Conservation of protein kinase a catalytic subunit sequences in the schistosome pathogens of humans

cAMP-dependent protein kinases (PKAs) are central mediators of cAMP signaling in eukaryotic cells. Previously we identified a cDNA which encodes for a PKA catalytic subunit (PKA-C) in Schistosoma mansoni (SmPKA-C) that is required for adult schistosome viability in vitro. As such, SmPKA-C could potentially represent a novel schistosome chemotherapeutic target. Here we sought to identify PKA-C subunit orthologues in the other medically important schistosome species, Schistosoma haematobium and Schistosoma japonicum, to determine the degree to which this potential target is conserved and could therefore be exploited for the treatment of all forms of schistosomiasis. We report the identification of PKA-C subunit orthologues in S. haematobium and S. japonicum (ShPKA-C and SjPKA-C, respectively) and show that PKA-C orthologues are highly conserved in the Schistosoma, with over 99% amino acid sequence identity shared among the three human pathogens we examined. Furthermore, we show that the recently published Schistosoma mansoni and S. japonicum genomes contain sequences encoding for several putative PKA substrates with homology to those found in Homo sapiens, Caenorhabditis elegans, and Saccharomyces cerevisiae.     

Bursts of transposition from non-long terminal repeat retrotransposon families of the RTE clade in Schistosoma mansoni

The genus Schistosoma is composed of blood flukes that infect vertebrates, from which three species are major causative agents of human schistosomiasis, a tropical disease that affects more than 200 million people. Current models of the recent evolution of Schistosoma indicate multiple events of migration and speciation from an Asian ancestral species. Transposable elements are important drivers of genome evolution and have been hypothesised to have an important role in speciation. In this work, we describe a comprehensive inventory of Schistosoma mansoni and Schistosoma japonicum retrotransposons, based on their recently published genomic data. We find a considerable difference in retrotransposon representation between the two species (22% and 13%, respectively). A large part of this difference can be attributed to higher representation of two previously described families of S. mansoni retrotransposons (SR2 and Perere-3/SR3), compared with the representation of their closest relative families in S. japonicum. A more detailed analysis suggests that these two S. mansoni families were the subject of recent bursts of transposition that were not paralleled by their S. japonicum counterparts. We hypothesise that these bursts could be a consequence of the evolutionary pressure resulting from migration of Schistosoma from Asia to Africa and their establishment in this new environment, helping both speciation and adaptation.     

Development of chiral praziquantel analogues as potential drug candidates with activity to juvenile Schistosoma japonicum

A series of chiral praziquantel analogues were synthesized and evaluated against Schistosoma japonicum both in vitro and in vivo. All compounds exhibited low to considerable good activity in vivo. Remarkably, worm reduction rate of R-3 was 60.0% at a single oral dose of 200 mg/kg against juvenile stage of Schistosoma japonicum. The target compounds displayed in vivo antischistosomal activity against both Schistosoma japonicum and Schistosoma mansoni. Furthermore, all R-isomers displayed stronger antischistosomal activity than S-isomers in vivo, indicating R-isomers were the active enantiomers, while S-isomers were less active ones. This structure activity relationship (SAR) could have important implications in further drug development for schistosomiasis.     

Sex-Biased Expression of MicroRNAs in Schistosoma mansoni

Schistosomiasis is an important neglected tropical disease caused by digenean helminth parasites of the genus Schistosoma. Schistosomes are unusual in that they are dioecious and the adult worms live in the blood system. MicroRNAs play crucial roles during gene regulation and are likely to be important in sex differentiation in dioecious species. Here we characterize 112 microRNAs from adult Schistosoma mansoni individuals, including 84 novel microRNA families, and investigate the expression pattern in different sexes. By deep sequencing, we measured the relative expression levels of conserved and newly identified microRNAs between male and female samples. We observed that 13 microRNAs exhibited sex-biased expression, 10 of which are more abundant in females than in males. Sex chromosomes showed a paucity of female-biased genes, as predicted by theoretical evolutionary models. We propose that the recent emergence of separate sexes in Schistosoma had an effect on the chromosomal distribution and evolution of microRNAs, and that microRNAs are likely to participate in the sex differentiation/maintenance process.     

Mitochondrial gene order change in Schistosoma (Platyhelminthes: Digenea: Schistosomatidae)

In the flatworm genus Schistosoma, species of which include parasites of biomedical and veterinary importance, mitochondrial gene order is radically different in some species. A PCR-based survey of 19 schistosomatid spp. established which of 14 Schistosoma spp. have the ancestral (plesiomorphic) or derived gene order condition. A phylogeny for Schistosoma was estimated and used to infer the origin of the gene order change which is present in all members of a clade containing Schistosoma incognitum and members of the traditionally recognised Schistosoma indicum, Schistosoma mansoni and Schistosoma haematobium spp. groups. Schistosoma turkestanicum, with the plesiomorphic gene order state, is sister to this clade. Common interval analysis suggests change in gene order, from ancestral to derived, consisted of two sequential transposition events: (a) nad1_nad3 to nad3_nad1 and (b) [atp6,nad2]_[nad3,nad1,cox1,rrnL,rrnS,cox2,nad6] to [nad3,nad1,cox1,rrnL,rrnS,cox2,nad6]_[atp6,nad2], where gene order of fragments within square brackets remain unchanged. Gene order change is rare in parasitic flatworms and is a robust synapomorphy for schistosome spp. that exhibit it. The schistosomatid phylogeny casts some doubt on the origin of Schistosoma (Asian or African), highlights the propensity for species to host switch amongst mammalian (definitive) hosts, and indicates the likely importance of snail (intermediate) hosts in determining and defining patterns of schistosome radiation and continental invasion. Mitogenomic sampling of Schistosoma dattai and Schistosoma harinasutai to determine gene order, and within key species, especially S. turkestanicum and S. incognitum, to determine ancestral ranges, may help discover the geographic origins of gene order change in the genus. Samples of S. incognitum from India and Thailand suggest this taxon may include cryptic species.     

Identification and Characterization of Novel MicroRNAs from Schistosoma japonicum

Background

Schistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs.

Methodology and Results

We have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18–26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host.

Conclusions

Authentic miRNAs were identified for the first time in S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development.     

Performance of a rapid immuno-chromatographic test (Schistosoma ICT IgG-IgM) for detecting Schistosoma-specific antibodies in sera of endemic and non-endemic populations

BackgroundSchistosomiasis, an acute and chronic parasitic disease caused by human pathogenic Schistosoma species, is a neglected tropical disease affecting more than 220 million people worldwide.For diagnosis of schistosomiasis, stool and urine microscopy for egg detection is still the recommended method, however sensitivity of these methods is limited. Therefore, other methods like molecular detection of DNA in stool, detection of circulating cathodic antigen in urine or circulating anodic antigen in urine and serum, as well as serological tests have gained more attention. This study examines the sensitivity and specificity of a rapid diagnostic test based on immunochromatography (Schistosoma ICT IgG-IgM, LD Bio, Lyon, France) for simultaneous detection of specific IgG and IgM antibodies in serum, against Schistosoma spp. in endemic and non-endemic populations.Methodology/Principal findingsFrozen banked serum samples from patients with confirmed schistosomiasis, patients with other helminth infections, patients with seropositive rheumatoid arthritis and healthy blood donors were used to assess the sensitivity and the specificity of the Schistosoma ICT IgG-IgM rapid diagnostic test.The test showed a sensitivity of 100% in patients with parasitologically confirmed schistosomiasis, irrespective of the species (S. mansoni, S. haematobium, S. japonicum, S. mekongi). In healthy blood donors and patients with rheumatoid factor positive rheumatoid arthritis from Europe, specificity was 100%. However, in serum samples of patients with other tissue invasive helminth infections, the test showed some cross-reactivity, resulting in a specificity of 85%.Conclusion/SignificanceWith its high sensitivity, the Schistosoma ICT IgG-IgM rapid diagnostic test is a suitable screening test for detection of Schistosoma specific antibodies, including S. mekongi. However, in populations with a high prevalence of co-infection with other tissue invasive helminths, positive results should be confirmed with other diagnostic assays due to the test’s imperfect specificity.     

The intestinal protozoan parasite Entamoeba histolytica contains 20 cysteine protease genes,of which only a small subset is expressed during in vitro cultivation

Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.     

In vitro cultivation of Schistosoma japonicum-parasites and cells

Schistosomiasis is a serious parasitic zoonosis caused by blood-dwelling flukes of the genus Schistosoma. Understanding functions of genes and proteins of this parasite is important for uncovering this pathogen's complex biology, which will provide valuable information to design new strategies for schistosomiasis control. Effective applications of molecular tools reported to investigate schistosome gene function, such as inhibitor studies and transgenesis, rely on the developments of in vitro cultivation system of this parasite and cells. Besides the in vitro culture studies dealing with Schistosoma mansoni, there are also numerous excellent studies about the in vitro cultivation of Schistosoma japonicum, which were performed by Chinese researchers and published in Chinese journals. Nearly every stage of the life-cycle of S. japonicum, including miracidia, mother sporocysts, cercariae, schistosomula, and egg-laying adult worms, was employed for developing in vitro cultivation methods, being accompanied by the introduction of several media and supplements that helped to improve culture conditions. It was not only possible to generate mother sporocysts from miracidia in vitro, but also to obtain adult worms from cercariae through in vitro cultivation. The main obstacles to complete the life cycle of S. japonicum in the lab are the transition from mother sporocysts to cercariae, and the production of fertilized and completely developed eggs by adult worms generated in vitro. With regard to cells from S. japonicum, besides established isolation protocols and morphological observations, media optimizations were conducted by using different chemical reagents, biological supplements and physical treatment. Among these, mutagens like N-methyl-N-nitro-N-nitrosoguanidine and the addition of extracellular matrix were found to be able to induce mitogenic activities. Although enzyme activities or the level of silver-stained nucleolar region associated protein in cultured cells indicated still suboptimal conditions, the achievements made point to the possibility of reaching the aim of establishing cell lines for S. japonicum. Both the improvements of the in vitro culture of larval and adult worms of S. japonicum as well as the access of cells of this parasite provide excellent advances for research on this important parasite in the future.     

Expression analysis of nel during zebrafish embryonic development

Nel is a multimeric extracellular glycoprotein which predominantly expressed in the nervous system and play an important role in neural development and functions. There are three nel paralogues included nell2a, nell2b, and nell3 in zebrafish, while systematic expression analysis of the nel family is still lacking. In this study, we performed a phylogenetic analysis on 7 species, in different species the nell2a are highly conserved, as is nell2b. Then, the expression profiles of nell2a, nell2b and nell3 were detected by in situ hybridization in zebrafish embryo, and the result showed that nel genes highly enriched in the central nervous system, but distributed in different regions of the brain. In addition, nell2a is also expressed in the olfactory pit, spinal cord, otic vesicle and retina (ganglion cell layer), nell2b was detected to express in gill arches, olfactory epithelium, olfactory pit, spinal cord, photoreceptor and retina (ganglion cell layer), it should be noted that the expression of nell3 is special, was only detected at 96 hpf in the brain and spinal cord of zebrafish. Overall, our results indicate that nell2a and nell2b genes are expressed in the nervous system and eyes of zebrafish embryo, while nell3 is expressed in different regions in the nervous system. The phylogenetic analysis also shows that nell3 sequences are significantly different from nell2a and nell2b. This study provides new evidence to better understand the role of nel in zebrafish embryo development.