoxide dismutase (SOD) isoenzyme analysis following starch-gel electrophoresis, and the isoenzyme patterns were compared with those obtained from other species of trichurids. MDH, ME, G6PD, LDH, and SOD isoenzyme patterns were identical for T. suis from both hosts. MDH isoenzyme patterns were characterized by the presence of one cathodic isoenzyme. ME, G6PD, and LDH isoenzyme patterns indicated the presence of three phenotypes, whereas the SOD isoenzyme pattern showed only one phenotype characterized by the existence of two (anodic and cathodic) bands. Different LDH and SOD isoenzyme patterns observed for T. suis, T. ovis, and T. skrjabini confirm once more that isoenzyme patterns have potential as a diagnostic tool for differentiation of different species of Trichuris.

Characterization of protease(s) in adults of Trichuris globulosa (Nematoda: Trichuridae).

The activity of protease(s) has been observed separately in male and female Trichuris globulosa. Different fractions showed optimum protease activity at 37 degrees C, pH 9.5 and 1.5 mg casein concentration. Excretory/secretory (E/S) products showed maximum protease activity, which might be helpful in the degradation of mucosal tissue of the host gut. Male parasites appear to feed extensively on connective tissue collagen/elastin, while females feed on both connective tissue and body/cellular fluids. Inhibition/activation studies revealed the presence of four kinds of protease in the E/S products, cytosolic- and membrane-bound fractions performing different functions.

The effect of disulfiram on egg shell formation in adult Trichuris muris.

The effect of disulfiram on egg shell morphology in the parasitic nematode Trichuris muris was studied in vitro and in vivo. Daily disulfiram treatment of mice infected with T. muris beginning 25 days after infection and continuing for 26 days resulted in the production of malformed eggs by adult female worms in all treated groups. In addition, significantly fewer adult worms were found at necropsy in mice treated with 5.0 or 7.5 mg/kg/day of disulfiram as compared with mice treated with 2.5 mg/kg/day or control mice. Adult worms collected from infected, untreated mice and placed in aerobic culture for 5 days in media containing 4 or 8 micrograms/ml of disulfiram released malformed eggs into the culture medium after 30 hr in culture. Oral inoculation of naive mice with malformed eggs did not result in T. muris infections in the mice. The results of these studies suggest that inhibition of phenol oxidase results in disruption of normal egg production by T. muris females and that the enzyme might be a useful target in the development of control strategies aimed at nematode parasites that rely on phenol oxidase for egg shell hardening.

Trichuris suis: thiol protease activity from adult worms.

Trichuris suis, the whipworm of swine, causes anemia, weight loss, anorexia, mucohemorrhagic diarrhea, and death in heavy infections. A zinc metalloprotease has been suggested to play a role in the severe enteric pathology associated with infection and the infiltration of opportunistic bacteria into deeper tissues in the swine colon. In this study, a thiol protease from gut extracts of adult T. suis and from excretory/secretory components (E/S) of adult worms was characterized using fluorogenic peptide substrates and protein substrate gels. The protease cleaved the fluorogenic substrate Z-Phe-Arg-AMC, and this cleavage was completely inhibited by the thiol protease inhibitors E-64, leupeptin, Z-Phe-Ala-CH2F, and Z-Phe-Arg-CH2F. Gelatin substrate gels and fluorescence assays using both the gut and the stichosome extracts and E/S revealed enhanced activity when 2 mM dithiothreitol or 5 mM cysteine was included in the incubation buffer, and optimal activity was seen over a pH range of 5.5 to 8.5. Incubation of gut extracts or E/S material with inhibitors of aspartic, serine, or metalloproteases had no effect on the cleavage of Z-Phe-Arg-AMC. Thiol protease activity was found in extracts of gut tissue but not in the extracts of stichocytes of adult worms. N-terminal amino acid sequencing of the protease revealed sequence homologies with cathepsin B-like thiol protease identified from parasitic and free-living nematodes.

Characterization of Trichuris skrjabini by isoenzyme gel electrophoresis: comparative study with Trichuris ovis.

Morphological and biometric studies were performed in Trichuris skrjabini (Baskakov, 1924) collected from the caecum of Capra hircus. The LDH (EC 1.1.1.27.), G6PD (EC 1.1.1.49.), GPI (EC 5.3.1.9.), MDH (EC 1.1.1.37) and malic enzyme (ME) (EC 1.1.1.40) isoenzymatic patterns of T. skrjabini were determined by starch gel electrophoresis. The G6PD and GPI isoenzymatic patterns of T. skrjabini displayed two anodic bands for both enzymes: one fast migration band and one band near the origin. This isoenzymatic pattern was interpreted as two gene loci encoding both enzymes. The LDH isoenzymatic pattern of T. skrjabini was characterized by the presence of a cathodically migrating band, while the MDH isoenzymatic pattern showed a very slow cathodic band. These two phenotypes were interpreted as the expression of a homozygous state of a gene locus for LDH and MDH in T. skrjabini. The ME isoenzymatic pattern was characterized by the presence of a single anodic band. Further, comparative isoenzymatic studies were carried out between T. skrjabini and T. ovis. The different G6PD, GPI, LDH, MDH and ME isoenzymatic patterns observed for both species allowed us to distinguish them and therefore to use isoenzymatic patterns as a diagnostic tool to differentiate species of Trichuris.

Localization of phenol oxidase in female Trichuris suis.

A phenol oxidase (E.C. 1.10.3.1) preparation from adult female Trichuris suis was assayed by both polarographic and spectrophotometric techniques. The T. suis enzyme oxidized most diphenols including 4-methylcatechol (4MC) and dihydroxyphenylalanine but did not oxidize tyrosine. The pH and temperature optima were 6.8 and 36 C, respectively. The Km measured using 4MC as a substrate ranged from 0.12 to 0.4 mM. The highest phenol oxidase activity was isolated in fractions from the adult females that were enriched in eggs relative to the activity in somatic tissue from females and all male tissues that were assayed. Phenol oxidase activity was localized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis substrate gels into 2 bands with M(r)'s of 44,000 and 53,000. An antibody to the 44,000 band recognized 2 bands of 40,000 and 45,000 M(r) on western blot analysis of the enzyme preparation. Immunocytochemical localization of anti-phenol oxidase antibody in serial cross sections of adult female worms indicates that the enzyme is found exclusively in the anterior part of the parasite in the proximal part of the uterus that is posterior to the junction with the stichosome. Eggs located in more distal parts of the reproductive system did not react with the antibody. The results indicate that a phenol oxidase is located in the fertilized eggs of adult female T. suis. It is likely that phenol oxidase contributes significantly to the chemical hardening process in the eggs when they pass out into the external environment. Inhibition of phenol oxidase may reduce the survivability of the eggs and thus minimize contamination of livestock facilities.

Characterization of peptidases of adult Trichuris muris.

Excretory/secretory (E/S) material of Trichuris muris was found to contain 2 major peptidases, M(r) 85 and 105 kDa, which degrade gelatin optimally at pH 6.0 in sodium dodecyl sulphate-polyacrylamide gels. The peptidases were inactivated by diisopropylfluorophosphate, leupeptin and soybean trypsin inhibitor, but were unaffected by inhibitors of aspartic-, cysteine- and metallo-peptidases, indicating that they are serine peptidases. Both enzymes were detectable within 5 h after incubation of worms in culture medium and showed a time-dependent increase in levels. Neither peptidase was detected in worm extracts, suggesting that they are activated during or following secretion from worms. Live worms degraded a radio-isotope labelled extracellular matrix protein substratum derived from mammalian cells. Aminopeptidase activities capable of catalysing hydrolysis of amino acyl aminomethylcoumarin (MCA) substrates and a Z-Phe-Arg-MCA-hydrolysing cysteine peptidase activity, were detected in extracts of adult worms but not in E/S material.

Trichuris suis: a zinc metalloprotease from culture fluids of adult parasites.

A zinc metalloendoprotease has been isolated from in vitro culture fluids of Trichuris suis adults. The protease was purified from total culture fluids by passage through a cation exchange high-pressure liquid chromatography column. The 45-kDa protease has a pH optimum of 7.0 and an isoelectric point of 8.0 and was localized to the stichosome of the parasites using immunohistochemistry techniques.

The occurrence of phenol oxidase activity in female Trichuris suis.

The body of Trichuris suis females maintained in vitro under a gas phase of 95% air 5% CO2 develops a brown pigment that is apparent after 1 day and intensifies with time. Development of the brown pigment is prevented by maintaining the parasites in an anaerobic gas phase (95% N2, 5% CO2), but tanning commences when worms are returned to aerobic conditions. Tanning was not observed in males. Intact female T. suis take up oxygen at a considerably higher rate than males. Supernatant fractions (10,000 g) and pellets from whole worm homogenates of females both convert dihydroxyphenylalanine (DOPA) to dopachrome, suggesting the presence of a phenol oxidase. About 70% of the total phenol oxidase activity in females was in the pellet and about 30% in the supernatant fraction. Homogenates of male worms contained minimal phenol oxidase activity. Polarographic assay of phenol oxidase activity confirmed the presence of this enzyme in female T. suis. Female homogenates oxidized both dihydroxyphenylalanine and 4-methylcatechol and to a lesser extent hydroquinone. This oxidation was inhibited (> 90%) by diethyldithiocarbamate. Males did not oxidize any of the substrates tested. These results suggest that an enzyme of the phenol oxidase type is present in female worms but is probably inactive because of low oxygen tensions in the swine colon. The function of this enzyme in T. suis is unknown but is most likely associated with tanning of eggshell proteins or other aspects of eggshell synthesis.

In vitro effect of albendazole and fenbendazole on the histochemical localization of some enzymes of Trichuris globulosa (Nematoda: Trichuridae).

Trichuris globulosa (Nematoda: Trichuridae) incubated in 10 micrograms/ml and 50 micrograms/ml concentrations of albendazole and fenbendazole in Tyrode's solution were stained for histoenzymic demonstration of various phosphatases and oxidoreductases. The intestine, muscles and bacillary band showed major alterations after the drug treatment. The strong reaction of the various mitochondrial enzymes and ATPase suggests a possible respiratory role of the bacillary band in this species. The most noticeable effect of these two drugs especially the higher concentrations on the intestine was the disruption of its epithelium with the release and scattering of the enzymic activity of the various enzymes such as SDH, GDH (only fenbendazole treatment), NADPH-D and NADH-D. The functional significance of these enzymes has been fully discussed.

Superoxide dismutase from Trichuris ovis--inhibition by benzimidazoles and pyrimidine derivatives.

Three superoxide dismutase isoenzymes of different cellular location were detected in an homogenate of Trichuris ovis. Each of these molecular forms was purified by differential centrifugation and precipitation with ammonium sulphate, followed by chromatography on DEAE-cellulose and Sephadex G-75 columns. The activity levels of the two molecular forms detected in the mitochondrial (one cyanide sensitive Cu-Zn-SOD and the other cyanide insensitive Mn-SOD) were higher than that of the superoxide dismutase detected in the cytoplasmic fraction (cyanide sensitive Cu-Zn-SOD). All molecular forms present evident differences to the SODs contained in the host liver. Molecular mass and some of the physical and chemical properties of the enzyme was determined for all three molecular forms. An inhibitory effect on the SOD of the parasite an the host was detected with a series of compounds, some of which markedly inhibited parasite enzyme but not host enzyme.