Gene_locus Report for: schma-ACHE1

Cholinesterase (ChE) function in schistosomes is essential for orchestration of parasite neurotransmission but has been poorly defined with respect to the molecules responsible. Interrogation of the S. mansoni genome has revealed the presence of three ChE domain-containing genes (Smche)s, which we have shown to encode two functional acetylcholinesterases (AChE)s (Smache1 -smp_154600 and Smache2 -smp_136690) and a butyrylcholinesterase (BChE) (Smbche1 -smp_125350). Antibodies to recombinant forms of each SmChE localized the proteins to the tegument of adults and schistosomula and developmental expression profiling differed among the three molecules, suggestive of functions extending beyond traditional cholinergic signaling. For the first time in schistosomes, we identified ChE enzymatic activity in fluke excretory/secretory (ES) products and, using proteomic approaches, attributed this activity to the presence of SmAChE1 and SmBChE1. Parasite survival in vitro and in vivo was significantly impaired by silencing of each smche, either individually or in combination, attesting to the essential roles of these molecules. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosome AChE inhibitor 2,2- dichlorovinyl dimethyl phosphate-dichlorvos (DDVP), whereas smbche1-silenced parasites displayed increased sensitivity to DDVP.

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

Acetylcholinesterase (AChE) on the surface of the parasitic blood fluke Schistosoma is the likely target for schistosomicidal anticholinesterases. Determination of the molecular structure of this drug target is key for the development of improved anticholinesterase drugs and potentially a novel vaccine. We have recently cloned the cDNA encoding the AChE from the human parasite Schistosoma haematobium and succeeded in expressing functional recombinant protein. We now describe the cloning and molecular characterisation of homologues from two other schistosome species-Schistosoma mansoni and Schistosoma bovis, which are important parasites of man and cattle, respectively, but which differ in their sensitivity to the therapeutic anticholinesterase metrifonate. Comparison of the deduced amino acid sequences revealed that the AChE from all three species posses a high degree of identity, with conservation of all of the residues known to be important for substrate binding and catalytic activity. Also conserved is a unique C-terminal domain which is unusual in that it lacks the consensus for GPI modification, even though the native protein is considered to be GPI-anchored. We have also established the AChE gene structures for all three species and cloned the complete gene for S. haematobium AChE. The gene structure is relatively complex, comprising nine coding exons; the location of the splice sites is identical in all three species, but the size of the introns varies considerably. The two C-terminal splicing sites that are conserved in all species are also present in Schistosoma, but a third C-terminal conserved splicing site which is located 11-13 amino acids upstream of the histidine of the catalytic triad in all invertebrate AChE genes characterised to date is absent. We discuss our findings in the context of the molecular phylogeny of the AChE genes and the potential application to the control of schistosomiasis.

Natural products have moved into the spotlight as possible sources for new drugs in the treatment of helminth infections including schistosomiasis. Surprisingly, insect-derived compounds have largely been neglected so far in the search for novel anthelminthics, despite the generally recognized high potential of insect biotechnology for drug discovery. This motivated us to assess the antischistosomal capacity of harmonine, an antimicrobial alkaloid from the harlequin ladybird Harmonia axyridis that raised high interest in insect biotechnology in recent years. We observed remarkably pleiotropic effects of harmonine on physiological, cellular, and molecular processes in adult male and female Schistosoma mansoni at concentrations as low as 5 muM in vitro. This included tegumental damage, gut dilatation, dysplasia of gonads, a complete stop of egg production at 10 muM, and increased production of abnormally shaped eggs at 5 muM. Motility was reduced with an EC50 of 8.8 muM and lethal effects occurred at 10-20 muM within 3 days of culture. Enzyme inhibition assays revealed acetylcholinesterase (AChE) as one potential target of harmonine. To assess possible effects on stem cells, which represent attractive anthelminthic targets, we developed a novel in silico 3D reconstruction of gonads based on confocal laser scanning microscopy of worms after EdU incorporation to allow for quantification of proliferating stem cells per organ. Harmonine significantly reduced the number of proliferating stem cells in testes, ovaries, and also the number of proliferating parenchymal neoblasts. This was further supported by a downregulated expression of the stem cell markers nanos-1 and nanos-2 in harmonine-treated worms revealed by quantitative real-time PCR. Our data demonstrate a multifaceted antischistosomal activity of the lady beetle-derived compound harmonine, and suggest AChE and stem cell genes as possible targets. Harmonine is the first animal-derived alkaloid detected to have antischistosomal capacity. This study highlights the potential of exploiting insects as a source for the discovery of anthelminthics.

Cholinesterase (ChE) function in schistosomes is essential for orchestration of parasite neurotransmission but has been poorly defined with respect to the molecules responsible. Interrogation of the S. mansoni genome has revealed the presence of three ChE domain-containing genes (Smche)s, which we have shown to encode two functional acetylcholinesterases (AChE)s (Smache1 -smp_154600 and Smache2 -smp_136690) and a butyrylcholinesterase (BChE) (Smbche1 -smp_125350). Antibodies to recombinant forms of each SmChE localized the proteins to the tegument of adults and schistosomula and developmental expression profiling differed among the three molecules, suggestive of functions extending beyond traditional cholinergic signaling. For the first time in schistosomes, we identified ChE enzymatic activity in fluke excretory/secretory (ES) products and, using proteomic approaches, attributed this activity to the presence of SmAChE1 and SmBChE1. Parasite survival in vitro and in vivo was significantly impaired by silencing of each smche, either individually or in combination, attesting to the essential roles of these molecules. Lastly, in the first characterization study of a BChE from helminths, evidence is provided that SmBChE1 may act as a bio-scavenger of AChE inhibitors as the addition of recombinant SmBChE1 to parasite cultures mitigated the effect of the anti-schistosome AChE inhibitor 2,2- dichlorovinyl dimethyl phosphate-dichlorvos (DDVP), whereas smbche1-silenced parasites displayed increased sensitivity to DDVP.

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

Acetylcholinesterase (AChE) on the surface of the parasitic blood fluke Schistosoma is the likely target for schistosomicidal anticholinesterases. Determination of the molecular structure of this drug target is key for the development of improved anticholinesterase drugs and potentially a novel vaccine. We have recently cloned the cDNA encoding the AChE from the human parasite Schistosoma haematobium and succeeded in expressing functional recombinant protein. We now describe the cloning and molecular characterisation of homologues from two other schistosome species-Schistosoma mansoni and Schistosoma bovis, which are important parasites of man and cattle, respectively, but which differ in their sensitivity to the therapeutic anticholinesterase metrifonate. Comparison of the deduced amino acid sequences revealed that the AChE from all three species posses a high degree of identity, with conservation of all of the residues known to be important for substrate binding and catalytic activity. Also conserved is a unique C-terminal domain which is unusual in that it lacks the consensus for GPI modification, even though the native protein is considered to be GPI-anchored. We have also established the AChE gene structures for all three species and cloned the complete gene for S. haematobium AChE. The gene structure is relatively complex, comprising nine coding exons; the location of the splice sites is identical in all three species, but the size of the introns varies considerably. The two C-terminal splicing sites that are conserved in all species are also present in Schistosoma, but a third C-terminal conserved splicing site which is located 11-13 amino acids upstream of the histidine of the catalytic triad in all invertebrate AChE genes characterised to date is absent. We discuss our findings in the context of the molecular phylogeny of the AChE genes and the potential application to the control of schistosomiasis.