Characterization of biochemical properties of a selenium-independent glutathione peroxidase of Cryptosporidium parvum.

Glutathione peroxidase (GPx; EC 1.11.1.9) is an important antioxidant enzyme that catalyses the reduction of organic and inorganic hydroperoxides to water in oxygen-consuming organisms, using glutathione as an electron donor. Here, we report the characterization of a GPx of Cryptosporidium parvum (CpGPx). CpGPx contained a standard UGU codon for cysteine instead of a UGA opal codon for seleno-cysteine (SeCys) at the active site, and no SeCys insertion sequence (SECIS) motif was identified within the 3'-untranslated region (UTR) of CpGPx, which suggested its selenium-independent nature. In silico and biochemical analyses indicated that CpGPx is a cytosolic protein with a monomeric structure. Recombinant CpGPx was active over a wide pH range and was stable under physiological conditions. It showed a substrate preference against organic hydroperoxides, such as cumene hydroperoxide and t-butyl hydroperoxide, but it also showed activity against inorganic hydroperoxide, hydrogen peroxide. Recombinant CpGPx was not inhibited by potassium cyanide or by sodium azide. The enzyme effectively protected DNA and protein from oxidative damage induced by hydrogen peroxide, and was functionally expressed in various developmental stages of C. parvum. These results collectively suggest the essential role of CpGPx for the parasite's antioxidant defence system.

Cryptostatin, a chagasin-family cysteine protease inhibitor of Cryptosporidium parvum.

Cysteine proteases of pathogenic protozoan parasites play pivotal roles in the life cycle of parasites, but strict regulation of their activities is also essential for maintenance of parasite physiology and interaction with hosts. In this study, we identified and characterized cryptostatin, a novel inhibitor of cysteine protease (ICP) of Cryptosporidium parvum. Cryptostatin showed low sequence identity to other chagasin-family ICPs, but 3 motifs (NPTTG, GXGG, and RPW/F motifs), which are evolutionarily conserved in chagasin-family ICPs, were found in the sequence. The overall structure of cryptostatin consisted of 8 ß-strands that progressed in parallel and closely resembled the immunoglobulin fold. Recombinant cryptostatin inhibited various cysteine proteases, including papain, human cathepsin B, human cathepsin L, and cryptopain-1, with K i's in the picomolar range. Cryptostatin was active over a wide pH range and was highly stable under physiological conditions. The protein was thermostable and retained its inhibitory activity even after incubation at 95°C. Cryptostatin formed tight complexes with cysteine proteases, so the complexes remained intact in the presence of sodium dodecyl sulfate and ß-mercaptoethanol, but they were disassembled by boiling. An immunogold electron microscopy analysis demonstrated diffused localization of cryptostatin within oocystes and meronts, but not within trophozoites, which suggests a possible role for cryptostatin in host cell invasion by C. parvum.

Molecular cloning and characterization of a M17 leucine aminopeptidase of Cryptosporidium parvum.

Leucine aminopeptidases (LAPs) are a group of metalloexopeptidases that catalyse the sequential removal of amino acids from the N-termini of polypeptides or proteins. They play an important role in regulating the balance between catabolism and anabolism in living cells. LAPs of apicomplexa parasitic protozoa have been intensively investigated due to their crucial roles in parasite biology as well as their potentials as drug targets. In this study, we identified an M17 leucine aminopeptidase of Cryptosporidium parvum (CpLAP) and characterized the biochemical properties of the recombinant protein. Multiple sequence alignment of the deduced amino acid sequence of CpLAP with those of other organisms revealed that typical amino acid residues essential for metal binding and active-site formation in M17 LAPs were well conserved in CpLAP. Recombinant CpLAP shared similar biochemical properties such as optimal pH, stability at neutral pHs, and metal-binding characteristics with other characterized LAPs. The enzyme showed a marked preference for Leu and its activity was effectively inhibited by bestatin. These results collectively suggest that CpLAP is a typical member of the M17 LAP family and may play an important role in free amino acid regulation in the parasite.

Identification and biochemical characterization of two novel peroxiredoxins in a liver fluke, Clonorchis sinensis.

We identified 2 novel genes encoding different 2-Cys peroxiredoxins (PRxs), designated CsPRx2 and CsPRx3, in Clonorchis sinensis, which invades the human hepatobiliary tracts. The CsPRx2 gene expression was temporally increased along with the parasite's development and its protein product was detected in almost all parts of adult worms including subtegument, as well as excretory-secretory products. Conversely, CsPRx3 expression was temporally maintained at a basal level and largely restricted within interior parts of various tissues/organs. The recombinant forms of CsPRx proteins exhibited reducing activity against various hydroperoxides in the presence of either thioredoxin or glutathione (GSH) as a reducing equivalent, although they preferred H2O2 and GSH as a catalytic substrate and electron donor, respectively. A steady-state kinetic study demonstrated that the CsPRx proteins followed a saturable, Michaelis-Menten-type equation with the catalytic efficiencies (kcat/Km) ranging from 103 to 104 M-1 s-1, somewhat lower than those for other PRxs studied (104-105 M-1 s-1). The expression patterns and histological distributions specific to CsPRx2 and CsPRx3 might suggest different physiological functions of the antioxidant enzymes in protecting the worms against oxidative damage.

Cryptopain-1, a cysteine protease of Cryptosporidium parvum, does not require the pro-domain for folding.

SUMMARY: Cryptosporidium parvum is an intracellular protozoan parasite that causes cryptosporidiosis in mammals including humans. In the current study, the gene encoding the cysteine protease of C. parvum (cryptopain-1) was identified and the biochemical properties of the recombinant enzyme were characterized. Cryptopain-1 shared common structural properties with cathepsin L-like papain family enzymes, but lacked a typical signal peptide sequence and contained a possible transmembrane domain near the amino terminus and a unique insert in the front of the mature domain. The recombinant cryptopain-1 expressed in Escherichia coli and refolded to the active form showed typical biochemical properties of cathepsin L-like enzymes. The folding determinant of cryptopain-1 was characterized through multiple constructs with or without different lengths of the pro-domain of the enzyme expressed in E. coli and assessment of their refolding abilities. All constructs, except one that did not contain the full-length mature domain, successfully refolded into the active enzymes, suggesting that cryptopain-1 did not require the pro-domain for folding. Western blot analysis showed that cryptopain-1 was expressed in the sporozoites and the enzyme preferentially degraded proteins, including collagen and fibronectin, but not globular proteins. This suggested a probable role for cryptopain-1 in host cell invasion and/or egression by the parasite.

Clonorchis sinensis omega-class glutathione transferases are reliable biomarkers for serodiagnosis of clonorchiasis and opisthorchiasis.

OBJECTIVES: To determine the potential for immunodiagnostic application of two recombinant forms of Clonorchis sinensis omega-class glutathione transferases (rCsGSTo1 and rCsGSTo2) against human small liver-fluke C. sinensis and Opisthorchis viverrini infections. METHODS: Specific antibody levels against rCsGSTo1 and rCsGSTo2 in patients' sera of egg-positive opisthorchiasis (n = 87) and clonorchiasis (n = 120), as well as those in sera from patients with other helminthic infections (n = 252) and healthy controls (n = 40) were retrospectively analysed by ELISA. RESULTS: We observed highly positive correlation coefficients between specific antibody levels against rCsGSTo1 and rCsGSTo2 and egg counts per gramme of faeces (EPG) of patients with opisthorchiasis (n = 87; r = 0.88 for rCsGSTo1 and r = 0.90 for rCsGSTo2). Sera from opisthorchiasis patients whose EPG counts >100 (n = 43) revealed high antibody titres against both antigens. Patients' sera with low EPG counts (<100, n = 44) also exhibited reliable sensitivities of 93.2% and 97.7% for rCsGSTo1 and rCsGSTo2, respectively. Sera from clonorchiasis patients showed sensitivities of 90% (108/120 samples) and 89.2% (107/120 sera) for rCsGSTo1 and rCsGSTo2. Overall diagnostic sensitivities for liver-fluke infections were 92.3% for rCsGSTo1 (191/207 samples) and 93.2% for rCsGSTo2 (193/207 samples). Specificities were 89.7% (rCsGSTo1) and 97.6% (rCsGSTo2). CONCLUSIONS: Detection of specific antibody levels against rCsGSTo1 or rCsGSTo2 might be promising for the serodiagnosis of patients infected with these two phylogenetically close carcinogenic liver-flukes.

Molecular cloning and immunological characterization of phosphoglycerate kinase from Clonorchis sinensis.

The parasite Clonorchis sinensis was determined to utilize a large amount of external glucose to carry its energy metabolism. Phosphoglycerate kinase (PGK), a glycolytic enzyme, found in many parasites, has been identified as one of the candidate molecules distinguished from human counterparts for vaccine and drug developments. A cDNA clone purified by screening a C. sinensis cDNA library using a heterologous cDNA probe encoded a putative peptide of 415 amino acids with over 60% identities with PGKs from a number of animals. The putative peptides revealed domains corresponding to 12 beta-sheets and inner loops forming a substrate-binding cleft of animal PGKs. The gene product was overexpressed in Escherichia coli and showed a PGK-like enzyme activity. A polyclonal antibody raised against the recombinant C. sinensis PGK was specific to native C. sinensis PGK and localized it to the muscular tissue and tegument of the adult flukes. The C. sinensis PGK elicited antibodies in C. sinensis-infected rabbits. Therefore, it is proposed that C. sinensis PGK could be used as an immunoreagent in the serodiagnosis for clonorchiasis.

Molecular cloning and characterization of a mu-class glutathione S-transferase from Clonorchis sinensis.

In biliary passages, Clonorchis sinensis causes epithelial hyperplasia and is assumed to promote carcinogenesis. Glutathione S-transferase (GST) is an antioxidant enzyme involved in phase II defense in trematodes. A clone (pcsGSTM1) encoding a GST was identified by screening a C. sinensis cDNA library with a PCR-synthesized cDNA probe. The predicted amino acid sequence encoded by pcsGSTM1 cDNA had a high degree of sequence identity and folding topology similar to the mu-class GSTs. The estimated molecular mass of the protein, 26 kDa, was consistent with an expression by pcsGSTM1 cDNA. The bacterially expressed recombinant csGSTM1 protein possessed an enzymatic GST activity and conjugated GSH to reactive carbonyls of lipid peroxidation. The recombinant csGSTM1 protein did not share antigenic epitope(s) with GSTs of Fasciola hepatica, Paragonimus westermani and Schistosoma japonicum. The csGSTM1 was identified to a mu-class GST in C. sinensis.

High prevalence of Gymnophalloides seoi infection in a village on a southwestern island of the Republic of Korea.

Gymnophalloides seoi (Digenea: Gymnophallidae), a new human intestinal trematode reported from a Korean woman who complained of epigastric discomfort, has been shown to be highly prevalent among the villagers of a southwestern island of the Republic of Korea. For the detection of human infections, fecal examinations were conducted on the inhabitants of a seashore village, where the first patient with a G. seoi infection had resided. Of 98 inhabitants examined, 70 (71.4%) were infected with various intestinal parasites; among them, G. seoi showed the highest rate of egg positivity 48 of 98 (49.0%). Individual worm burdens of G. seoi, as measured by collection of adult flukes after anthelmintic treatment and purgation, ranged from 106 to 26,373 specimens (average per infected case = 3,326). There was no sex-related difference in the prevalence of G. seoi, and the age distribution of the infected cases showed a relatively even pattern. This study confirms that human infection by G. seoi is not an incidental one and provides the first evidence of its high prevalence on this Korean island.

A DNA vaccine encoding a fatty acid-binding protein of Clonorchis sinensis induces protective immune response in Sprague-Dawley rats.

Clonorchis sinensis, the Chinese liver fluke, resides chronically in the biliary tract, and fatty acid-binding protein (FABP) is known to play an important role in the intracellular transport of long-chain fatty acids obtained from the host. Although FABP has stimulated considerable interest as a vaccine candidate, the nature of C. sinensis FABP (CsFABP) remains unclear. We investigated the immunogenicity and protective efficacy of a DNA vaccine encoding CsFABP. The intradermal injection of plasmid DNA carrying the CsFABP gene (pcDNA3.1-FABP) into Sprague-Dawley (SD) rats induced both humoural and cellular immune responses. Animals injected with pcDNA3.1-FABP developed FABP-specific antibody, which is dominance of IgG2a in sera. In addition, the DNA vaccine elicited the production of IFN-gamma, but not the production of IL-4 in spleen cells stimulated with recombinant FABP. Moreover, pcDNA3.1-FABP induced a significant level of protection, decreased worm burden (40.9%, P<0.05) in SD rats against C. sinensis metacerariae challenge. These results suggest that pcDNA3.1-FABP induces a typical T helper-1-dominated immune response and it is a good candidate for use in future clonorchiasis vaccination studies.

Molecular determination of the origin of acephalic cysticercus.

Acephalic cysticercus (Ac), a rarely developed multilobulated and nonencysted form of larval Taenia, causes hydrocephalus or adhesive arachnoiditis in the ventricles and subarachnoidal space that often lead to fatal outcome in affected patients. Ac has been proposed to originate from T. solium on the basis of morphological features, while no molecular data supporting the presumption have been available. In the present study, we investigated the immunological properties as well as molecular characteristics of Ac that was obtained surgically from 6 patients. Immunoblotting of the cyst fluid from Ac samples demonstrated the constitutive expression of a T. solium metacestode (TsM) 10 kDa protein. Specific antibodies against the truncated 10 kDa protein, which appears to be species specific for TsM cysticercosis, were detected in both serum and cerebrospinal fluid samples of Ac patients. Nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) genes of Ac were almost identical to those of T. solium but differed substantially from those of the other Taenia species. In phylogenetic analysis, Ac clustered with T. solium in a well-supported clade. Our results strongly suggest that Ac may have originated from T. solium.

Mixed infections with Opisthorchis viverrini and intestinal flukes in residents of Vientiane Municipality and Saravane Province in Laos.

Faecal examinations for helminth eggs were performed on 1869 people from two riverside localities, Vientiane Municipality and Saravane Province, along the Mekong River, Laos. To obtain adult flukes, 42 people positive for small trematode eggs (Opisthorchis viverrini, heterophyid, or lecithodendriid eggs) were treated with a 20-30 mg kg(-1) single dose of praziquantel and purged. Diarrhoeic stools were then collected from 36 people (18 in each area) and searched for helminth parasites using stereomicroscopes. Faecal examinations revealed positive rates for small trematode eggs of 53.3% and 70.8% (average 65.2%) in Vientiane and Saravane Province, respectively. Infections with O. viverrini and six species of intestinal flukes were found, namely, Haplorchis taichui, H. pumilio, H. yokogawai, Centrocestus caninus, Prosthodendrium molenkampi, and Phaneropsolus bonnei. The total number of flukes collected and the proportion of fluke species recovered were markedly different in the two localities; in Vientiane, 1041 O. viverrini (57.8 per person) and 615 others (34.2 per person), whereas in Saravane, 395 O. viverrini (21.9 per person) and 155207 others (8622.6 per person). Five people from Saravane harboured no O. viverrini but numerous heterophyid and/or lecithodendriid flukes. The results indicate that O. viverrini and several species of heterophyid and lecithodendriid flukes are endemic in these two riverside localities, and suggest that the intensity of infection and the relative proportion of fluke species vary by locality along the Mekong River basin.

[Infection status of Pseudorasbora parva collected from the Sunam stream with metacercariae of Metorchis orientalis].

This study revealed the infection status of P. parva with the metacercariae of M. orientalis in the Sunam stream of the Nakdong River, a well-known focus of Clonorchis sinensis. A total of 60 P. parva were divided into 4 groups by the size (80-90, 70-79, 60-69 and 50-59 mm) and were digested by pepsin-HCl solution in a 36 degrees C incubator. The metacercariae of M. orientalis were collected and counted under a stereomicroscope. A total of 3,885 metacercariae were found, and average number/fish in each group were 143, 54, 23 and 40. They were 175.3 x 155.4 microns in average size and had thick cyst wall(13.1 microns in average). By the results, it is proved that the life cycle of M. orientalis is actively maintained in the Sunam stream of the Nakdong River.

Life history of Echinoparyphium recurvatum (Trematoda: Echinostomatidae) in Korea.

The present study was performed to observe characteristics of the life history of Echinoparyphium recurvatum under both natural and laboratory conditions in Korea. A batch of Radix auricularia coreana was collected from Sunamchon, one of the stream of West Naktonggang (River), in Kangso-gu, Pusan during August and September 1992. Out of 106 snails examined by crushing, 52 (49.0%) were infected with larval E. recurvatum, i.e. rediae, cercariae and metacercariae. Cercariae naturally shed from snails encysted in the snails of same species and loaches, but not in mud-snails. Adult worms were detected from chicks and ducks experimentally infected with metacercariae, but not from rats and mice. The average recovery rate of adults from chicks was 13.1%. Rediae were sac-like, 2.437 x 0.317 mm in average size, with a muscular pharynx and a brownish cecum which reached the anterior half of the body. Cercariae consisted of a spindle-shaped body (0.262 x 0.129 mm in average) and a rod-like tail (0.528 x 0.056 mm in average). In the cercarial body, 45 collar spines were observed on the head crown, and double rows of excretory ducts with fine granules were laterally arranged between the pharynx and the ventral sucker. Metacercariae were spherical, 0.144 x 0.142 mm in average size, with thick hyaline outer and thin elastic inner walls, and many excretory granules. Adults were slender and more attenuated in the anterior end, 2.760 x 0.550 mm in average size, and had 45 collar spines including four end group spines on both ventral corners. From the above results, it was confirmed that R. auricularia coreana plays a pivotal role in the life cycle of E. recurvatum as the first and/or second intermediate hosts in Korea.

[Transmission electron microscopic ultrastructure of the tegument of Fibricola seoulensis].

An electron microscopic study was performed to observe the ultrastructure of the tegument of F. seoulensis. The outer surface of the tegument was covered with a trilaminated plasma membrane. The electron-dense cytoplasmic layer was 2.5 microns wide in the anterior portion and contained numerous vacuoles, mitochondria and granular materials in its matrix. The basement layer was 330 nm wide or so, and its numerous extensions protruded into the cytoplasmic layer. The sensory organ was composed of a small vesicle of 1.7 x 1.1 microns in dimensions, which possessed a cilium of 1.2 x 0.19 micron in size. The pharynx was composed of the epithelial layer of about 0.5 micron wide, well developed muscle layer and basement layer. The tegument of the oral sucker was composed of a cytoplasmic layer of 0.4-0.5 micron width, a narrow basement layer, a well developed muscle layer and tegumental cells. Some kinds of secretory granules that seemed to be originated from the cells of the oral sucker were observed in the parenchymal portions of the adjacent cells. The tribocytic organ consisted of numerous microvilli. The microvilli were 5 nm wide and heptalaminated. Two types of secretory granules originated from the gland cells of tribocytic organ were observed in the tegument and parenchyme. The tegumental cells were irregular in shape, and of which nuclei were multifarious.

Infection status with trematode metacercariae in pond smelts, Hypomesus olidus.

Many Koreans usually eat raw pond smelts, Hypomesus olidus, in the winter. This study was performed to evaluate the infection status with trematode metacercariae in pond smelts from January 1998 through February 1999. Among 1,305 fish collected, 459 were purchased from wholesale dealers in Chinchon-gun, Chungchongbuk-do, and the rest of them were caught with a casting net in Soyangho (Lake), Taehoman (Bay) and Paekkokchosuchi (Pond). Seven species of trematode metacercariae including two unidentified ones were detected from 1,305 pond smelts. The number of detected trematode metacercariae according to the species are as follow: Clonorchis sinensis 8, Holostephanus nipponicus 7, Cyathocotyle orientalis 24, Diplostomum sp. 14, and Metorchis orientalis 7. From the above results, it was confirmed that H. olidus plays a role as the second intermediate host of some kinds of trematode including C. sinensis in Korea. Our report shows possible clonorchiasis caused by eating raw pond smelts.

A human case of gastric anisakiasis by Pseudoterranova decipiens larva.

A case of gastric anisakiasis due to the larva of Pseudoterranova decipiens was confirmed by gastroendoscopic examination in April 23, 1991. The patient, residing in Pusan, was a 42-year-old housewife, who complained of severe epigastric pain and recalled that the symptom suddenly attacked her about 6 hours after eating raw Sebastes inermis. In the gastroendoscopic examination performed about 9 hours after the onset of the symptom, a long whitish nematode larva penetrating the gastric mucosa in the greater curvature of mid-body was found and removed with a biopsy forcep. The nematode was 29. 73 x 0.94 mm in size, had an intestinal cecum reaching over mid-level of the ventriculus and was identified as the 4th stage larva of P. decipiens.

Identification of larval Gnathostoma obtained from imported Chinese loaches.

We observed the morphological characteristics and identified the species of gnathostome larvae obtained from the imported Chinese loaches. The early third-stage larvae (EL3) were collected from viscera of the loaches and a part of them were infected to mice. The advanced third-stage larvae [AdL3] were recovered from the mice at 4 weeks post-infection, both larval worms were fixed with 10% formalin, cleared in alcohol-glycerin solution, mounted with glycerin-jelly, and observed. A total of 369 EL3 were collected from viscera of 9,493 Chinese loaches. The whole body of EL3 was covered with about 190 transverse rows of minute cuticular spines and 0.624 x 0.101 mm in average size. A pair of lips were protruded at the anterior end, and the muscular esophagus and brownish intestine were followed. The characteristic head bulb was provided with 4 rows of hooklets. The average number of hooklets in the respective row was 36.7, 39.5, 41.6 and 44.3 posteriorly. AdL3 was 2.660 x 0.346 mm in average size, and retained the esophagus (about 0.755 mm length) and cervical sac (about 0.355 mm length). The average number of hooklets in the respective row on the head bulb was 39.0, 41.9, 43.9 and 45.6, posteriorly. On the basis of the morphological characteristics, they were identified as the third-stage larvae of Gnathostoma hispidum.

[Infection status with trematode metacercariae in the fresh-water fish from Chunamchosuchi (pond), Uichang-gun, Kyongsangnam-do, Korea].

The present study was performed to analyze the infection status of trematode metacercariae in fishes caught from Chunamchosuchi (pond) located in Uichang-gun, Kyongsangnam-do. A total of 130 freshwater fish of 5 species was collected by a fish net and fish traps from November, 1995 to May, 1996. They were examined under a stereomicroscope after artificial digestion with pepsin-HCl solution. A total of 8 species of metacercaria, i.e. Clonorchis sinensis, Echinochasmus japonicus, Cyathocotyle orientalis, Diplostomum sp., Metorchis orientalis, Holostephanus nipponicus, Exorchis oviformis and unidentified echinostome, was detected from them. The metacercariae of C. sinenesis were found in 8/20 (40.0%) Acanthorhodeus asmussi, 20/20 (100%) Culter brevicauda, 31/45 (68.9) Cultriculus eigenmanni and 21/25 (84.0%) Pseudorasbora parva, and the average number of metacercariae detected in each fish species were 1.9, 31.7, 15.3, and 73.0. From the above results, it was confirmed that fresh-water fishes from Chunamchosuchi (pond) were highly infected with metacercariae of avian trematode, i.e. C. orientalis, H. nipponicus, M. orientalis, E. japonicus and Diplostomum sp., and 4 species of fish, P. parva, C. brevicauda, C. eigenmanni and A. asmussi, were infected with metacaecariae of C. sinensis.

Susceptibility of some vertebrate hosts to infection with early third-stage larvae of Gnathostoma hispidum.

Susceptibility of some vertebrates was examined to the early third-stage larvae (EL3) of Gnathostoma hispidum. The larvae collected from the Chinese loaches were infected to 4 silk carps, 3 snake heads, 3 bullfrogs, 5 mice and 9 albino rats. No worms were detected in fish, silk carps and snake heads. In 3 bullfrogs fed 30 larvae, a total of 9 EL3 was recovered in the gastrointestinal tract (8 larvae) and liver (one). In 5 mice infected with 50 larvae, a total of 37 (74.0%) advanced third-stage larvae (AdL3) was recovered from the muscle (31 larvae), liver (5 larvae) and kidney at 4 weeks after infection. In 9 albino rats infected with 115 larvae, a total of 40 (34.8%) AdL3 was found in the muscle. The mammalian hosts were found susceptible to the EL3 of G. hispidum from Chinese loaches.