Biomedical subjects

Paul J Brindley

Publications and source records attributed to Paul J Brindley.

At least 19 recordsLinked to original sources

PubMed2006

Both sense and antisense strands of the LTR of the Schistosoma mansoni Pao-like retrotransposon Sinbad drive luciferase expression.

Long terminal repeat (LTR) retrotransposons, mobile genetic elements comprising substantial proportions of many eukaryotic genomes, are so named for the presence of LTRs, direct repeats about 250-600 bp in length flanking the open reading frames that encode the retrotransposon enzymes and structural proteins. LTRs include promotor functions as well as other roles in retrotransposition. LTR retrotransposons, including the Gypsy-like Boudicca and the Pao/BEL-like Sinbad elements, comprise a substantial proportion of the genome of the human blood fluke, Schistosoma mansoni. In order to deduce the capability of specific copies of Boudicca and Sinbad LTRs to function as promotors, these LTRs were investigated analytically and experimentally. Sequence analysis revealed the presence of TATA boxes, canonical polyadenylation signals, and direct inverted repeats within the LTRs of both the Boudicca and Sinbad retrotransposons. Inserted in the reporter plasmid pGL3, the LTR of Sinbad drove firefly luciferase activity in HeLa cells in its forward and inverted orientation. In contrast, the LTR of Boudicca did not drive luciferase activity in HeLa cells. The ability of the Sinbad LTR to transcribe in both its forward and inverted orientation represents one of few documented examples of bidirectional promotor function.

Animals↗

PubMed2006

Reconstruction and in silico analysis of the MAPK signaling pathways in the human blood fluke, Schistosoma japonicum.

At present, little is known about signal transduction mechanisms in schistosomes, which cause the disease of schistosomiasis. The mitogen-activated protein kinase (MAPK) signaling pathways, which are evolutionarily conserved from yeast to Homo sapiens, play key roles in multiple cellular processes. Here, we reconstructed the hypothetical MAPK signaling pathways in Schistosoma japonicum and compared the schistosome pathways with those of model eukaryote species. We identified 60 homologous components in the S. japonciumMAPK signaling pathways. Among these, 27 were predicted to be full-length sequences. Phylogenetic analysis of these proteins confirmed the evolutionary conservation of the MAPK signaling pathways. Remarkably, we identified S. japonicum homologues of GTP-binding protein beta and alpha-I subunits in the yeast mating pathway, which might be involved in the regulation of different life stages and female sexual maturation processes as well in schistosomes. In addition, several pathway member genes, including ERK, JNK, Sja-DSP, MRAS and RAS, were determined through quantitative PCR analysis to be expressed in a stage-specific manner, with ERK, JNK and their inhibitor Sja-DSP markedly upregulated in adult female schistosomes.

Amino Acid Sequence↗

PubMed2006

New perspectives on host-parasite interplay by comparative transcriptomic and proteomic analyses of Schistosoma japonicum.

Schistosomiasis remains a serious public health problem with an estimated 200 million people infected in 76 countries. Here we isolated ~ 8,400 potential protein-encoding cDNA contigs from Schistosoma japonicum after sequencing circa 84,000 expressed sequence tags. In tandem, we undertook a high-throughput proteomics approach to characterize the protein expression profiles of a number of developmental stages (cercariae, hepatic schistosomula, female and male adults, eggs, and miracidia) and tissues at the host-parasite interface (eggshell and tegument) by interrogating the protein database deduced from the contigs. Comparative analysis of these transcriptomic and proteomic data, the latter including 3,260 proteins with putative identities, revealed differential expression of genes among the various developmental stages and sexes of S. japonicum and localization of putative secretory and membrane antigens, enzymes, and other gene products on the adult tegument and eggshell, many of which displayed genetic polymorphisms. Numerous S. japonicum genes exhibited high levels of identity with those of their mammalian hosts, whereas many others appeared to be conserved only across the genus Schistosoma or Phylum Platyhelminthes. These findings are expected to provide new insights into the pathophysiology of schistosomiasis and for the development of improved interventions for disease control and will facilitate a more fundamental understanding of schistosome biology, evolution, and the host-parasite interplay.

Amino Acid Sequence↗

PubMed2006

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum.

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.

Animals↗

PubMed2006

Transduction of Schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped Moloney murine leukemia retrovirus.

Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of schistosome gene function and expression. The Moloney murine leukemia retroviral (MMLV) vector pLNHX was modified to incorporate EGFP or luciferase reporter genes under control of schistosome endogenous gene promoters from the spliced leader RNA and HSP70 genes. These constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) were utilized along with GP2-293 cells to produce replication incompetent retrovirus particles pseudotyped with the VSVG envelope. Exposure of several developmental stages, including sporocysts, of Schistosoma mansoni to these virions was facilitated by incubation with polybrene and/or by centrifugation. The early stages of binding and uptake of virus to the parasite tegument were demonstrated by the immunofluorescence colocalization of VSVG envelope and retroviral capsid proteins. Southern hybridization analysis indicated the integration of proviral forms of the MMLV constructs in genomic DNA isolated from the virus exposed schistosomes. Furthermore, analysis of RNA isolated from virus treated parasites demonstrated the presence of transcripts encoding reporter transgenes. Together these results indicated productive transduction by VSVG pseudotyped MMLV of cultured schistosomes, and suggest a tractable route forward towards heritable schistosome transgenesis.

Animals↗

PubMed2006

The dingo non-long terminal repeat retrotransposons from the genome of the hookworm, Ancylostoma caninum.

Members of the retrotransposable element (RTE) clade of non-long terminal repeat (LTR) retrotransposon are widely distributed among eukaryote taxa, with representatives known from Caenorhabditis elegans, mammals, mosquitoes, schistosomes, and other taxa. An RTE retrotransposon has not, however, been characterized in detail from a parasitic nematode. Here, we characterize two discrete copies of an RTE-like non-LTR retrotransposon from the genome of the dog hookworm, Ancylostoma caninum. The elements were named dingo-1 and dingo-2. The full-length dingo-1 and dingo-2 elements were 3421 and 3171bp in length, respectively. They exhibited 54% nucleotide sequence identity to one another across their entire length and 40%/58% amino-acid sequence identity/similarity across their open reading frames. dingo-1 and dingo-2 exhibited hallmark structures and sequences of non-LTR retrotransposons of the RTE family including a single open reading frame encoding apurinic-apyrimidinic endonuclease (EN) and reverse transcriptase (RT), in that order. Phylogenetic analyses targeting the RT and the EN domains both confirmed that dingo-1 and dingo-2 were members of the RTE clade and that they were closely related to RTE-1 from C. elegans, to BDDF from Bos taurus and to SR2 from Schistosoma mansoni. Dot blot hybridization indicated that as many as 100-1000 copies of dingo-1 reside within the genome of A. caninum, while detection by RT-PCR of transcripts encoding dingo-like elements suggested that dingo-1 and -2 may be retrotranspositionally active within the genome of A. caninum. The dingo elements are the first retrotransposons to be characterized from a hookworm genome.

Ancylostoma↗

PubMed2006

Antimicrosporidial activities of fumagillin, TNP-470, ovalicin, and ovalicin derivatives in vitro and in vivo.

Therapies for microsporidiosis in humans are limited, and fumagillin, which appears to be the most broadly effective antimicrosporidial drug, is considered to be moderately toxic. The purpose of this study was to apply an in vitro drug screening assay for Encephalitozoon intestinalis and Vittaforma corneae and an in vivo athymic mouse model of V. corneae infection to assess the efficacy of TNP-470 (a semisynthetic analogue of fumagillin), ovalicin, and eight ovalicin derivatives. TNP-470, ovalicin, and three of the ovalicin derivatives inhibited both E. intestinalis and V. corneae replication by more than 70% in vitro. Another three of the ovalicin derivatives inhibited one of the two microsporidian species by more than 70%. None of the treated athymic mice survived the V. corneae infection, but they did survive statistically significantly longer than the untreated controls after daily treatment with fumagillin administered at 5, 10, and 20 mg/kg of body weight subcutaneously (s.c.), TNP-470 administered at 20 mg/kg intraperitoneally (i.p.), or ovalicin administered at 5 mg/kg s.c. Of two ovalicin derivatives that were assessed in vivo, NSC 9665 given at 10 mg/kg i.p. daily also statistically significantly prolonged survival of the mice. No lesions associated with drug toxicity were observed in the kidneys or livers of uninfected mice treated with these drugs at the highest dose of 20 mg/kg daily. These results thus support continued studies to identify more effective fumagillin-related drugs for treating microsporidiosis.

Animals↗

PubMed2006

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.

Animals↗

PubMed2005

Characterization of SR3 reveals abundance of non-LTR retrotransposons of the RTE clade in the genome of the human blood fluke, Schistosoma mansoni.

BACKGROUND: It is becoming apparent that perhaps as much as half of the genome of the human blood fluke Schistosoma mansoni is constituted of mobile genetic element-related sequences. Non-long terminal repeat (LTR) retrotransposons, related to the LINE elements of mammals, comprise much of this repetitive component of the schistosome genome. Of more than 12 recognized clades of non-LTR retrotransposons, only members of the CR1, RTE, and R2 clades have been reported from the schistosome genome. RESULTS: Inspection of the nucleotide sequence of bacterial artificial chromosome number 49_J_14 from chromosome 1 of the genome of Schistosoma mansoni (GenBank AC093105) revealed the likely presence of several RTE-like retrotransposons. Among these, a new non-LTR retrotransposon designated SR3 was identified and is characterized here. Analysis of gene structure and phylogenetic analysis of both the reverse transcriptase and endonuclease domains of the mobile element indicated that SR3 represented a new family of RTE-like non-LTR retrotransposons. Remarkably, two full-length copies of SR3-like elements were present in BAC 49-J-14, and one of 3,211 bp in length appeared to be intact, indicating SR3 to be an active non-LTR retrotransposon. Both were flanked by target site duplications of 10-12 bp. Southern hybridization and bioinformatics analyses indicated the presence of numerous copies (probably >1,000) of SR3 interspersed throughout the genome of S. mansoni. Bioinformatics analyses also revealed SR3 to be transcribed in both larval and adult developmental stages of S. mansoni and to be also present in the genomes of the other major schistosome parasites of humans, Schistosoma haematobium and S. japonicum. CONCLUSION: Numerous copies of SR3, a novel non-LTR retrotransposon of the RTE clade are present in the genome of S. mansoni. Non-LTR retrotransposons of the RTE clade including SR3 appear to have been remarkably successful in colonizing, and proliferation within the schistosome genome.

Animals↗

PubMed2005

The molecular biology of schistosomes.

Twenty years ago, an article by Carter and Colley was published in an early issue of this journal. The report outlined pioneering studies by several laboratories into schistosome molecular biology and molecular genetics. To commemorate that prescient report and, in like fashion, to provide a brief (and non-comprehensive) synopsis of progress in this field up to the present time, I will outline some key aspects of the molecular biology of schistosomes that have been reported in the intervening years.

Animals↗

PubMed2005

Identification of an astacin-like metallo-proteinase transcript from the infective larvae of Strongyloides stercoralis.

Strongyloides stercoralis, an important nematode pathogen of humans, is transmitted by contact with soil contaminated with the microscopic larvae of the parasite. We determined the cDNA sequence and deduced amino acid structure of a metallo-proteinase that is abundantly transcribed expressed by infective stage larvae of S. stercoralis. This deduced structure of the enzyme revealed a multi-domain protein that included an NH2-terminal peptidase. This peptidase consisted of a signal peptide, a pro-enzyme region, and a mature peptidase domain that included the metal ion co-ordinating motifs, HETSHALGVIH and SIMHY ("Met-turn"), characteristic of the catalytic active site of members of the metzincin superfamily of zinc metallo-endopeptidases. It was phylogenetically and structurally similar to astacin from the digestive gland of the crayfish Astacus astacus, to the HCH-1 peptidase of Caenorhabditis elegans required for hatching and migration of a post-embryonic neuroblast, and to the morphogenetically important peptidases, bone morphogenetic protein-1 (BMP-1) and Drosophila tolloid. In addition, the Strongyloides enzyme, designated strongylastacin, includes a central epidermal growth factor (EGF) domain followed by a carboxyl CUB (complement sub component C1r/C1s/embryonic sea urchin protein Uegf/bone morphogenetic protein) domain. Inspection of the dbEST database revealed the presence of at least 9 transcript clusters that are related to greater or lesser extent to strongylastacin; based on these expressed sequence tags, strongylastacin was expressed only in the infective third stage larvae, whereas other transcript clusters were expressed both in filariform and rhabditiform stages or only in the rhabditiform stage. Based on the deduced sequence, structure, and expression profile, strongylastacin is the probable candidate for the zinc-dependent metalloprotease, Ss40, known to be deployed by larvae of S. stercoralis to penetrate human skin to initiate infection.

Amino Acid Motifs↗

PubMed2005

The Sinbad retrotransposon from the genome of the human blood fluke, Schistosoma mansoni, and the distribution of related Pao-like elements.

BACKGROUND: Of the major families of long terminal repeat (LTR) retrotransposons, the Pao/BEL family is probably the least well studied. It is becoming apparent that numerous LTR retrotransposons and other mobile genetic elements have colonized the genome of the human blood fluke, Schistosoma mansoni. RESULTS: A proviral form of Sinbad, a new LTR retrotransposon, was identified in the genome of S. mansoni. Phylogenetic analysis indicated that Sinbad belongs to one of five discreet subfamilies of Pao/BEL like elements. BLAST searches of whole genomes and EST databases indicated that members of this clade occurred in species of the Insecta, Nematoda, Echinodermata and Chordata, as well as Platyhelminthes, but were absent from all plants, fungi and lower eukaryotes examined. Among the deuterostomes examined, only aquatic species harbored these types of elements. All four species of nematode examined were positive for Sinbad sequences, although among insect and vertebrate genomes, some were positive and some negative. The full length, consensus Sinbad retrotransposon was 6,287 bp long and was flanked at its 5'- and 3'-ends by identical LTRs of 386 bp. Sinbad displayed a triple Cys-His RNA binding motif characteristic of Gag of Pao/BEL-like elements, followed by the enzymatic domains of protease, reverse transcriptase (RT), RNAseH, and integrase, in that order. A phylogenetic tree of deduced RT sequences from 26 elements revealed that Sinbad was most closely related to an unnamed element from the zebrafish Danio rerio and to Saci-1, also from S. mansoni. It was also closely related to Pao from Bombyx mori and to Ninja of Drosophila simulans. Sinbad was only distantly related to the other schistosome LTR retrotransposons Boudicca, Gulliver, Saci-2, Saci-3, and Fugitive, which are gypsy-like. Southern hybridization and bioinformatics analyses indicated that there were about 50 copies of Sinbad in the S. mansoni genome. The presence of ESTs representing transcripts of Sinbad in numerous developmental stages of S. mansoni along with the identical 5'- and 3'-LTR sequences suggests that Sinbad is an active retrotransposon. CONCLUSION: Sinbad is a Pao/BEL type retrotransposon from the genome of S. mansoni. The Pao/BEL group appears to be comprised of at least five discrete subfamilies, which tend to cluster with host species phylogeny. Pao/BEL type elements appear to have colonized only the genomes of the Animalia. The distribution of these elements in the Ecdysozoa, Deuterostomia, and Lophotrochozoa is discontinuous, suggesting horizontal transmission and/or efficient elimination of Pao-like mobile genetic elements from some genomes.

Amino Acid Motifs↗

PubMed2005

Phylogenetic relationships of methionine aminopeptidase 2 among Encephalitozoon species and genotypes of microsporidia.

This report describes the characterization and phylogenetic analysis of the deduced amino acid sequences of methionine aminopeptidase 2 (MetAP-2) enzymes from microsporidian species and genotypes of the genus Encephalitozoon. Fragments of DNA encoding 318 to 335 amino acid residues of the MetAP-2 genes were isolated from genomic DNA prepared from cultured spores of Encephalitozoon hellem, Encephalitozoon intestinalis, and Encephalitozoon cuniculi genotypes I-III. Sequence comparisons of the deduced amino acid residues indicated that the microsporidian sequences are MetAP-2-like rather than MetAP-1-like. Alignments demonstrated that the new Encephalitozoon sequences included sequences and structures conserved in eukaryotic MetAP-2s, including the five conserved, active site residues, Asp, Asp, His, Glu, and His, considered to be critical for catalysis and for coordinating the cation (e.g., cobalt) co-factor, and included residues known to interact with the antibiotic, fumagillin. The primary structure of the Encephalitozoon MetAP-2s, however, showed some dissimilarity with human and yeast MetAP-2s, including the absence of the NH2-terminal polylysine tract. Phylogenetic comparison of these Encephalitozoon MetAP-2s with orthologues from related species and from other informative taxa confirmed that the MetAP-2s of these Encephalitozoon species and strains are closely related to each other and cluster with MetAP-2s.

Amino Acid Sequence↗

PubMed2005

Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth.

Schistosoma mansoni is an important flatworm parasite of man that has remained intractable to experimental analyses of gene function. We have developed an approach for using dsRNA to target schistosome transcripts for RNA interference, and used it to address the role of cathepsin B (SmCB1), a cysteine protease that has been proposed to play a central role in hemoglobin digestion in the schistosome gut. Electroporation of 3 h old larval schistosomes with SmCB1-specific dsRNA (SmCB1-dsRNA) resulted in a greater than 10-fold reduction in SmCB1 transcript levels that persisted for >20 days. RNAi mediated reductions in transcript levels led to associated reductions in SmCB1 enzyme activity. Schistosomes treated with SmCB1-dsRNA were viable and developed intestinal heme pigmentation indicative of hemoglobin digestion, but showed significant growth retardation when compared to control parasites, indicating that SmCB1 function is not essential for hemoglobin digestion but is necessary for normal parasite growth. This effect on growth was apparent when parasites were maintained in culture or introduced into mammalian hosts. The report sheds new light on the role of SmCB1 and provides a template for using RNAi to examine gene function in the mammal-parasitic stages of schistosomes during early development in vitro and in vivo.

Animals↗

PubMed2005

Antimicrosporidial activity of (fluoro)quinolones in vitro and in vivo.

Microsporidia are a cause of emerging and opportunistic infections in humans and animals. Although two drugs are currently being used to treat microsporidiosis, concerns exist that albendazole is only selective for inhibiting some species of microsporidia that infect mammals, and fumagillin appears to have been found to be toxic. During a limited sequence survey of the Vittaforma corneae genome, a partial gene encoding for the ParC topoisomerase IV subunit was identified. The purpose of this set of studies was to determine if fluoroquinolones, which target topoisomerase IV, exert activity against Encephalitozoon intestinalis and V. corneae in vitro, and whether these compounds could prolong survival of V. corneae-infected athymic mice. Fifteen fluoroquinolones were tested. Of these, norfloxacin and ofloxacin inhibited E. intestinalis replication by more than 70% compared with non-treated control cultures, while gatifloxacin, lomefloxacin, moxifloxacin, and nalidixic acid (sodium salt) inhibited both E. intestinalis and V. corneae by at least 60% at concentrations not toxic to the host cells. These drugs were tested in vivo also, where gatifloxacin, lomefloxacin, norfloxacin, and ofloxacin prolonged survival of V. corneae-infected athymic mice (P < 0.05), whereas moxifloxacin and nalidixic acid failed to prolong survival. Therefore, these results support continued studies for evaluating the efficacy of the fluoroquinolones for treating microsporidiosis and for characterizing the target(s) of these fluoroquinolones in the microsporidia.

Animals↗

PubMed2005

Therapeutic strategies for human microsporidia infections.

Over the past 20 years, microsporidia have emerged as a cause of infectious diseases in AIDS patients, organ transplant recipients, children, travelers, contact lens wearers and the elderly. Enterocytozoon bieneusi and the Encephalitozoon spp., Encephalitozoon cuniculi, Encephalitozoon hellem and Encephalitozoon intestinalis, are the most frequently identified microsporidia in humans, and are associated with diarrhea and systemic disease. The microsporidia are small, single-celled, obligately intracellular parasites that have been identified in water sources, as well as in wild, domestic and food-producing farm animals, thereby raising concerns for waterborne, foodborne and zoonotic transmission. Current therapies for microsporidiosis include albendazole, a benzimidazole that inhibits microtubule assembly and is effective against several microsporidia, including the Encephalitozoon spp., although it is less effective against Encephalitozoon bieneusi. Fumagillin, an antibiotic and antiangiogenic compound produced by Aspergillus fumigatus, is more broadly effective against Encephalitozoon spp. and E. bieneusi; however, is toxic when administered systemically to mammals. Recent studies are also focusing on compounds that target the microsporidia polyamines (e.g., polyamine analogs), methionine aminopeptidase 2 (e.g., fumagillin-related compounds), chitin inhibitors (e.g., nikkomycins), topoisomerases (e.g., fluoroquinolones) and tubulin (e.g., benzimidazole-related compounds).

Antifungal Agents↗

PubMed2004

Organization and functional analysis of the Schistosoma mansoni cathepsin D-like aspartic protease gene promoter.

We have cloned a 969-bp fragment of genomic DNA that spans 821 bp of the 5' untranslated region, exon 1, a short intron, and part of exon 2 of the Schistosoma mansoni cathepsin D gene by inverse PCR. Inspection of this sequence revealed the presence of two TATA-box motifs, two inverted CCAAT-box (inverted NF-Y) motifs and sequences with homology to binding sites for the transcription factors, AP-1 and NF-Y. This sequence and deletion variants were cloned into reporter gene constructs, in order to examine the ability of these putative regulatory sequences to drive heterologous reporter gene activity. PCR products were cloned into the luciferase reporter vector pXP2. These reporter gene constructs were used to transform HeLa cells which were cultured and examined for luciferase activity. Additionally, HeLa cells transiently transfected with an EGFP reporter plasmid driven by the putative promoter from the S. mansoni cathepsin D gene were examined for EGFP transcripts and fluorescence. The 5' untranslated region of the S. mansoni cathepsin D gene, from position -772 to +40 (translation start ATG), included functional regulatory sequences capable of driving luciferase and EGFP expression, whereas shorter fragments from position -264 or -185 to +40 were insufficient to drive reporter activities.

5' Flanking Region↗

PubMed2004

Genomic organization of the Schistosoma mansoni aspartic protease gene, a platyhelminth orthologue of mammalian lysosomal cathepsin D.

Schistosomes are considered the most important of the helminth parasites of humans in terms of morbidity and mortality. Schistosomes employ proteolytic enzymes to digest host hemoglobin from ingested human blood, including a cathepsin D-like, aspartic protease that is overexpressed in the gut of the adult female schistosome. Because of its key role in parasite nutrition, this enzyme represents a potential intervention target. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cathepsin D gene locus of Schistosoma mansoni. Using the cDNA encoding S. mansoni cathepsin D as a probe, we isolated several positive bacterial artificial chromosomes (BAC) from a BAC library that represents an approximately 8-fold coverage of the schistosome genome. Sequencing of BAC clone 25-J-24 revealed that the cathepsin D gene locus was approximately 13 kb in length, and included seven exons interrupted by six introns. The exons ranged in length from 49 to 294 bp, and the introns from 30 to 5025 bp. The genomic organization of schistosome cathepsin D was similar in sequence, structure and complexity to human cathepsin D, including to a greater or lesser extent the conservation of all six exon/intron boundaries of the schistosome gene. It was less similar to aspartic protease genes of the nematodes Caenorhabditis elegans and Haemonchus contortus, and dissimilar to those of plasmepsins from malarial parasites. Examination of the introns revealed the presence of endogenous mobile genetic elements including SR2, the ASL-associated retrotransposon, and the SINE-like element, SMalpha. Phylogenetically, schistosome cathepsin D appeared to be more closely related to mammalian cathepsin D than to other sub-families of eukaryotic aspartic proteases known from mammals. Taken together, these features indicated that schistosome cathepsin D is a platyhelminth orthologue of mammalian lysosomal cathepsin D.

Amino Acid Sequence↗