Comparison of gene representation between diploid and triploid Paragonimus westermani by expressed sequence tag analyses

Expressed sequence tag (EST) analysis of the diploid and triploid Paragonimus westermani genes was done to have a rapid and informative outlook of the gene-expression profiles of the parasites. Totals of 506 and 505 ESTs were generated from the diploid and triploid P. westermani cDNA libraries. Based on the BLASTx search results of the diploid P. westermani ESTs, 308 (60.9%) matched significantly with formerly identified genes and 198 (39.1%) showed no significant homology in the GenBank database. A similar homology pattern was shown from the triploid EST BLASTx search results with 346 (68.5%) sharing homology with previously identified genes and 159 (31.5%) showing no significant homology. The EST data from both libraries were analyzed and grouped into 9 categories. Comparison of the 2 EST pools revealed high similarities among the categories of the significantly matched genes. Single genes matched repeatedly were also observed in the 2 EST data. Some genes were found that are not yet characterized in P. westermani; these genes were matched by both the diploid and triploid ESTs. Further study of these genes may provide us with more understanding on the parasite's biology and their specific functions in the 2 strains.     

The origin of the triploid in Paragonimus westermani on the basis of variable regions in the mitochondrial DNA

Triploid, parthenogenetic forms of the lungfluke, Paragonimus westermani, occur in Japan, Korea and China. The origin(s) of triploidy has been debated over the years. Sequences of two regions in the mitochondrial DNA, i.e. partial lrRNA (16S), and a portion of the non-coding region, were obtained from natural populations of P. westermani. All triploid individuals (Japan, Korea, China) and a single tetraploid individual (China) had identical sequences in the 16S region studied. Some sequence variation was observed among diploids, with those from Taiwan being distinct from the remainder. Both neighbour joining and parsimony trees using the 16S region placed diploid individuals from southwestern Japan close to the triploids and the tetraploid. The fragment amplified from the mitochondrial non-coding region showed dimorphism. One form (type A) consisted of 239 bp comprising two identical tracts of 70 bp separated by a tract of 93 bp. The second form (Type B) consisted of only a single 70 bp tract. All diploid individuals from Taiwan, China and Korea possessed type A, while those from Japan were polymorphic; individuals from Oita and Hyogo had type B, those from Chiba had type A, but both types were found in Mie. On the other hand, all of the triploid individuals and two tetraploid individuals possessed type B. Both the form present in the non-coding region and the 16S sequence suggest an affinity between a south-eastern group of diploid populations in Japan and the triploid form. A possible mechanism responsible for the origin of the triploid is discussed.     

Molecular phylogeographic studies on Paragonimus westermani in Asia

The lung fluke, Paragonimus westermani (Kerbert, 1878), is widely distributed in Asia, and exhibits much variation in its biological properties. Previous phylogenetic studies using DNA sequences have demonstrated that samples from north-east Asia form a tight group distinct from samples from south Asia (Philippines, Thailand, Malaysia). Among countries from the latter region, considerable molecular diversity was observed. This was investigated further using additional DNA sequences (partial mitochondrial cytochrome c oxidase subunit 1 (COI) and the second internal transcribed spacer of the nuclear ribosomal gene repeat (ITS2)) from additional samples of P. westermani. Phylogenies inferred from these again found three or four groups within P. westermani, depending on the method of analysis. Populations of P. westermani from north-east Asia use snail hosts of the family Pleuroceridae and differ in other biological properties from populations in south Asia (that use snail hosts of the family Thiaridae). It is considered that the populations we sampled can be divided into two species, one in north-east Asia and the other in south Asia.     

Molecular characterization of two myoglobins of Paragonimus westermani

Myoglobins (Mbs), globin proteins, are present in high concentrations in trematodes. In Paragonimus westermani, 2 cDNAs were found to encode Mbs. The first clone, Pwmyo1, codes a total of 149 amino acids with a calculated mass of 16.6 kDa. The second, Pwmyo2, encodes a 146-amino acid protein with a calculated mass of 16.2 kDa. The predicted secondary structures showed the presence of 8 helices, which is the basic characteristic of Mbs. Sequence alignment revealed a high homology with the other trematode Mbs. The 2 clones contained the characteristic tyrosyl residues at helical positions B10 and distal E7, which are substitutions that have been previously shown to contribute to the high oxygen affinity of Mbs. Polyclonal antibodies against the recombinant Mbs were raised with no cross-reactivity observed. Immunolocalization revealed the proteins to be distributed generally throughout the parenchymal tissues, but absent from the tegument and reproductive organs. The cell mass of the eggs of the worm stained positive to Pwmyo2 but not Pwmyo1, suggesting the stage-specific expression of these Mbs.     

Serum IgE levels in rats infected with Paragonimus westermani]

Paragonimus westermani is a common fluke in Korea. The present study aimed to determine serum total IgE and specific IgG levels in experimental paragonimiasis of rats. Each Wistar rat was inoculated orally with 20-25 metacercariae of P. westermani from Cambaroides similis. Before and after infection (1, 2, 3, 4, 6, 8 weeks) of P. westermani, the blood was collected from the retro-orbital venous plexus of rats and kept serum at -70 degrees C. Serum total IgE and specific IgG levels were determined by the capture and conventional enzyme-linked immunosorbent assay, respectively. The results were as follows; 1. Serum IgE values were increased to 0.18 +/- 0.042 at 2 weeks, 0.28 +/- 0.151 at 4 weeks and 0.43 +/- 0.055 at 8 weeks after infection. The absorbances of non-infected rats ranged 0.07 +/- 0.021-0.12 +/- 0.025. 2. Specific IgG values were slightly increased at 3 weeks (0.20 +/- 0.032) and gradually increased up to 8 weeks (0.31 +/- 0.067) after infection. The absorbances of non-infected rats ranged 0.11 +/- 0.035-0.18 +/- 0.019. The present results suggested that P. westermani could elevate serum IgE and specific IgG antibodies in Wistar rats which were not a good definitive host.     

Paragonimus westermani: biochemical and immunological characterizations of paramyosin

Paramyosin of the helminth parasite is a muscle protein that plays multifunctional roles in host-parasite relationships. In this study, we have cloned a gene encoding Paragonimus westermani paramyosin (PwPmy) and characterized biochemical and immunological properties of the recombinant protein. The recombinant PwPmy (rPwPmy) was shown to bind both human immunoglobulin G (IgG) and collagen. The protein was constitutively expressed in various developmental stages of the parasite and its expression level increased progressively as the parasite matured. Immunohistological analysis revealed that PwPmy was mainly localized in subtegumental muscle, tegument and cells surrounding the oral sucker, intestine, and ovary of the parasite. Sera from patients with paragonimiasis showed antibody reactivity against rPwPmy, and IgG1 and IgG4 were predominant. Immunization of mice with rPwPmy also induced high IgG responses. Biochemical and immunological characterization of PwPmy may provide valuable information for the further study to develop a vaccine or a chemotherapeutic agent for paragonimiasis.     

Antibody-dependent rat macrophage-mediated damage to the excysted metacercariae of Paragonimus westermani in vitro

An in vitro immune effector mechanism against the target excysted metacercariae of Paragonimus westermani was demonstrated in the rat system. Peritoneal exudate cells, mainly macrophages from normal rats, showed adherence to and killing of excysted metacercariae of P. westermani in the presence of complement-independent serum from rats infected with Paragonimus metacercariae. These reactions were specific for the excysted metacercariae, as tissue-migrating juvenile worms were not affected. Damage of excysted metacercariae of P. westermani due to antibody and macrophages was assessed by morphological observation, by cell adherence reaction and by the use of vital dyes. Trypan blue dye exclusion proved to be a reliable indicator of judging metacercarial viability. Electron microscopic studies demonstrated that macrophages reacted with fuzzy material on the tegumental surface and fine structures in the syncytium of the parasites. The tubular tunnels formed between the basement membrane and muscle layers of the damaged parasites were also noticeable. The relevance of these findings to cellular immunity in the early paragonimiasis was discussed.     

Early cysteine protease activity in excretory bladder triggers metacercaria excystment of Paragonimus westermani

The cysteine proteases of Paragonimus westermani metacercaria are known to initiate metacercaria excystment. However, their secretory sites, such as the intestine, tegument, and excretory bladder are not well defined. In this study, the metacercariae were labeled with bromodeoxyuridine (BrdU) to distinguish the initial activation sites. BrdU was labeled mainly at the excretory bladder and the excretory granules of the metacercariae and the newly excysted juvenile worms. This result shows that early 'defecation' of the proteases from the excretory bladder may accelerate the excystment of P. westermani metacercariae.     

Differential expression of Paragonimus westermani eggshell proteins during the developmental stages

Eggs of trematode parasites are comprised of numerous vitelline cells and one fertilized ovum, and are encapsulated within a protein shell provided by the vitellocytes. In this study, we isolated two full-length cDNA clones that showed substantial levels of sequence identity with trematode-specific eggshell precursor proteins from the human lung fluke, Paragonimus westermani. These cDNAs, designated Pw-Vit20 (868-bp-long) and Pw-Vit36 (883-bp-long), shared a 76% identity with one another at the nucleotide level, and each encoded a 261-amino acid (aa) polypeptide. The deduced aa sequences contained a N-terminal hydrophobic segment, as well as a sequence motif of Gly-Gly-Gly-Tyr-Asp-Asn/Thr-Tyr-Gly-Lys/Gln, which is highly homologous with the eggshell proteins of Fasciola hepatica. With the high frequencies of tyrosine, glycine and lysine, the positions occupied by tyrosine, which has been proved to be converted into dihydroxyphenylalanine, were well preserved. Pw-Vit20 and Pw-Vit36 were found to be monoexonic genes with variably diverged variants scattered into multiple genomic loci. Their protein products were localized in the vitelline follicles and eggshells. Expression of Pw-Vit20 was restricted to the egg and adult stages, thus suggesting a critical involvement of Pw-Vit20 in the parasite's fecundity activity. Conversely, Pw-Vit36 was constitutively expressed in the metacercariae and juvenile stages in the vitelline follicles and ducts, which suggested that the prepositioning of stem or primordial vitelline cells within the juveniles prior to sexual maturation. Pw-Vit36 might acquire a unique or additional function relevant to the maturation and/or development of the vitelline cells/follicles during the evolutionary period of P. westermani. Differential biological implications of multiple eggshell precursor proteins may provide insight into the molecular mechanism of eggshell formation and the developmental process of the vitelline follicles in the parasitic trematode.     

Metacercarial infections of Paragonimus westermani in freshwater crabs sold in markets in Seoul

Status of metacercarial infections of Paragonimus westermani was observed in freshwater crabs, which were purchased at 3 markets in its peak season of 1990. All of 85 crabs were Eriocheir japonicus. No other species of Eriocheir were found. When crushed muscle and viscera was examined individually, the infection rate was 11.8%; and mean number of metacercariae was 2.1 per infected crab. Unless adequately cooked, freshwater crabs are still potential sources of human paragonimiasis.     

Immunohistochemical study on the antigenicity of body compartments of Paragonimus westermani

Production of circulating specific antibodies to the lung fluke (Paragonimus westermani) by its host is well known and used in various kinds of immunodiagnostic methods. However, it has not been well documented which compartments (or structures) of the lung fluke are most responsible for the production of specific antibodies. The present immunohistochemical study was undertaken to demonstrate the antigenicity of each body compartment of P. westermani such as suckers, tegument, spines, vitelline glands, intestine, reproductive organs (male and female), and eggs. Indirect immunoperoxidase (IP) stain technique was applied, using formalin-fixed, paraffin-embedded lung tissues of P. westermani-infected cats sectioned in 4 microns thickness as the antigen and cat antisera (11-20 weeks of infection) as the primary antibody. Peroxidase-conjugated goat anti-cat IgG was used as the secondary antibody and diaminobenzidine (DAB) as the coloring agent. Strong yellow or yellowish brown staining was regarded positive. The primary and secondary antibody dilutions were made at 1:500-1:2,000 and 1:200-1:500 respectively, and IP stain was repeated 10 times for each dilution. A consistent result obtained was that the intestinal epithelial border, intestinal content, vitelline glands, and eggs scattered around the worm capsule showed strong positive staining, while uterine eggs and some parenchymal portions showed weak positive reaction. On the other hand, the suckers, tegument, spines, subtegumental cells, cytoplasm of intestinal epithelial cells, male reproductive organs, and ovary revealed negative staining. The body compartments showing higher antigenicity were, in the decreasing order, the intestinal epithelial border, intestinal content, eggs in the worm capsule, vitelline glands, uterine eggs, and parenchymatous portions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of antigenic specificities of Paragonimus westermani developmental stages using immunoblot technique

Serodiagnosis of parasitic infections is widely used, since parasites or their eggs are not always detected by ordinary methods. The sensitive tests such as ELISA are highly dependent on the purity of antigens used. To solve this problem, many workers have tried to find species-specific components of antigens. The present study was performed to determine the antigenic profile of crude saline extracts of 3, 5, 8 and 12-week old P. westermani worms, which were collected from experimentally infected cats, based on SDS-PAGE and immunoblot technique. The results were as follows: 1. The SDS-PAGE showed at least 30 protein bands ranging from 229 kDa to 10 kDa molecular weight. The protein components of P. westermani changed chronologically during its developmental period. The 229 kDa band was recognized only in 12-week old worms (SEP12). 2. Analysis by ELISA showed a significant increase in antibody levels at 3 weeks in infected cats using crude saline extract antigens (SEP3, SEP5, SEP8, SEP12). 3. By EITB using SEP3 and SEP5, infected cats recognized major protein bands with molecular weight of 60, 35, 28, 25 or 21 kDa at 3-12 weeks of infection, and 3 additional antigens, 19, 13 and 10 kDa, were detected at 8-12 weeks of infections. 4. Using SEP8, 5 antigens, 91, 85, 31, 25 and 21 kDa, were consistently detected by all infected sera tested. In addition, 3 antigens of 19, 13 and 10 kDa were detected at 8-12 weeks of infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular weight of major component proteins in crude saline extract of adult Paragonimus westermani

When the component proteins in crude saline extract of 13-week old adult Paragonimus westermani were observed by non-denaturing discontinuous-polyacrylamide gel electrophoresis (Disc-PAGE), 8 distinct bands were clearly recognized. Molecular weight (MW) of each band protein, numbered in sequence from cathodal side which appeared in 10% separating gel, was measured first by Ferguson plot utilizing different gel concentrations from 10% to 4.5%. MW of band 1 protein (known as egg protein) was 440 kDa. And MW of other band proteins were: 386 kDa in band 2, 17.4 kDa in band 3, 17 kDa in band 4, 14.3 kDa in band 5, 46 kDa in band 6, 38 kDa in band 7 and 23 kDa in band 8. When the proteins in the crude extract were separated into fractions by molecular sieve chromatography through 1.6(phi) X 70 cm sized Sephacryl S-300 Superfine column and revisualized by Disc-PAGE in 8% gel, the sequence of eluted proteins was band 1, band 2, band 6, band 7 and bands 3, 4, 5 and 8. This elution profile confirmed MW of each band protein in the crude extract as measured by Ferguson plot.     

Infection Status of Freshwater Crabs and Crayfish with Metacercariae of Paragonimus westermani in Korea

The present study investigated the infection status of Paragonimus westermani metacercariae in freshwater crabs (n = 363) and crayfish (n = 31) from October 2007 to October 2008 using the crush method. All of the freshwater crabs, Eriocheir japonicus, were negative for P. westermani metacercariae while 10 (32.3%) of the 31 examined crayfish were positive. The 10 positive crayfish were caught in Haenam, Jeollanam-do, and there were 8-59 (mean 28.4) metacrcariae per infected crayfish. These results suggest that P. westermani metacerariae are still transmitted by crayfish enzootically in southern Korea, and that freshwater crabs may transmit metacercariae only on rare occasions.     

Genomes of Paragonimus westermani and related species: current state of knowledge

There have been few investigations of genomes of Paragonimus westermani and related species. Most studies have focussed on questions such as the identities of species and relationships among them, origins and relationships of strains with different ploidy states, and the characterisation of genes producing immunologically significant proteins. In the context of these questions, work on the karyotypes, nuclear and mitochondrial genomes is reviewed.     

Characterization and classification of five cysteine proteinases expressed by Paragonimus westermani adult worm

Three new members of the cysteine proteinase gene family of Paragonimus westermani have been isolated and classified. Comparisons of the predicted amino acid sequences of PwCP2 (U69121), PwCP4 (U56958), and PwCP5 (U33215) were performed with those of the previously reported PwCP1 (U69120) and PwCP3 (U56865) sequence. The amino acid alignment showed conservation of the cysteine, histidine, and asparagine residue that form the catalytic triad. With 57 cysteine proteinases including PwCP1-5, we conducted phylogenetic analysis using neighbor joining method (NJ). A resultant unrooted tree revealed that PwCP1-5 were clustered with cruzipain-like or cathepsin L-like cysteine proteinases. More detailed phylogenetic analyses with a reduced alignment set (22 cysteine proteinases) were performed by NJ and maximum parsimony (MP) methods. The results showed coincidently that PwCP1, 2, 3, and 4 belonged to the group of previously reported cruzipain-like cysteine proteinases (bootstrapping values of 97 and 100% in the MP and NJ trees) but PwCP5 to cathepsin L-like cysteine proteinases (the value of 76 and 100% in MP and NJ trees). Within the cruzipain-like clade, PwCP2 and 4 were found to be the most closely related. PwCP 2, 3, and 4 have five of six cruzipain signature sequences known previously, whereas PwCP5 do not have any cruzipain sequences in the corresponding sites. We found that two signature candidate sites (Gly 174, Asn 175--human cathepsin L numbering) for cathepsin L-like group are conserved in PwCP5, which are conserved within cathepsin L-like group and also different from those of cruzipain and other cysteine proteinase groups. PwCP5 has three-residue insertion (hydrophilic residues, Ser-Tyr-Gly) within the position corresponding to S3 subsite of SmCL2. Compared to the two-residue insertion (Tyr-Gly) in SmCL2, the three-residue insertion appeared in PwCP5 may bring bigger difference in substrate specificity between PwCP1-4 (cruzipain) and PwCP5 (cathepsin L-like). Such presumption is quite plausible considering extremely lower amino acid sequence similarity (18.2%) between PwCP1-4 and PwCP5. The present study is worthy of reporting one another case, the third organism after Schistosoma mansoni and Schistosoma japonicum, which has the two kinds of genes encoding both the cruzipain and cathepsin L-like cysteine proteinases. In addition, the fact that most of cysteine proteinases from P. westermani are cruzipain-like type implies strongly that a new powerful drug for paragonimiasis could be designed and developed if we focus on the exploration of anti-agents against P. westermani cruzipain-like cysteine proteinases.