Infectious disease. Medical helminthology in the 21st century.
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Animals↗
Biomedical subjects
P T LoVerde
Publications and source records attributed to P T LoVerde.
Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer.
Smad proteins are essential intracellular signal transducers of the transforming growth factor-beta (TGF-beta) superfamily. The TGF-beta superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70- and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmTbetaR-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmTbetaR-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-beta(1). These data indicate that the SmSmad2 responds to the TGF-beta signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-beta effect.
Amino Acid Sequence↗
Evaluation of Schistosoma mansoni retinoid X receptor (SmRXR1 and SmRXR2) activity and tissue distribution.
Recently, we reported the identification of cDNA's encoding retinoid X receptor (RXR) homologues in Schistosoma mansoni. RXRs are known to be involved in the regulation of genes important for homeostasis and development. Previous studies indicated that SmRXR1 plays a role in the regulation of the female-specific gene, p14. Herein, we report that SmRXR2 also binds to cis-elements present in the p14 upstream region when evaluated in yeast reporter strains. SmRXR2 shows a pattern of recognition of cis-sequences present in the p14 gene upstream region different than SmRXR1. However, the SmRXR2 C (DNA binding) domain binds promiscuously in electrophoretic mobility shift assays to cis-elements of the p14 upstream region. The SmRXRs differ in their ability to activate transcription. The N-terminal A/B domain of SmRXR1 is necessary and sufficient for autonomous transcription activation function (AF) in yeast. SmRXR2 does not exhibit an equivalent autonomous AF. SmRXR1 and SmRXR2 fail to dimerize when investigated both in the yeast two-hybrid system and in immunoprecipitation experiments. In situ hybridization experiments using paraffin sections of adult worms demonstrate that SmRXR1 and SmRXR2 exhibit both common and unique cell type distribution which indicates that SmRXR1 and SmRXR2 both play a role in regulating gene expression in certain cells, yet each plays a distinct role in modulating the expression of genes in other cell types. Both SmRXR1 and SmRXR2 localize to vitelline cells. These studies provide a solid basis for improving our understanding of RXRs and their importance in female-specific gene regulation.
Animals↗
Exposure to Schistosoma mansoni infection in a rural area of Brazil. I: water contact.
The study of water contact patterns in rural Brazil presents unique challenges due to widely dispersed settlement patterns, the ubiquity of water contact sites, and the privatization of water resources. This study addresses these challenges by comparing the two most widely used methods of assessing water contact behaviour: direct observation and survey. The results of a 7-day direct observation of water contact were compared with water contact surveys administered 1 week after and then 1 year after the direct observation study. The direct observation study recorded a water contact rate higher than reported by other investigators (3.2 contacts per person per day); however, 75% of these contacts were for females and consisted mainly of domestic activities occurring around the household. A comparison of the frequency of water contact activities between the direct observation and the two surveys revealed several important points. First, no significant differences were found between methods for routine water contact activities (e.g. bathing), indicating that participants were able to accurately self-report some types of water contact activities. Second, significant differences were found in the recording of water contact activities that took place outside the observation area, indicating that direct observation may under-report water contact activities in areas where contact sites are dispersed widely. Third, significant differences between the direct observation and the survey method were more common for males than for females, indicating that the combination of widespread water contact sites and gender-specific division of labour may result in under-reporting of male contacts by direct observation methods. In short, despite the limitations in the recording of duration and body exposure, the survey method may more accurately record the frequency of water contact activities than direct observation methods in areas of widely dispersed water contact sites. Hence, surveys may be more suitable for the unique challenges of water contact in rural areas of Brazil.
Adolescent↗
Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library.
A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen.
Animals↗
Identification and functional characterization of a member of the PUR-alpha family from Schistosoma mansoni.
Schistosoma mansoni p14 gene encodes an eggshell precursor that is expressed only in vitelline cells of mature female worms in response to a male stimulus. The upstream region of the p14 gene contains several potential cis-acting regulatory sequences. We used the upstream region of the p14 gene as bait in a yeast-one-hybrid screen of a S. mansoni cDNA library to identify interacting proteins. We report the identification and characterization of a cDNA (S. mansoni PUR-alpha (SmPUR-alpha)) encoding a protein homologous to single-stranded DNA transcription activator PUR-alpha, that binds to the p14 upstream region and activates transcription of the HIS3 reporter gene in yeast. SmPUR-alpha has a predicted molecular mass of 30 kDa and shares an overall homology of 63% with mammalian PUR-alpha. The DNA binding domain of SmPUR-alpha is highly conserved. We show by gel shift assays that GST-SmPUR-alpha binds to oligonucleotides comprising the p14 upstream region. SmPUR-alpha binds preferentially to single-stranded DNA and also binds RNA. Unlike the mammalian homologue, SmPUR-alpha exhibits little specificity for the PUR element GGn, but shows strong preference for a sequence containing alternating pyrimidines. Our data support that SmPUR-alpha is a single-copy gene and through reverse transcriptase-polymerase chain reaction and in situ hybridization, we show that SmPUR-alpha is constitutively transcribed in many cell types and thus likely plays a role as a general transcription activator in schistosomes.
Amino Acid Sequence↗
Schistosoma mansoni phosphoenolpyruvate carboxykinase, a novel egg antigen: immunological properties of the recombinant protein and identification of a T-cell epitope.
In schistosomiasis mansoni, hepatic granulomatous inflammation surrounding parasite eggs is mediated by CD4(+) T helper (Th) cells sensitized to schistosomal egg antigens (SEA). We previously showed that a prominent lymphoproliferative response of CD4(+) Th cells from schistosome-infected C57BL/6 (BL/6) mice was directed against a 62-kDa component of SEA. A partial amino acid sequence of the 62-kDa component was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Based on this sequence, a cDNA clone containing the entire coding region of PEPCK was identified, and the full recombinant Schistosoma mansoni PEPCK (rSm-PEPCK) of 626 amino acids was purified from a prokaryotic expression system. rSm-PEPCK strongly stimulated a specific T-cell hybridoma, 4E6, as well as CD4(+) Th cells from SEA-immunized BL/6 mice and from infected BL/6, CBA, and BALB/c mice. In the infected mice, rSm-PEPCK elicited significant gamma interferon production as well as, to a lesser extent, production of interleukin-2 and -5. In BL/6 and BALB/c mice, the CD4(+) Th cell response to rSm-PEPCK was greater than that directed against the egg antigen Sm-p40; conversely, CBA mice responded better to Sm-p40 than to Sm-PEPCK. A 12-amino-acid region (residues 398 to 409: DKSKDPKAHPNS) was demonstrated to contain a T-cell epitope; synthetic peptides containing this epitope significantly stimulated specific hybridoma 4E6 and polyclonal CD4(+) Th cells. The identification and characterization of immunogenic egg components will contribute to the understanding and possible control of T-cell-mediated schistosomal disease.
Amino Acid Sequence↗
Transient expression of DNA and RNA in parasitic helminths by using particle bombardment.
Parasitic helminths (worms belonging to several metazoan phyla) cause considerable morbidity and mortality in humans. They are an important veterinary problem, and they result in significant economic losses in animal grazing and agriculture. Experimental studies on parasitic helminths have been limited by a lack of parasite cell lines and methods for molecular genetic analyses. We evaluated particle bombardment (biolistics) as a strategy to introduce and express nucleic acids in these multicellular parasites. By using embryos of the parasitic nematode Ascaris as a model, we developed methods to introduce and express both DNA and RNA during several stages of Ascaris embryogenesis. Biolistic transfection will facilitate experimental strategies in Ascaris embryos complementing other biochemical tools available (e.g., in vitro whole-cell embryo extracts for transcription, RNA processing, and translation). Transfection experiments with adult schistosomes further suggest that the biolistic strategy should be applicable to a variety of other parasitic helminths. The development of these methods provides molecular genetic tools to study gene expression and the biology of a variety of types and developmental stages of important helminth parasites.
Animals↗
RXR-2, a member of the retinoid x receptor family in Schistosoma mansoni.
A cDNA encoding a second full-length member of the Schistosoma mansoni RXR family (SmRXR-2) was identified. The nucleotide sequence of SmRXR-2 translates into a protein of 784 amino acids with a pI of 7.63 and an approximate mass of 78kDa making it the largest reported RXR to date. Phylogenetic tree analysis provides evidence that SmRXR-2 is the most ancient full-length RXR identified. SmRXR-2 exhibits unique sequence features compared with other RXRs. RT-PCR results demonstrate that the SmRXR-2 gene is constitutively expressed and thus must play multiple roles throughout schistosome development in the vertebrate host.
Amino Acid Sequence↗
Characterization of the Ras homologue of Schistosoma mansoni.
Ras is a member of a super-family of guanine-binding or G-proteins. Ras functions as a molecular switch in the transduction of signals generated by the activation of a variety of cell surface receptors and relays the signals to downstream effectors. Little is known about signal transduction in schistosomes. In order for Schistosoma mansoni to survive different immune responses triggered by the host as well as to migrate from the site of penetration at the skin to the final destination in portal circulation, they must receive signals from the host environment and respond to them in a way that allows their survival. We have isolated the schistosome Ras cDNA by using sequence information of the schistosome Ras homologue submitted to the Genbank database. Analysis of the encoded peptide revealed 81% identity and 92% similarity with K-Ras from various species. Ras is a single copy gene as determined by quantitative hybridization experiments. The cDNA was cloned into pGEX-4T and the expressed peptide was used to generate specific antibody reagents. Affinity purified antibodies identified a 23 kDa native protein that localizes to the subtegument. Ras is not associated with the tegument. Ras is expressed in all the developmental stages of the parasite. However, Ras is over-expressed in female worms compared to males. Schistosome Ras was also shown to be post-translationally modified by addition of farnesyl isoprenoid moiety to the cysteine residue in the C-terminal box. Using a schistosome extract in vitro SmRas farnesylation was inhibited by the farnesyl transferase inhibitor, FTI-277, at concentrations comparable to those required to inhibit K-Ras processing. These initial studies on signal transduction in schistosomes should provide a solid basis for improving our understanding of schistosome-host interactions.
Alkyl and Aryl Transferases↗
Identification of a cDNA encoding a retinoid X receptor homologue from Schistosoma mansoni. Evidence for a role in female-specific gene expression.
Schistosoma mansoni, a multicelluar eukaryotic blood fluke, is a major cause of morbidity worldwide in humans. The study of female parasite growth, development, and gene regulation is important because the eggs produced are responsible for the pathogenesis observed in schistosomiasis. p14, an eggshell precursor gene expressed only in sexually mature females in response to a male stimulus, is a model for female-specific gene regulation. The upstream region of the p14 gene shares sequences present in insect genes known to be regulated in a sex-, temporal-, and tissue-specific manner by members of the steroid receptor superfamily. Herein, we report the identification and characterization of a cDNA that encodes the S. mansoni (Sm) RXR homologue. Sequence analysis predicts and Western blot analysis confirms the synthesis of a 74-kDa protein, the largest member of the RXR family reported to date. We show by electrophoretic mobility shift assay analysis that SmRXR binds to cis-elements of the p14 gene including a direct repeat that follows the "3-4-5" rule of binding elements recognized by members of the steroid receptor superfamily. Furthermore, we demonstrate that SmRXR can act as a transcription activator in the yeast one-hybrid system. Through quantitative reverse transcriptase-polymerase chain reaction, we show that the SmRXR gene is constitutively expressed and thus must play multiple roles throughout the schistosome life cycle.
Amino Acid Sequence↗
Familial resemblance in humoral immune response to defined and crude Schistosoma mansoni antigens in an endemic area in Brazil.
This study addressed whether the humoral immune response to crude and defined Schistosoma mansoni antigens aggregates within families. The sample included 155 siblings from 42 nuclear families in Brazil. Sera examined by ELISA for antibody isotypes reactive to defined schistosome antigens and crude schistosome antigens (soluble adult worm antigen preparation and soluble egg antigen) demonstrated that there was a difference in sibling-pair correlations between defined and crude S. mansoni antigens. In contrast to the finding with crude antigens, egg-positive sibling pairs showed significant familial resemblance for all IgG subclasses and IgE to adult-stage antigens Smp20.8 and Smp50. Only the IgE and IgG4 isotypes showed familial resemblance to the egg-stage antigen, Smp40. Egg-negative sibling pairs showed significant familial resemblance only for IgE and IgG4 to Smp40. That both the IgE and IgG4 response to defined S. mansoni antigens showed familial resemblance is interesting in light of the converging evidence for the role of IgE and IgG4 in human susceptibility and resistance to reinfection.
Adolescent↗
The R-type pyocin of Pseudomonas aeruginosa C is a bacteriophage tail-like particle that contains single-stranded DNA.
Pseudomonas aeruginosa R-type pyocin particles have been described as bacteriocins that resemble bacteriophage tail-like structures. Because of their unusual structure, we reexamined whether they contained nucleic acids. Our data indicated that pyocin particles isolated from P. aeruginosa C (pyocin C) contain DNA. Probes generated from this DNA by the random-primer extension method hybridized to distinct bands in restriction endonuclease-digested P. aeruginosa C genomic DNA. These probes also hybridized to genomic DNA from 6 of 18 P. aeruginosa strains that produced R-type pyocins. Asymmetric PCR, complementary oligonucleotide hybridization, and electron microscopy indicated that pyocin C particles contained closed circular single-stranded DNA, approximately 4.0 kb in length. Examination of total intracellular DNA from mitomycin C-induced cultures revealed the presence of two extrachromosomal DNA molecules, a double-stranded molecule and a single-stranded molecule, which hybridized to pyocin DNA. Sequence analysis of 7,480 nucleotides of P. aeruginosa C chromosomal DNA containing the pyocin DNA indicated the presence of pyocin open reading frames with similarities to open reading frames from filamentous phages and cryptic phage elements. We did not observe any similarities to known phage structural proteins or previously characterized pseudomonal prt genes expressing R-type pyocin structural proteins. These studies demonstrate that pyocin particles from P. aeruginosa C are defective phages that contain a novel closed circular single-stranded DNA and that this DNA was derived from the chromosome of P. aeruginosa C.
Amino Acid Sequence↗
Characterization of Sm20.8, a member of a family of schistosome tegumental antigens.
Two cDNA clones each encoding a 20.8-kDa protein (Sm20.8) were identified from the human blood fluke Schistosoma mansoni sporocyst and adult worm cDNA expression libraries by antibodies derived from rabbits vaccinated with irradiated cercariae and purified over an NP-40 extract of 3h schistosomula. Each identified cDNA has an open reading frame encoding a protein of 181 amino acids and shows homology (29-30%) with Sm21.7, Sm22.6, and Sj22.6, previously identified as belonging to a family of soluble schistosome tegumental antigens. An EF-hand calcium-binding motif is found in Sm20.8 protein in two different positions. However, neither motif binds 45calcium (45Ca) Recombinant Sm20.8 showed immunoreactivity with sera from infected humans and rabbits vaccinated with irradiated cercariae. Polyclonal rabbit sera against the Sm20.8 recognized the native protein in an extract of infected snail (sporocyst), cercariae, 3 hour schistosomules (3 h NP-40) and an adult worm preparation but not in uninfected snail tissue or eggs. Further demonstration that Sm20.8 was expressed in the different developmental stages of the parasite was by RT-PCR. Confocal microscopy demonstrates that Sm20.8 localizes to the tegument of adult worms and 3 h np-40. The IgG fraction specific to Sm20.8 mediated complement killing of schistosomules in vitro by 34%. Vaccination of mice with naked DNA containing the Sm20.8 gene and subsequently challenged with cercariae showed 30% reduction in worm burden compared to controls.
Amino Acid Sequence↗
Cloning and characterization of Schistosoma mansoni fructose-1,6-bisphosphate aldolase isoenzyme.
A Schistosoma mansoni cercarial cDNA expression library, constructed in lambda gt11, was screened using the IgG fraction of sera taken from rabbits vaccinated with irradiated cercariae. A positive cDNA clone (1,431 base pairs) was selected and characterized. The amino acid sequence predicted from the cDNA sequence identified a polypeptide of 363 amino acids that showed significant homology to different family members of the enzyme fructose-1,6-bisphosphate aldolase (EC 1.4.2.13). The identity was 66% and 65% with human C and A isoenzymes, respectively. Active sites and substrate-binding determinant analysis suggest that the isolated enzyme in terms of function resembles type A aldolase. The recombinant protein expressed in the vector pGEX-2T was found to be active enzymatically. Antibodies raised against the purified recombinant protein recognized a 40-kDa band in extracts from cercariae, schistosomula (5 and 25 days), adult worms, and eggs. Using immunocytochemistry, aldolase localized to the tegumental region of the adult worms.
Amino Acid Sequence↗
Schistosoma mansoni: the developmental regulation and immunolocalization of antioxidant enzymes.
Antioxidant enzymes from S. mansoni, cytosolic Cu-Zn superoxide dismutase (CT-SOD), signal-peptide-containing SOD (SP-SOD), glutathione peroxidase (GPX), and glutathione transferase (GST) were compared for their relative levels of transcript expression throughout development in a semiquantitative reverse transcriptase-polymerase chain reaction assay. All of the antioxidant enzymes exhibited a similar pattern of developmental regulation. Adult worms have the highest level of specific mRNA compared with larval stages. GST shows the highest level of expression, being approximately 10-fold more abundant than CT-SOD and SP-SOD and 100-fold more abundant than GPX. This order of expression was nearly consistent for all the developmental stages studied. To localize the antioxidant enzymes, immunofluorescence staining was performed on 3-hr schistosomula and adult worms. GPX, SP-SOD, and CT-SOD were all found to be associated with the adult tegument and gut epithelium. SP-SOD was also associated with organelle and cell membranes of parenchymal cells and interestingly with the spines of adult worms. Schistosomula, on the other hand, showed little immunofluorescence. These studies further demonstrate the developmental regulation of antioxidant enzymes and localize them to the host-parasite interface, supporting the notion that they have a role in allowing adult worms to evade immune attack.
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Cloning the gene encoding Schistosoma mansoni p50, an immunophilin.
A 2.2-kb fragment of genomic DNA encoding Schistosoma mansoni immunophilin p50 (Smp50) was identified on a 14-kb genomic clone. The sequence of Smp50 reveals seven exons interrupted by six small introns ranging from 28-35 bp in size. The transcription start point, defined by primer extension analysis of schistosome RNA, begins at 30 bp upstream from the start AUG codon. Smp50 lacks a TATA box and appears to be a single-copy gene.
Animals↗
Identification of the telomeres on Schistosoma mansoni chromosomes by FISH.
The telomeres (molecular termini) of chromosomes play an important role in maintaining the structural integrity of the chromosome. Using a deoxyoligomer that contains the core sequence TTAGGG, we identified by fluorescence in situ hybridization (FISH) the telomeres on all 8 pairs of Schistosoma mansoni mitotic metaphase chromosomes. Our results indicate that S. mansoni exhibits a telomere sequence typical of other species.
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