Schistosoma japonicum: analysis of eggshell protein genes, their expression, and comparison with similar genes from other schistosomes.

As the egg of Schistosoma japonicum plays a central role in transmission and in pathogenesis, we sought to understand the molecular biology of egg formation. In this study we characterized an eggshell protein gene of S. japonicum and compared it with similar genes from S. mansoni and S. haematobium. To initiate studies on the eggshell protein genes of S. japonicum, a cloned genomic fragment containing an entire copy of a S. haematobium eggshell protein gene was used to identify three EcoRI hybridizing fragments of 2.6, 2.0, and 1.3 kbp in S. japonicum genomic DNA and to isolate three independent genomic clones from a S. japonicum genomic library. Two genomic clones, SJ 4-1 and SJ 3-1, contain at least two copies of the gene. The DNA sequence of a 2.0-kbp EcoRI fragment of clone SJ 3-1 showed two open reading frames (ORF), one of which showed a strong homology to the chorion proteins of insects. This ORF had 207 amino acids with a calculated molecular size of 18.5 kDa. The predicted peptide was glycine (50%) and tyrosine (10%) rich like other described schistosome eggshell proteins. Primer extension and the dideoxynucleotide sequence of the mRNA defined the cap site of the RNA and positioned the putative TATA and CAAAT elements and other cis-acting elements. Northern analysis demonstrated that eggshell protein mRNA was only detected in mature female parasites. The appearance of the female-specific mRNA was dependent on pairing with the male parasite and increased with egg production (as determined by hybridization intensity). A comparison of the DNA and deduced protein sequences of eggshell protein genes from S. japonicum with those of similar genes from S. mansoni and S. haematobium indicated that the genes are highly conserved, with S. mansoni and S. haematobium genes being more similar to each other than either is to S. japonicum.     

Schistosoma japonicum: the pathology of experimental infection

The pathology of experimental schistosomiasis japonica is reviewed and compared with the pathology of schistosomiasis japonica in man and to some aspects of schistosomiasis mansoni and schistosomiasis haematobia in experimental animals. The induction of granulomas around Schistosoma japonicum eggs depends upon cell mediated immunity, as do the reactions to Schistosoma mansoni and Schistosoma haematobium eggs. However, the modulation of the reaction to S. japonicum eggs can be greatly influenced by antibody, while antibody has no effect on the granulomas around S. mansoni eggs. Adult worm pairs of S. japonicum tend to cluster in the mesenteric venules, and most eggs are laid in a few sites. This leads to large, focal intestinal lesions similar to the discrete lesions produced by S. haematobium in the intestine and urinary tract but in contrast to the widespread, diffuse lesions produced by S. mansoni. Comparison with S. japonicum infection in humans is limited chiefly by our scant knowledge of the pathology produced by S. japonicum in infected persons. Most such comparisons are, in any case, limited by the marked differences in the reactions of various experimental host species to the infection and by differences in the reaction of a given host species to different strains of the parasite.     

Schistosoma mansoni,S. haematobium,and S. japonicum: early events associated with penetration and migration of schistosomula through human skin

Migratory pattern of schistosomula of Schistosoma mansoni, S. haematobium, and S. japonicum through human skin were analyzed in skin organ cultures. These studies showed that the schistosomula of S. mansoni and S. haematobium has similar migratory patterns through human skin. During the first 24h after infection nearly 90% of S. mansoni and S. haematobium schistosomula were present only in the epidermis. Majority of the schistosomula were found in the dermis only after 48h and they appear to reach the dermal vessels around 72h after infection. Migratory pattern of S. japonicum on the other hand was significantly different from the other two species in that over 90% of the parasites had already reached the dermis within the first 24h and schistosomula were present in the dermal vessels within 2h after infection. Analysis of the cytokine pattern at 8h after infection by a macro gene array and RT-PCR analysis showed that out of 24 different cytokines analyzed only IL-1ra, IL-10, and TNF-alpha were increased in the human skin following infections with S. mansoni and S. haematobium, whereas, after infection with S. japonicum there was significant increases in IL-1beta, IL-1ra, IL-2, IL-6, IL-8, IL-10, IL-15, IL-18, and TNF-alpha. Immunohistochemical analysis of epidermal sheets showed focal accumulation of HLA-DR(+) cells in areas where schistosomula of S. mansoni had entered the human skin.     

Interspecific variation within the 'hypervariable' region of the 18S ribosomal RNA gene among species of Schistosoma Weinland, 1858 (Digenea)

The 'hypervariable' region of the 18S ribosomal DNA gene, comprising 344 base pairs, was sequenced for five species of the genus Schistosoma (S. intercalatum, S. margrebowiei, S. mattheei, S. malayensis and S. rodhaini), representing three of the schistosome species groups. Heterobilharzia americana was also included as an outgroup. The sequences were compared with previously published sequences (S. haematobium, S. japonicum, S. mansoni and S. spindale). The analysis confirmed that the Asian schistosomes were distantly related to the other groups of schistosomes and showed that the amount of variation within the S. japonicum group (1.49%) was greater than that detected within the S. mansoni and S. haematobium groups (0% in each case).     

Identification of the Boudicca and Sinbad retrotransposons in the genome of the human blood fluke Schistosoma haematobium

Schistosomes have a comparatively large genome, estimated for Schistosoma mansoni to be about 270 megabase pairs (haploid genome). Recent findings have shown that mobile genetic elements constitute significant proportions of the genomes of S. mansoni and S. japonicum. Much less information is available on the genome of the third major human schistosome, S. haematobium. In order to investigate the possible evolutionary origins of the S. mansoni long terminal repeat retrotransposons Boudicca and Sinbad, several genomes were searched by Southern blot for the presence of these retrotransposons. These included three species of schistosomes, S. mansoni, S. japonicum, and S. haematobium, and three related platyhelminth genomes, the liver flukes Fasciola hepatica and Fascioloides magna and the planarian, Dugesia dorotocephala. In addition, Homo sapiens and three snail host genomes, Biomphalaria glabrata, Oncomelania hupensis, and Bulinus truncatus, were examined for possible indications of a horizontal origin for these retrotransposons. Southern hybridization analysis indicated that both Boudicca and Sinbad were present in the genome of S. haematobium. Furthermore, low stringency Southern hybridization analyses suggested that a Boudicca-like retrotransposon was present in the genome of B. truncatus, the snail host of S. haematobium.     

Schistosoma mansoni, S. haematobium, and S. japonicum: identification of genus- and species-specific antigenic egg glycoproteins

Immunoreactive egg glycoproteins of Schistosoma mansoni, S. haematobium, and S. japonicum which are genus- and species-specific, or react with sera of patients infected with other parasites, have been identified. Egg proteins were labeled with Iodine-125, and the concanavalin A-binding glycoproteins were immunoprecipitated with sera of patients infected with one of four species of Schistosoma or Trichinella spiralis, Taenia solium, Echinococcus granulosus, Entamoeba histolytica, or Wuchereria bancrofti. These immunoprecipitates were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Despite the strikingly different patterns of glycoproteins of the African species, the antibody immune responses of patients infected with S. mansoni and S. haematobium were found to be so similar that differentiation could not be established. In contrast, sera of patients infected with S. japonicum, S. mekongi, or parasites not of the genus Schistosoma, immunoprecipitated fewer of the major S. mansoni or S. haematobium glycoproteins. Likewise, antibody immune responses of patients infected with the Oriental schistosomes (S. japonicum and S. mekongi) could not be differentiated. Only a few quantitative differences were noted between our S. mansoni egg glycoprotein extract and a standardized soluble egg antigen extract. This study provides an explanation for the extensive cross-reactivity observed in diagnostic assays which utilize various fractions of schistosomal egg extracts as the antigen.     

Molecular mechanism of introduction of the hidden break into the 28S rRNA of insects: implication based on structural studies.

We determined the structure around the gap region between 28S alpha and 28S beta rRNA of Bombyx mori, a lepidopteran insect, to know the introduction mechanism of the hidden break, an interruption of the phosphodiester bond specific to the 28S rRNA of protostomes. Sequence analysis and S1 nuclease mapping suggested that a stretch of 30 nucleotides is excised from the mid region of the 28S rRNA to generate the hidden break. The length of excluded stretch was very various among three insects so far studied. However, the gap responsible for the hidden break was located in a fixed position in the 28S rRNA irrespective of insect species. It was suggested that extremely AU-rich sequence including the specific UAAU tract forming a loop can be a signal for the introduction of the hidden break. The same signal seemed also involved in splitting the dipteran 5.8S rRNA.     

The complete mitochondrial genomes of Schistosoma haematobium and Schistosoma spindale and the evolutionary history of mitochondrial genome changes among parasitic flatworms

Complete mitochondrial genome sequences for the schistosomes Schistosoma haematobium and Schistosoma. spindale have been characterized. S. haematobium is the causative agent of urinary schistosomiasis in humans and S. spindale uses ruminants as its definitive host; both are transmitted by freshwater snail intermediate hosts. Results confirm a major gene order rearrangement among schistosomes in all traditional Schistosoma species groups other than Schistosoma japonicum; i.e., species groups S. mansoni, S. haematobium, and S. indicum. These data lend support to the 'out of Asia' (East and Southeast Asia) hypothesis for Schistosoma. The gene order change involves translocation of atp6-nad2-trnA and a rearrangement of nad3-nad1 relative to other parasitic flatworm mt genomes so far sequenced. Gene order and tRNA secondary structure changes (loss and acquisition of the DHU and/or TPsiC arms of trnC, trnF, and trnR) between mitochondrial genomes of these and other (digenean and cestode) flatworms were inferred by character mapping onto a phylogeny estimated from nuclear small subunit rRNA gene sequences of these same species, in order to find additional rare genomic changes suitable as synapomorphies. Denser and wider taxon sampling of mt genomes across the Platyhelminthes will validate these putative characters.     

The timing of reproductive cell development and movement in Schistosoma mansoni, S. japonicum, and S. haematobium, using techniques of autoradiography and transplantation.

Tritiated thymidine was incorporated into gonial and vitelline cells of male and female Schistosoma mansoni, S. japonicum, and S. haematobium after 2 hr of incubation. Spermatogonial cells developed into labeled mature sperm after 6 days of transplantation in all species. Insemination of females was detected on the same day indicating copulation is frequent. Oogonia labeled in the initial incubation matured into primary oocytes after 7 days of transplantation for S. mansoni and S. haematobium and after 6 days in S. japonicum. Cytoplasmic thymidine label was observed after 2-hr incubations in the primary oocytes of all species. Initial label in the vitellaria was scattered and heavy. After 3 days of transplantation, label was considerably diluted and by day 6 was undetectable in vitelline glands. These times for development and movement of reproductive cells are considered to be standards against which the effects of stressful conditions on the reproductive system can be assessed.     

Comparative glucose utilization rates in separated and mated schistosomes

The rate of phosphorylation of 2-deoxyglucose (2DG) was determined by sequential pulsing of schistosomes (Schistosoma mansoni, S. japonicum, and S. haematobium) with 3H- and 14C-labeled 2-deoxy-D-glucose. Subsequent column chromatographic separation of the neutral [3H]2DG and [14C]2DG from the 3H- and 14C-labeled 2-deoxy-D-glucose 6-phosphate permitted estimation of the quantity of [3H]2DG phosphorylated in 2 min, and the proportion of [14C]2DG phosphorylated in 1 min; thus a phosphorylation rate was determined from a single tissue sample. The relative phosphorylation rate of 2-[3H]2DG to D-1-[14C]glucose (i.e., the phosphorylation coefficient) was also measured in male and female schistosomes. It was demonstrated that even though 2DG is taken up more rapidly than glucose, it is phosphorylated at a much slower rate in both S. mansoni and S. japonicum. In both of these species, mated males phosphorylate 2DG and glucose at a greater rate than do unmated males. Similarly, mated females phosphorylate and consume more glucose than do separated females. In contrast, the phosphorylation coefficient is greater in separated than in mated schistosomes. Intraspecific comparisons suggest that, at reduced substrate concentrations, glucose utilization rates are higher in S. japonicum, intermediate in S. mansoni, and lower in S. haematobium.     

Transcriptional organization of the 5.8S ribosomal RNA cistron in Xenopus laevis ribosomal DNA.

Hybridization of purified, 32p-labeled 5.8S ribosomal RNA from Xenopus laevis to fragments generated from X. laevis rDNA by the restriction endonuclease, EcoRI, demonstrates that the 5.8S rRNA cistron lies within the transcribed region that links the 18S and 28S rRNA cistrons.     

Ribosomal genes of the loach Misgurnus fossilis L. Isolation and restriction analysis

The ribosomal DNA of the teleost fish--loach has been isolated from sperm DNA by CsCl density gradient centrifugation. The rDNA sediments on density gradients by two heavy satellites beta = 1.715 and greater than 1.720. The DNA of the first satellite (1.715) was separated and treated by restrictases EcoRI and BamHI. It was shown that there are two EcoRI-sites in rDNA of loach, locating in 18S and 28S rRNA coding sequences. From tandem of repeating ribosomal genes EcoRI cuts out the fragment with homogeneous length-3 megadaltons (constant fragment) and heterogeneous population of fragments 11-13 megadaltons (major) and 7-8 megadaltons (minor fraction). The constant fragment contains mostly 28S coding sequence, and the heterogeneous fragment--18S coding sequence. The data indicate that the ribosomal genes of the loach as well as other higher eucaryotes were organized in genome as tandem of repeating units with heterogeneous length (10-16 megadaltons, 14.5-24 kb) due to heterogeneity of the length of nontranscribed spacer.     

Regular distribution of length heterogeneities within non-transcribed spacer regions of cloned and genomic rDNA of Saccharomyces cerevisiae.

A length difference of about 50 bp in the EcoRI fragment B of the rDNA from two different strains of Saccharomyces cerevisiae has been mapped in detail by sequencing of cloned fragments. This 2.4 kb EcoRI fragment contains the start of the 35S rRNA gene at one end and the 5S rRNA gene in the middle flanked by non-transcribed spacers, NTS1 and NTS2. The difference appeared as short deletions or insertions in five regularly spaced regions within the 1 kb NTS1, 3' to the 5S rRNA gene. The same regions of heterogeneities were displayed when all available sequence data of the NTS1 were compared. Four of the variable regions are located 160-170 bp apart, indicating that they might represent linker sequences between phased nucleosomes. Two variant clones, differing in the length of one subfragment of NTS1, were isolated for each strain. In both cases these represented the major variants among chromosomal NTS1 as revealed by sequencing of genomic fragments.     

Africa or Asia, which is the evolutionary origin of human schistosomes?]

The origin and the evolution of Schistosomatidae species, due to their medical importance (responsible of the second most important human parasitosis after malaria), arouse a great interest. A combination of phylogenetic studies using several molecular markers has provided support for the traditional grouping and evolutionary inferences derived from morphological and biological data. The genus Schistosoma, which comprises all species parasitizing Man, is generally split into four evolutionary lineages (mansoni, haematobium, indicum and japonicum lineages). The group of African schistosomes (including mansoni and haematobium lineages) appears very divergent from the japonicum lineage. Recent phylogenetic studies using partial 28S rDNA sequencing and including Orientobilharzia turkestanicum from Iran, an Asian parasite of livestock, found, unexpectedly, that this species nested among Schistosoma species, thus rendering the latter paraphyletic, and suggested an Asian origin for the Schistosoma genus. The present work re-examines the question of the geographical origin of human schistosomes by analysing a new genomic marker (ITS2) as well as by including the use of O. turkestanicum originating from northeastern China. Our results are in agreement with previous work using 28S, in demonstrating that Schistosoma is not monophyletic. However, O. turkestanicum, whatever the method of analysis used (distance or parsimony), was grouped with members of the japonicum group to the exclusion of African Schistosoma species. Then, our data argue strongly for the need for further phylogenetic study including new taxa and new genomic sequences before definitely concluding either an Asian or African origin for the genus Schistosoma.     

Isolation and characterization of surface antigens from Schistosoma mansoni. II. Antigenicity of radiolabeled proteins from adult worms.

Adult Schistosoma mansoni were radiolabeled in vitro with 125I Bolton-Hunter reagent. Surface membrane antigens were solubilized with non-ionic detergent, then reacted with infection or normal serum. The antigen-antibody complexes were then precipitated with staphylococcal protein A immunoadsorbent, eluted with urea and SDS, and fractionated by SDS-PAGE. The results indicated the presence of 6 to 8 tegument antigens, depending on the type of antisera used. Human antisera to S. japonicum and S. haematobium reacted with some but not all of the antigens identified with human S. mansoni infection serum; this implies the presence of species-specific tegument antigens. The molecular weights of the radiolabeled antigens ranged from 10,000 to 100,000. A large (greater than 100,000) molecular weight glycoprotein and an uncharacterized lipid fraction appeared to be precipitated nonspecifically. Immunoprecipitation methods with anti-mouse IgG and anti-mouse whole serum failed to detect the presence of hostlike antigens in the labeled extracts. Several of the labeled proteins from S. mansoni were found to react with serum from patients infected with either S. haematobium or with S. japonicum.     

Isolation and characterization of Chinese hamster ribosomal DNA clones

We have examined the ribosomal RNA (rRNA) genes of the Chinese hamster ovary (CHO) cell line. A partial EcoRI library of genomic CHO DNA was prepared using lambda Charon-4A. We isolated two recombinants containing the region transcribed as 45S pre-rRNA and 13 kb of external spacer flanking 5' and 3' to the transcribed region. These sequences show restriction site homology with the vast majority of the genomic sequences complementary to rRNA. In addition to this form of rDNA, Southern blot analysis of EcoRI-cut CHO genomic DNA reveals numerous minor fragments ranging from 2 to 19 kb which are complementary to 18S rRNA. We isolated one clone which contains the 18S rRNA gene and sequences 5' which appear to contain length heterogeneity within the non-transcribed spacer region. We have nine additional cloned EcoRI fragments in which the homology with 18S rRNA is limited to a 0.9-kb EcoRI-HindIII fragment. This EcoRI-HindIII fragment is present in each of the cloned EcoRI fragments, and is flanked on both sides by apparently nonribosomal sequences which bear little restriction site homology with each other or the major cloned rDNA repeat.     

Schistosoma haematobium: analysis of eggshell protein genes and their expression

Eggshell protein genes of Schistosoma mansoni that encode a 14 kDa protein have been shown to be highly conserved and expressed in a sex-, tissue-, and temporal-specific manner. To initiate studies on the eggshell protein genes of S. haematobium, a cDNA probe, pSMf 61-46, representing a S. mansoni eggshell protein mRNA was used to screen a S. haematobium genomic library. Of the seven independent recombinant clones isolated, two (lambda SH 2-1 and lambda SH 6-1) were analyzed and compared to those of S. mansoni. lambda SH 2-1 and lambda SH 6-1 each contain a different genomic copy of the gene encoding a 19.8 and 17.6 kDa protein, respectively. This is due to an additional 78 bp present in the coding region of lambda SH 2-1 relative to lambda SH 6-1. The rest of the coding sequences are identical, and the 5' and 3' untranslated regions are nearly identical. The deduced amino acid sequences of S. haematobium eggshell proteins are very rich in glycine (47 and 50%) when compared to 43.5% glycine in the protein encoded by S. mansoni. Long stretches of glycines, as many as 15 in a row, occur in the S. haematobium sequence. DNA comparison of the eggshell protein genes of the two schistosome species yielded an overall homology of 83.1%. The homology is much higher in the 5' and 3' untranslated regions than in the protein-coding regions. Genomic clones of both species contained second open reading frames, which appeared to be kept open as a consequence of the amino acid composition of the other. There are no introns in S. haematobium or S. mansoni eggshell protein genes, and the genomic Southern data indicated a similar arrangement of these genes in the genome of both species. Primer extension experiments and dideoxynucleotide sequencing of the RNA determined the mRNA cap site sequence as ATCAT and ATCAC in lambda SH 2-1 and lambda SH 6-1, respectively. Northern blot analysis determined the size of the mRNA to be about 1.0 kp. Expression of the RNA from these genes appears to be regulated in a manner similar to the corresponding genes in S. mansoni. mRNA is found only in mature females and first appears at 70 days after infection of hamsters. DNA sequence comparisons of the 5' flanking regions of S. haematobium and S. mansoni eggshell protein genes to each other and to those of silkmoth and Drosophila revealed several short sequence elements that are shared.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mating interactions between Schistosoma haematobium and S. mansoni

Schistosoma haematobium and S. mansoni are two medically important schistosomes, commonly occurring sympatrically in Africa and so potentially able to infect the same human host. Experiments were designed to study the mating behaviour of these two species in mixed infections in hamsters. Analysis of the data obtained showed that both heterospecific and homospecific pairs readily form. No significant difference was seen between the two species in their ability in forming pairs, however, S. mansoni showed a greater homospecific mate preference. Analysis of the data using the Mantel-Haenszel test suggests that mating competition does occur between S. haematobium and S. mansoni, the former being the more dominant species. Both species appeared to be able to change mate, with S. haematobium showing a greater ability in taking S. mansoni females away from S. mansoni males when introduced into a pre-established S. mansoni infection highlighting the competitiveness of S. haematobium. The significance of the results is discussed in relation to the epidemiological consequences occurring in Senegal, and other areas where both species are sympatric.