Comparison between site-specific DNA binding proteins of male and female Schistosoma mansoni

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.     

Lack of DNA methylation in Schistosoma mansoni

Schistosoma mansoni genomic DNA from male and female adult worms was subjected to restriction by the isoschizomeric endonucleases HpaII and MspI, which display different sensitivities with respect to cytosine methylation. The digested DNA was hybridized with 13 S. mansoni probes. Southern blot analysis showed that there were no observable differences in the restriction patterns of the two isoschizomers and that the patterns were identical in male and female parasites. Adenine methylation was also ruled out since no differences were observed with DpnI, Sau3A1, or MboI restriction enzymes. The methylation-dependent restriction endonuclease McrBC, which cleaves DNA containing methylcytosine and will not cleave unmethylated DNA, did not digest S. mansoni genomic DNA. These results demonstrate that the genome of adult S. mansoni is not methylated.     

Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins

PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol''s replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication.     

Schistosoma mansoni: eggshell formation is regulated by pH and calcium

The protein precursors of the schistosome eggshell are synthesized and packaged into secretory vesicles in the vitelline cells. These vesicles appear to contain an emulsion of eggshell precursor material. Evidence is presented to show that these secretory vesicles are acidic as in other systems and that this acidity stabilizes the emulsion and prevents the eggshell cross-linking reactions from occurring. Alkalinizing treatments trigger eggshell formation within the secretory vesicles as shown by (1) the induction of autofluorescence and (2) by electron microscopy which shows that the eggshell precursors have aggregated within the secretory vesicles into spherical particles bearing microspines. These aggregates formed in the secretory vesicles were isolated and shown to have the same protease resistance and amino acid composition as authentic eggshell. The calcium ionophore A23187 induces scattered autofluorescence in intact female worms which electron micrographs show to be due to exocytosis of eggshell material. Based on these observations we propose a model for the formation of schistosome eggshell and suggest that it may apply to all trematodes in which the eggshell precursors are present as stable emulsions in the secretory vesicles of the vitelline cells.     

Populational structure of Schistosoma mansoni assessed by DNA microsatellites

DNA microsatellites were used as molecular markers to analyse the population structure of the laboratory LE strain and of 10 field isolates of Schistosoma mansoni, the aetiologic agent of schistosomiasis. Out of 16,000 DNA sequences analysed in databases, 622 microsatellite loci were identified in 481 sequences (3.0%). The AT repetitions were the most frequent, followed by AAT and AC. Six loci showing perfect repetitions were selected and used in the polymerase chain reaction to evaluate polymorphisms in the number of repeats. Two groups of worms were studied. The first group consisted of 78 individuals, 39 of each sex, of the LE strain. The second group of worms consisted of 10 field isolates: seven from humans and three from snails. Four of the six loci were polymorphic, containing 11-17 alleles per locus. No linkage disequilibrium was observed among loci and none of the loci was sex linked. In both groups of worms, a significant deviation from Hardy-Weinberg equilibrium was observed. The observed heterozygosity was always lower than the expected one. The polymerase chain reaction primers were S. mansoni specific. The LE strain showed a lower total number of alleles or a lower average number of alleles/polymorphic locus than the field isolates, suggesting that 41 years of laboratory maintenance exerted selective pressure on the LE strain. The S. mansoni populations from the field were most genetically undifferentiated (R(ST)<0.027), suggesting a high gene flow among them. Our results showed the usefulness of microsatellites for population analysis of S. mansoni, offering a new alternative for a better understanding of schistosomiasis epidemiology.     

Immunocytochemistry of cytoskeletal proteins in adult Schistosoma mansoni

Antisera to vertebrate actin and actin-binding proteins were used to characterize the cytoskeleton of adult Schistosoma mansoni. Actin, alpha-actinin and tropomyosin immunoreactivities were detected in the cytoplasm of the apical tegument. Antiserum to alpha-actinin bound to the tegumental spines and this protein may be involved in cross-linking of spine actin filaments. Actin, alpha-actinin and tropomyosin antisera bound to the musculature. Strongest immunoreactivity was seen in the parenchyma. Antisera to actin, alpha-actinin, tropomyosin and spectrin bound to parenchyma cells including those of the tubercles, suggesting that these proteins are located in muscle cell bodies. The distribution of cytoskeletal proteins is discussed in relation to tegumental repair processes.     

Characterization of proteins and immunogens released by adult Schistosoma mansoni

The proteins released in vitro by metabolically radiolabeled adult Schistosoma mansoni were identified by 2-dimensional gel electrophoresis. To determine the origin of these proteins, adult worms were fractionated into surface membrane, tegument, and remaining body components, and the electrophoretic patterns of the proteins in the 3 fractions were compared to those of the released proteins. The immunogens present in these fractions then were identified by immunoprecipitation with sera from humans infected with S. mansoni. This analysis indicated that essentially all of the proteins released from the worm were immunogenic, whereas most of the major membrane and tegumental proteins were not reactive with the immune sera. Thus, it appears that the adult worm is defended against immune attack by detection of the host's antibody response against released proteins rather than against proteins-exposed on the worm's surface.     

Schistosoma mansoni: stage-specific expression of muscle-specific genes

It was previously shown that an antigen preparation termed 9B obtained from Schistosoma mansoni cercarial extracts partially (34%) protects mice from challenge infection with cercariae (R. Tarrab-Hazdai et al., J. Immunol. 135, 2772, 1985). To characterize some of the proteins which comprise this preparation, rabbit antibodies to the 9B antigen preparation were used to screen cDNA libraries of cercariae and adult worms. We isolated and sequenced cDNA clones encoding three proteins: calcium-binding protein, paramyosin, and myosin. The calcium-binding protein was previously shown to be expressed in cercariae but not in sporocysts or adult worms (D. Ram et al., Mol. Biochem. Parasitol. 34, 167, 1989). Northern blots showed the presence of paramyosin and myosin mRNAs in sporocysts and adult worms but not in cercariae. Antibodies to paramyosin detected the protein in sporocysts and adult worms as well as in cercariae. These findings explain, in part, the protective activity of the 9B antigen preparation against challenge infection.     

A 14-kDa Schistosoma mansoni polypeptide is homologous to a gene family of fatty acid binding proteins

The complete nucleotide sequence encoding a Schistosoma mansoni protein termed Sm14 was determined from cDNA clones propagated in bacteriophage lambda gt11 in Escherichia coli. The 14.8-kDa protein bears significant homologies with a family of related polypeptides which bind hydrophobic ligands. Members of this group of cytosolic proteins were originally identified based on their affinity for long chain fatty acids. The purified recombinant protein exhibited an affinity to fatty acids, in contrast to a mutant lacking 16 N-terminal amino acids. Immunofluorescence experiments show that tubercles, which are structures located on the dorsal surface of adult male schistosome and known to contain lipids, are stained using antibodies raised to the beta-galactosidase fusion protein. A regular staining pattern is also evident in the muscle layers as well as in the body of the parasite. As the schistosome cannot synthesize fatty acids de novo and is dependent on the uptake of lipids from serum, the available data support a role for Sm14 in the transport of fatty acids.     

Characterization of excretory-secretory proteins synthesized in vitro by Schistosoma mansoni primary sporocysts

Excretory-secretory (E-S) products released by larval schistosomes have been implicated in the interference of host snail defense systems. Because of the potentially important role that E-S products play in the parasite-host relationship, total and newly synthesized E-S proteins from in vitro-cultured Schistosoma mansoni primary sporocysts were characterized using incorporation of [35S]methionine followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Total E-S protein decreased more than 5-fold from day 1 to day 3 of culture and remained constant until day 8 when protein concentrations began to increase. Release of newly synthesized protein, however, increased from day 1 through day 8. Both silver staining and fluorography of SDS-PAGE-separated E-S products revealed a wide variety of polypeptides ranging in Mr from 13 to greater than 200 kDa. The dynamics of the release of individual polypeptides, both total and newly synthesized, varied over time. Although certain polypeptides decreased in concentration, others remained constant or increased with time in culture. Culture conditions were found to be important for sporocyst viability and growth, and for release of newly synthesized proteins. Sporocysts cultured in medium containing fetal bovine serum (complete) grew significantly larger and had a significantly greater viability than did sporocysts cultured in medium lacking serum (incomplete). Also, sporocysts cultured in complete medium synthesized and released significantly more protein than did sporocysts cultured in incomplete medium. These sporocysts continued to produce a 54-kDa polypeptide, whereas sporocysts in incomplete medium stopped producing this protein by day 3 of culture. The present study has shown that S. mansoni primary sporocysts, cultured in vitro, synthesize and secrete a wide variety of glycoproteins and that the type and quantity of glycoproteins released are dependent on culture conditions.     

Differential binding of hemolymph proteins from schistosome-resistant and -susceptible Biomphalaria glabrata to Schistosoma mansoni sporocysts

Humoral factors have been associated with resistance of Biomphalaria glabrata to infection by Schistosoma mansoni. The goal of this study was to determine which serum (cell-free hemolymph) proteins bind to the surface of S. mansoni sporocysts. For this, 125I-labeled serum from schistosome-resistant (10-R2) and -susceptible (M-line) B. glabrata was incubated with sporocysts, washed, and then subjected to SDS-PAGE and autoradiography. Other samples examined included radiolabeled 10-R2 and M-line serum, sporocysts incubated with unlabeled serum followed by incubation with radiolabeled serum, and radiolabeled sporocysts. Results indicated that many polypeptides in the serum from both strains of B. glabrata were radiolabeled. Dominating both profiles were bands in the 90-210-kDa range. However, some differences between the serum of the 2 snail strains were observed with M-line serum having several radiolabeled polypeptides in the 31-40- and 66-85-kDa range that were absent in serum from 10-R2 B. glabrata. When sporocysts were incubated with radiolabeled serum, 3 polypeptides (116, 180, 210 kDa) from both snail strains bound to the surface of the parasite. Further, a 55-kDa polypeptide bound to sporocysts incubated with 10-R2 serum but did not bind to those parasites incubated with M-line serum. Preincubation of sporocysts with unlabeled serum prior to incubation with radiolabeled serum significantly inhibited the uptake of radiolabeled proteins. This differential binding of serum polypeptides from different strains of B. glabrata may be important in determining resistance or susceptibility of the snail to larval schistosome infection.     

Characterization of developmentally regulated epitopes of Schistosoma mansoni egg glycoprotein antigens

The chemical and antigenic composition of a major group of concanavalin A-binding glycoprotein antigens of Schistosoma mansoni eggs was examined by the use of monoclonal antibodies. The individual glycoproteins of this group each displayed a very wide range of apparent m.w. and had isoelectric points of less than 5 when analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. These glycoproteins could be chemically labeled with 125Iodine by two different methods and biosynthetically labeled with 35S-methionine during in vitro synthesis by isolated eggs. By using five monoclonal antibodies, the individual egg glycoproteins were shown to share several antigenic determinants when analyzed by radioimmunoprecipitation and solid-phase binding assays. These epitopes were present in a high level, and the degree of expression appeared to be developmentally regulated. In addition, Schistosoma haematobium and Schistosoma japonicum eggs contained antigenically related glycoprotein antigens. Preliminary evidence suggests that the epitopes involve carbohydrate moieties.     

Using structural motif templates to identify proteins with DNA binding function

This work describes a method for predicting DNA binding function from structure using 3-dimensional templates. Proteins that bind DNA using small contiguous helix–turn–helix (HTH) motifs comprise a significant number of all DNA-binding proteins. A structural template library of seven HTH motifs has been created from non-homologous DNA-binding proteins in the Protein Data Bank. The templates were used to scan complete protein structures using an algorithm that calculated the root mean squared deviation (rmsd) for the optimal superposition of each template on each structure, based on C_α backbone coordinates. Distributions of rmsd values for known HTH-containing proteins (true hits) and non-HTH proteins (false hits) were calculated. A threshold value of 1.6 Å rmsd was selected that gave a true hit rate of 88.4% and a false positive rate of 0.7%. The false positive rate was further reduced to 0.5% by introducing an accessible surface area threshold value of 990 Ų per HTH motif. The template library and the validated thresholds were used to make predictions for target proteins from a structural genomics project.     

Schistosoma mansoni: immunoblot analysis of adult worm proteins

Proteins of adult Schistosoma mansoni were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assayed in immunoblots for reactions with individual mouse sera. Four weeks after a heavy infection with a few hundred cercariae, IgG antibodies directed predominantly against a protein of 31 kDa were detected. The protein was only weakly recognized by antibodies of mice harboring a 4-week-old light infection with about 60 cercariae. After 6 weeks or more, mice infected with either dose formed antibodies, not only against the 31-kDa protein and a 67-kDa protein, but also against a number of other components. While reactions with the 31- and 67-kDa proteins occurred with sera of all individual mice of four different strains, the reactions with other components were less consistently observed. Mice vaccinated with a heavy or light dose of 20,000-rad-irradiated cercariae did not form antibodies detectable in the blotting system. However, in immunofluorescence assays with living skin schistosomula, but not lung schistosomula, antibodies against the larval surface were detected with all sera obtained 4 weeks after infection or vaccination. In addition, immunofluorescence studies using the same sera and sectioned adult parasites demonstrated the presence of antibodies against the parasite surface in all sera except those obtained from mice exposed to a light infection with normal cercariae. Mice infected in this latter way were the only animals that did not develop a significant resistance against a challenge infection 4 weeks after exposure to normal or irradiated cercariae. The presence of an immunofluorescent reaction against the schistosome gut always coincided with a reaction of the sera with the 31-kDa protein in the immunoblots. Although a role in immune resistance could not be ascribed to any of the proteins reacting in the immunoblots, the data demonstrate important differences in the antibody specificities induced by various infection schemes.     

Acquisition of host proteins by the tegument of Schistosoma mansoni recovered from rats

Four-week-old Schistosoma mansoni collected from experimentally infected albino rats were shown by a fluorescent antibody technique to possess several serum proteins of host origin, including IgM, IgG, complement and red blood cell components. The capacity of these rat-derived worms to acquire and shed host proteins was also demonstrated in vitro with fluoresceinated antibody-labelled worms in the presence of xenogeneic molecules. The implications of these results are discussed in relation to the mechanisms of parasite evasion and "self-cure" phenomenon.     

Compositional correlations in the nuclear genes of the flatworm Schistosoma mansoni

We have investigated the genome organization in the flatworm Schistosoma mansoni. First, we analyzed the compositional distributions of the three codon positions. Second, we investigated the correlations that exist between (1) the GC levels of exons against flanking regions, (2) the GC levels of third codon positions against flanking regions, (3) the dinucleotide frequencies of exons against flanking regions, and (4) the GC levels of 5 against 3 regions. The modality of the distribution of third codon positions, together with the significant correlations found, leads us to propose that the nuclear genome of this species is compositionally compartmentalized.