Molecular characterization of thioester-containing proteins in Biomphalaria glabrata and their differential gene expression upon Schistosoma mansoni exposure.

Schistosomiasis is a disease caused by trematode parasites of the genus Schistosoma that affects approximately 200 million people worldwide. Schistosomiasis has been a persistent problem in endemic areas as there is no vaccine available, currently used anti-helmintic medications do not prevent reinfection, and most concerning, drug resistance has been documented in laboratory and field isolates. Thus, alternative approaches to curtail this human disease are warranted. Understanding the immunobiology of the obligate intermediate host of these parasites, which include the freshwater snail Biomphalaria glabrata, may facilitate the development of novel methods to stop or reduce transmission to humans. Molecules from the thioester-containing protein (TEP) superfamily have been shown to be involved in immunological functions in many animals including corals and humans. In this study we identified, characterized, and compared TEP transcripts and their expression upon S. mansoni exposure in resistant and susceptible strains of B. glabrata snails. Results showed the expression of 11 unique TEPs in B. glabrata snails. These transcripts present high sequence identity at the nucleotide and putative amino acid levels between susceptible and resistant strains. Further analysis revealed differences in several TEPs' constitutive expression levels between resistant and susceptible snail strains, with C3-1, C3-3, and CD109 having higher constitutive expression levels in the resistant (BS90) strain, whereas C3-2 and TEP-1 showed higher constitutive expression levels in the susceptible (NMRI) strain. Furthermore, TEP-specific response to S. mansoni miracidia exposure reiterated their differential expression, with resistant snails upregulating the expression of both TEP-4 and TEP-3 at 2 h and 48 h post-exposure, respectively. Further understanding the diverse TEP genes and their functions in invertebrate animal vectors will not only expand our knowledge in regard to this ancient family of immune proteins, but also offer the opportunity to identify novel molecular targets that could aid in the efforts to develop control methods to reduce schistosomiasis transmission.

Advances in gastropod immunity from the study of the interaction between the snail Biomphalaria glabrata and its parasites: A review of research progress over the last decade.

This review summarizes the research progress made over the past decade in the field of gastropod immunity resulting from investigations of the interaction between the snail Biomphalaria glabrata and its trematode parasites. A combination of integrated approaches, including cellular, genetic and comparative molecular and proteomic approaches have revealed novel molecular components involved in mediating Biomphalaria immune responses that provide insights into the nature of host-parasite compatibility and the mechanisms involved in parasite recognition and killing. The current overview emphasizes that the interaction between B. glabrata and its trematode parasites involves a complex molecular crosstalk between numerous antigens, immune receptors, effectors and anti-effector systems that are highly diverse structurally and extremely variable in expression between and within host and parasite populations. Ultimately, integration of these molecular signals will determine the outcome of a specific interaction between a B. glabrata individual and its interacting trematodes. Understanding these complex molecular interactions and identifying key factors that may be targeted to impairment of schistosome development in the snail host is crucial to generating new alternative schistosomiasis control strategies.

Molluscan cells in culture: primary cell cultures and cell lines.

In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

Transcriptome analysis of Schistosoma mansoni larval development using serial analysis of gene expression (SAGE).

SUMMARY: Infection of the snail, Biomphalaria glabrata, by the free-swimming miracidial stage of the human blood fluke, Schistosoma mansoni, and its subsequent development to the parasitic sporocyst stage is critical to establishment of viable infections and continued human transmission. We performed a genome-wide expression analysis of the S. mansoni miracidia and developing sporocyst using Long Serial Analysis of Gene Expression (LongSAGE). Five cDNA libraries were constructed from miracidia and in vitro cultured 6- and 20-day-old sporocysts maintained in sporocyst medium (SM) or in SM conditioned by previous cultivation with cells of the B. glabrata embryonic (Bge) cell line. We generated 21 440 SAGE tags and mapped 13 381 to the S. mansoni gene predictions (v4.0e) either by estimating theoretical 3' UTR lengths or using existing 3' EST sequence data. Overall, 432 transcripts were found to be differentially expressed amongst all 5 libraries. In total, 172 tags were differentially expressed between miracidia and 6-day conditioned sporocysts and 152 were differentially expressed between miracidia and 6-day unconditioned sporocysts. In addition, 53 and 45 tags, respectively, were differentially expressed in 6-day and 20-day cultured sporocysts, due to the effects of exposure to Bge cell-conditioned medium.

Antioxidant gene expression and function in in vitro-developing Schistosoma mansoni mother sporocysts: possible role in self-protection.

The ability of the larval forms of Schistosoma mansoni to invade and parasitize their molluscan host, Biomphalaria glabrata, is determined by a multitude of factors. In this study we sought to elucidate the possible mechanisms by which the invading larvae are able to counteract the potentially harmful oxidative environment presented by the host upon initial miracidial infection. This was attempted by examining the gene expression profile of parasite antioxidant enzymes of the linked glutathione-(GSH) thioredoxin (Trx) redox pathway during early intramolluscan larval development. Three such enzymes, the peroxiredoxins (Prx1, Prx2 and Prx3) were examined as to their activity and sites of expression within S. mansoni miracidia and in vitro-cultured mother sporocysts. Results of these studies demonstrated that the H(2)O(2)-reducing enzymes Prx1 and 2 are upregulated during early mother sporocyst development compared to miracidia. Immunolocalization studies further indicated that Prx1 and Prx2 proteins are expressed within the apical papillae of miracidia and tegumental syncytium of sporocysts, and are released with parasite excretory-secretory proteins (ESP) during in vitro larval transformation. Removal of Prx1 and Prx2 from larval ESP by immunoabsorption significantly reduced the ability of ESP to breakdown exogenous H(2)O(2), thereby directly linking ESP Prx proteins with H(2)O(2)-scavenging activity. Moreover, exposure of live sporocysts to exogenous H(2)O(2)stimulated an upregulation of Prx1 and 2 gene expression suggesting the involvement of H(2)O(2)-responsive elements in regulating larval Prx gene expression. These data provide evidence that Prx1 and Prx2 may function in the protection of S. mansoni sporocysts during the early stages of infection.

Signal transduction in larval trematodes: putative systems associated with regulating larval motility and behaviour.

The multi-host lifestyle of parasitic trematodes necessitates their ability to communicate with their external environment in order to invade and navigate within their hosts' internal environment. Through recent EST and genome sequencing efforts, it has become clear that members of the Trematoda possess many of the elaborate signal transduction systems that have been delineated in other invertebrate model systems like Drosophila melanogaster and Caenorhabditis elegans. Gene homologues representing several well-described signal receptor families including receptor tyrosine kinases, receptor serine tyrosine kinases, G protein-coupled receptors and elements of their downstream signalling systems have been identified in larval trematodes. A majority of this work has focused on the blood flukes, Schistosoma spp. and therefore represents a narrow sampling of the diverse digenean helminth taxon. Despite this fact and given the substantial evidence supporting the existence of such signalling systems, the question then becomes, how are these systems employed by larval trematodes to aid them in interpreting signals received from their immediate environment to initiate appropriate responses in cells and tissues comprising the developing parasite stages? High-throughput, genome-wide analysis tools now allow us to begin to functionally characterize genes differentially expressed throughout the development of trematode larvae. Investigation of the systems used by these parasites to receive and transduce external signals may facilitate the creation of technologies for achieving control of intramolluscan schistosome infections and also continue to yield valuable insights into the basic mechanisms regulating motility and behaviour in this important group of helminths.

Pharmacological and autoradiographical characterization of serotonin transporter-like activity in sporocysts of the human blood fluke, Schistosoma mansoni.

The present study focuses on the role of the biogenic monoamine serotonin (5-hydroxytryptamine) in the biology of sporocyst stages of the human blood fluke, Schistosoma mansoni, and its importance during obligate development within its snail host Biomphalaria glabrata. Based on previous work demonstrating that snails infected with S. mansoni have reduced levels of 5-hydroxytryptamine, we hypothesized that sporocysts actively transport this molecule from the host milieu. Intact sporocysts isolated in vitro take up exogenous 5-hydroxytryptamine via a high-affinity mechanism (K(m)=1.4 micromol l(-1)), and this serotonin transporter-like activity is dependent upon extracellular Na(+) and Cl(-) and is highly sensitive to previously characterized serotonin transporter inhibitors. Autoradiography suggests that transported [(3)H]5-hydroxytryptamine localizes within the body of the sporocyst, and in many cases is found in apical gland cells. Moreover, serotonin transporter-like activity is absent in free-swimming miracidia, the infective stage for the snail host, and the increase in larval serotonin transporter-like activity after miracidium-to-sporocyst transformation is accompanied by a corresponding decrease in steady-state levels of transcripts for tryptophan hydroxylase, the rate-limiting enzyme in serotonin biosynthesis. Overall our data suggest that S. mansoni larvae express surface-exposed serotonin transporter-like molecules, and that the transition from free-living miracidium to parasitic mother sporocyst is characterized by an increased dependence upon exogenous 5-hydroxytryptamine.

Carbohydrate inhibition of Biomphalaria glabrata embryonic (Bge) cell adhesion to primary sporocysts of Schistosoma mansoni.

Due to shared characteristics with snail haemocytes, the Biomphalaria glabrata embryonic (Bge) cell line has been used as a model in vitro system for the study of snail-trematode interactions. In this study, Bge cells were used to characterize the adherence of snail host cells to schistosome primary sporocysts, and to test the effect of carbohydrates as inhibitors of this behaviour. Bge cells bound to the surface of >90% of Schistosoma mansoni sporocysts and, based on a semi-quantitative cell adhesion scale of 1-4, exhibited a cell adhesion index (CAI) of 2.45. This cellular adhesion was significantly inhibited in the presence of selected carbohydrate-containing substances. Fucoidan was the most potent inhibitor, reducing Bge cell-binding prevalence to approximately 50% and the CAI to 1.6. Other inhibitory compounds included mannose-6-phosphate, heparin, dextran sulfate, and various forms of the polysaccharide carrageenan. Fluoresceinated-fucoidan was found to attach to Bge cells confirming their ability to associate with sugar moieties. These results were further supported by the specific binding of surface biotinylated Bge cell proteins to sporocyst tegumental glycoproteins ranging from 40 to 120 kDa. N-linked tegumental carbohydrates appeared to represent ligands for Bge cell proteins since N-glycosidase treatment of blotted tegumental glycoproteins completely abolished biotinylated Bge protein binding. We hypothesize that the involvement of lectins as potential host cellular receptors in snail cell-sporocyst interactions, and suggest that negatively charged (mainly sulfated) carbohydrate moieties may represent the schistosome surface ligand(s).

Protein kinase C regulation of cell spreading in the molluscan Biomphalaria glabrata embryonic (Bge) cell line.

Cellular adhesion and spreading are critical components involved in the processes of cell and tissue development, and immune responses in molluscs, but at present, little is known regarding the signaling pathways involved in these basic cellular functions. In the present study, the molluscan Biomphalaria glabrata embryonic (Bge) cell line was used as an in vitro model to study the signal transduction pathways regulating molluscan cell adhesion and spreading behavior. Western blot analysis using antibodies specific to mitogen-activated protein kinase (MAPK) revealed the presence of an MAPK-like immunoreactive protein in Bge cells, that was phosphorylated upon exposure to phorbol myristate acetate (PMA). Moreover, Bge cell treatment with inhibitors of protein kinase C (PKC), Ras and MAPK kinase (Mek) suppressed PMA-induced expression of activated MAPK, suggesting that PKC-, Ras- and Mek-like molecules may be acting upstream of MAPK. Similarly, in vitro Bge cell-spreading assays were performed in conjunction with the same panel of inhibitors to determine the potential involvement of PKC, Ras and Mek in cellular adhesion/spreading. Results revealed a similar pattern of inhibition of cell-spreading behavior strongly implying that the Bge cell spreading also may be regulated through a MAPK-associated signal transduction pathway(s) involving proteins similar to PKC, Ras and Mek.

Larval Schistosoma mansoni excretory-secretory glycoproteins (ESPs) bind to hemocytes of Biomphalaria glabrata (Gastropoda) via surface carbohydrate binding receptors.

Flow cytometric analysis of circulating blood cells (hemocytes) of Biomphalaria glabrata, molluscan intermediate host of Schistosoma mansoni, revealed the presence of 2 overlapping hemocyte subpopulations, designated R1 and R2. R1 hemocytes are characterized by their smaller size, reduced granularity, and the presence of the BGH1 surface epitope, whereas R2 cells are larger, more granulated, and generally lack the BGH1 cell marker. Both hemocyte subpopulations bound fluorescent dye (Oregon Green)-conjugated excretory-secretory glycoproteins (fESPs), although the specific fESP binding signal (geometric mean value), after correction for cellular autofluorescence, was greater in the R1 hemocyte subpopulation compared to that of the R2 subset. Partial inhibition of fESP binding to hemocytes consistently was achieved using various glycoconjugates (mucin, asialo-mucin, asialo-fetuin, heparin) and polysaccharides (fucoidan, dextran sulfate 8000), suggesting the involvement of hemocyte carbohydrate-binding receptors (CBRs) in reactions with ESP-associated glycans. Although sulfation of carbohydrate ligands contributed significantly to ESP blocking activity of some inhibitory polysaccharides and heparin, other sulfated proteoglycans (chondroitins A and B, heparan sulfate) were noninhibitory, indicating that charge alone was not solely responsible for the observed inhibition of hemocyte binding by fESPs. A similar blocking effect by desialylated glycoproteins (asialo-mucin, asialo-fetuin) further supports the contention that ESP-hemocyte interactions are mediated primarily through CBRs. The glycoconjugate inhibitors of ESP binding were only partially effective over a range of concentrations and their glycan moieties (oligosaccharides or long-chain polymers) comprised a diversity of major sugar groups, suggesting that hemocyte CBRs and S. mansoni larval ESPs likely represent a multiple receptor-ligand system. Previously reported findings of differential effects of ESPs on a variety of in vitro hemocyte functions are consistent with such a hypothesis.

Receptor-ligand interactions and cellular signalling at the host-parasite interface.

Although the effects of trematode infection on snail host physiology or host responses on parasite development have been well described in the literature, very little is known regarding the underlying mechanisms and specific molecules responsible for mediating those effects. It is presumed that many host-parasite interactions are communicated through receptor-mediated events, in particular those involving haemocytic immune responses to invading parasites, larval motility and migration through host tissues, and larval acquisition of host molecules either as nutrients or critical developmental factors. The intent of this chapter is to review current knowledge of molecules (both receptors and their ligands or counter-receptors) involved in molecular communication at the interface between larval trematodes, especially the mother or primary sporocyst stage, and host cells/tissues in intimate proximity to developing larvae. Information to date suggests that the molecular exchange at this interface is a highly complex and dynamic process, and appears to be regulated in specific cases. Topics discussed will focus on snail cell receptor interactions with the sporocyst tegument and its secretions, host cell-cell and cell-substrate adhesion receptors and their related signal transduction pathways, and sporocyst tegumental surface receptors and ligands involved in the binding of soluble host molecules.

Schistosoma mansoni: effects of serotonin and serotonin receptor antagonists on motility and length of primary sporocysts in vitro.

The effects of serotonin (5-hydroxytryptamine; 5-HT) on in vitro transformed primary sporocysts of Schistosoma mansoni were investigated. Serotonin treatment significantly increased parasite motility (percentage of motile sporocysts) and length at concentrations as low as 1 microM. These effects were mimicked by the 5-HT agonist tryptamine, albeit with 10- to 100-fold less potency. The effects of 10 microM 5-HT on sporocyst motility were observed within 15 min posttreatment and on parasite length by 6 h posttreatment, and both effects were stable for up to 48 h. Receptor antagonists with varying affinities for defined vertebrate neurotransmitter receptor subtypes were examined for their effects on parasite behavior in the absence and presence of 10 microM 5-HT. In the absence of 5-HT, only methiothepin significantly inhibited normal parasite growth after 48 h of incubation. In the presence of 10 microM 5-HT, the serotonin receptor antagonists mianserin, ketanserin (both at 100 microM), and methiothepin (at 10 microM) significantly inhibited 5-HT-induced lengthening of primary sporocysts, while 3-tropanyl-indole-3-carboxylate and chlorpromazine had no significant effect. The effects of these same drugs on parasite motility were also examined. In the absence of 5-HT, 10 microM chlorpromazine increased parasite motility, while the other antagonists had no effect. When sporocysts were treated with 10 microM 5-HT for 2 h in the continued presence of antagonist, 100 microM mianserin, ketanserin, 3-tropanyl-indole-3-carboxylate, and 10 microM methiothepin inhibited 5-HT induced increases in parasite motility, while 10 microM chlorpromazine had no effect. These results show that primary sporocysts of S. mansoni exhibit behavioral responses to serotonin much like adult stages of this parasite. Furthermore, these responses appear to be mediated via receptors with pharmacological similarities to those previously described in adult worms.

Monoamines and their metabolites in the freshwater snail Biomphalaria glabrata.

Metabolism of the major monoamines and their functions were studied in the freshwater snail Biomphalaria glabrata. In both juvenile and adult snails, the plasma (cell-free hemolymph) appears to act as a reservoir for most of these monoamines and their metabolites including among others, L-dopa and dopamine as major constituents. Significant quantities of L-tryptophan, precursor of indoleamines, also was found in the plasma. L-dopa, serotonin, homovanillic acid and dopamine were prominently represented in the central nervous system of the snail, while serotonin and its metabolites, 5-hydroxyindole acetic acid and 5-hydroxytryptophol were found in the ovotestis. Catecholamines such as L-dopa, dopamine and homovanillic acid were identified in the albumen gland. Functional aspects of both dopamine and serotonin were studied using in vitro cultures of albumen glands, the site of perivitelline fluid and galactogen synthesis in B. glabrata. Dopamine was found to stimulate the release of secretory proteins when exogenously added to gland cultures and this process was inhibited by chlorpromazine, a dopamine receptor antagonist. Similarly, exogenous serotonin stimulated in vitro protein secretion by albumen glands. Thus, these results suggest that monoamines may play important roles in regulating reproductive activity of this snail and provides an excellent model for studying neurotransmitter function and metabolism in molluscs.

Flukes without snails: advances in the in vitro cultivation of intramolluscan stages of trematodes.

In vitro cultivation of parasitic helminths, including the digenetic trematodes, has long been a valuable tool in medical and veterinary parasitology, permitting and/or facilitating the development of diagnostic reagents, chemotherapeutic agents, and vaccines and providing insights into naturally complex host-parasite interactions. In vitro cultivation of the intramolluscan stages of trematodes has been particularly challenging, given the ontogenic complexities involved in the production of multiple larval generations from germinal tissues through an asexual "budding" process. Recently, however, advanced larval development has been achieved by incorporating the Biomphalaria glabrata embryonic (Bge) cell line into cocultivation systems. Most notably, the entire intramolluscan cycle (from miracidium to cercaria) has been completed for the human blood fluke Schistosoma mansoni, while significant primary sporocyst development has been attained for several other digeneans including S. japonicum and Fascioloides magna. Here we review recent advances in the cultivation of several larval trematode species and discuss the potential use of this culture system for addressing fundamental questions of host-parasite compatibility.

Cloning of a beta integrin subunit cDNA from an embryonic cell line derived from the freshwater mollusc, Biomphalaria glabrata.

A cDNA encoding an integrin subunit was cloned and structurally characterized from an embryonic cell line derived from Biomphalaria glabrata, snail intermediate host of the human blood fluke Schistosoma mansoni. Cells of the B. glabrata embryonic (Bge) snail cell line were initially tested for their sensitivity to the integrin-specific tetrapeptide inhibitor Arg-Gly-Asp-Ser (RGDS). Washed Bge cells when exposed to 0.5 to 2.0mM of RGDS were significantly inhibited in their ability to spread on a glass substrate. Spreading inhibition was specific, since a control peptide Arg-Gly-Glu-Ser (RGES) did not have the same effect. RT-PCR was performed using previously reported degenerate oligonucleotide primers to the ligand binding domain (LBD) of known beta integrin subunits and Bge cDNA. A 137 bp fragment was amplified, TA-cloned, sequenced, and the na and deduced aa sequences were compared with other beta integrins. Databank analysis showed that the 137 bp product shared >/=55.6% aa similarity to other beta integrin LBDs. Southern and northern blot analyses using the 137 bp sequence as a probe revealed binding to Bge genomic DNA restriction fragments and to an approximately 8 kb poly-(A)+RNA transcript, respectively. An exact 5' primer synthesized to the 137 bp product and an oligo-d(T) primer then were used to amplify from Bge cDNA, a partial beta integrin sequence of 2285 bp that contained a 1971 bp ORF. The remaining upstream coding region was obtained using 5' RACE methods. The complete ORF, consisting of 2364 bp, encoded a 788 aa sequence with shared similarity to other known beta integrins (44.6-61.5%). Sequence and structural comparisons, which include a characteristic LBD, a series of three homologous cysteine-rich repeats, membrane proximal sequence (LLTFIHD), cytoplasmic NPXY motifs, and predicted domain lengths of the molluscan protein, clearly identifies it as an integrin homologue. This report represents the first cloning of a cDNA putatively encoding an integrin subunit from molluscan cells, and establishes the Bge cell line as a model for studying cellular adhesion in molluscs at the molecular level.

Biomphalaria glabrata embryonic (Bge) cell line supports in vitro miracidial transformation and early larval development of the deer liver fluke, Fascioloides magna.

A Bge cell co-culture system, previously shown to support the in vitro production of daughter sporocysts from mother sporocysts of Schistosoma mansoni and S. japonicum, has proven capable of supporting the in vitro development of intramolluscan stages of the deer liver fluke, Fascioloides magna. Miracidia commenced transforming within 4 h of incubation with Bge cells, and had completely shed their epidermal plates within 18-24 h. Redial stages were visible inside in vitro-transformed mother sporocysts after 12-16 days of co-culture with Bge cells, and emerged as fully-developed larvae starting at 14-20 days post-cultivation. Rediae survived over 60 days of in vitro culture, and reached a maximum size of 150-170 microns. Although particulate material was visible in their cecae, rediae were not observed to actively feed on Bge cells. Bge cells did not attach to or encapsulate larval stages at any time throughout the incubation period. Unlike Schistosoma spp., in which a high percentage of miracidia spontaneously shed their ciliated epidermal plates and transformed into mother sporocysts in Chernin's balanced saline solution (CBSS), transformation of F. magna was dependent on Bge cell products. Less than 5% of F. magna miracidia transformed spontaneously in either CBSS or Bge medium with 10% fetal bovine serum (complete or C-Bge). However, incubation of miracidia in either Bge cell-conditioned C-Bge medium or a greater than 30 kDa fraction concentrated from conditioned CBSS increased transformation rates to 67 and 83%, respectively. This secretory Bge cell factor(s) appeared to be protein in nature since its activity was completely abrogated by heat or proteinase K treatments. Overall, these results demonstrate that Bge cells are required for stimulating in vitro miracidial transformation and supporting early larval development of a fasciolid trematode under culture conditions. This is the first report of in vitro development of rediae from miracidia for a digenetic trematode.

Transfection and heat-inducible expression of molluscan promoter-luciferase reporter gene constructs in the Biomphalaria glabrata embryonic snail cell line.

Studies were initiated to begin developing a genetic transformation system for cells derived from the freshwater gastropod, Biomphalaria glabrata, an intermediate host of the human blood fluke Schistosoma mansoni. Using a 70-kD heat-shock protein (HSP70) cDNA probe obtained from the B. glabrata embryonic (Bge) cell line, we cloned from Bge cells a complete HSP70 gene including a 1-kb genomic DNA fragment in its 5'-flanking region containing sequences indicative of a HSP promoter. Identified in the 5'-half (416 nucleotides) of this genomic fragment were TATA and CAAT boxes, two putative transcription initiation sites, and a series of palindromic DNA repeats with shared homology to the heat-shock element consensus sequence (Bge HSP70(0.5k) promoter). The 3'-half of this upstream flanking region was comprised of a 508-base intron located immediately 5' of the ATG start codon. To determine the functionality of the putative snail promoter sequence, Bge HSP promoter/luciferase (Luc) reporter gene constructs were introduced into Bge cells by N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP)-mediated transfection methods, and assayed for Luc activity 48 hr following a 1.5-hr heat-shock treatment (40 degrees C). Compared with control vectors or the Bge HSP70(0.5k/1.0k) promoter constructs at 26 degrees C, a 10- to 300-fold increase in Luc expression was obtained only in the Bge HSP70 promoter/Luc-transfected cells following heat-shock. Results of transfection experiments demonstrate that the Bge HSP70(0.5k) DNA segment contains appropriate promoter sequences for driving temperature-inducible gene expression in the Bge snail cell line. This report represents the first isolation and functional characterization of an inducible promoter from a freshwater gastropod mollusc. Successful transient expression of a foreign reporter gene in Bge cells using a homologous, inducible promoter sequence now paves the way for development of methods for stable integration and expression of snail genes of interest into the Bge cell line.

Integrin-like RGD-dependent binding mechanism involved in the spreading response of circulating molluscan phagocytes.

Circulating phagocytic cells (hemocytes) of the snail Biomphalaria glabrata, intermediate host of the human blood fluke Schistosoma mansoni, were treated with the tetrapeptide, arg-gly-asp-ser (RGDS), an integrin-specific adhesion inhibitor, and assessed for their ability to adhere and spread on uncoated and snail plasma protein-coated glass slides. Although cells were capable of adherence, RGDS significantly inhibited the spreading ability of hemocytes in both a time and RGDS concentration-dependent fashion regardless of plasma protein coating. The inhibition of hemocyte spreading by RGDS was a specific response, since treatment of cells with a glutamic acid-substituted control peptide (RGES) did not exert the same inhibitory effect. A comparison of RGDS-responses between hemocytes of two strains of B. glabrata, one resistant (R; 13-16-R1 strain) and the other susceptible (S; NMRI strain) to infection by S. mansoni, revealed several snail strain-specific differences. At concentrations of 0.5 mM RGDS, R snail hemocyte spreading was unaffected, whereas a significant depression of spreading was seen in cells of the S snail. Moreover, we observed that R strain hemocytes spread more rapidly on homologous plasma-coated surfaces than the S snail strain following peptide pretreatment and removal. These data suggest that hemocytes from S and R snails may differ either in the number of RGDS-binding receptors or in their affinity for the RGDS peptide. In order to identify the type(s) of integrin-like RGD-binding receptors that may be present on the surface of snail immunocytes, washed hemocytes were placed on various mammalian extracellular matrix proteins and evaluated for their spreading function in the presence of specific or non-specific peptides. Hemocyte aggregation or clumping was observed on all test protein substrates, and this aggregation behavior was specifically inhibited by RGDS. Thus, RGD-binding receptors appear to play a critical role in cellular motility on matrix-coated surfaces and/or cell-cell binding. Our data provide functional evidence for an integrin-like receptor on circulating phagocytes of snails, and for an RGD-binding mechanism involved in cell-substrate interactions.

Heat-shock response in a molluscan cell line: characterization of the response and cloning of an inducible HSP70 cDNA.

Sublethal heat-shock of cells of the Bge (Biomphalaria glabrata embryonic) snail cell line resulted in increased or new expression of metabolically labeled polypeptides of approximately 21.5, 41, 70, and 74 kDa molecular mass. Regulation of this response appeared to be at the transcriptional level since a similar protein banding pattern was seen upon SDS-PAGE/fluorographic analysis of polypeptides produced by in vitro translation of total RNA from cells subjected to heat shock. Using a yeast (Saccharomyces cerevisiae) 70-kDa heat-shock protein (HSP70) probe to screen a cDNA library from heat-treated Bge cells, we isolated a full-length cDNA clone encoding a putative Bge HSP70. The cDNA was 2453 bp in length and contained an open reading frame of 1908 bp encoding a 636-amino-acid polypeptide with calculated molecular mass of 70,740 Da. Comparison of a conserved region of 209 amino acid residues revealed > 80% identity between the deduced amino acid sequence of Bge HSP70 and that of yeast (81%), the human blood fluke Schistosoma mansoni (for which B. glabrata serves as intermediate host) (81%), Drosophila (81%), human (84%), and the marine gastropod Aplysia californica (88%, 90%). In addition to the extensive sharing of sequence homology, the identification of several eukaryotic HSP70 signature sequences and an N-linked glycosylation site characteristic of cytoplasmic HSPs strongly support the identity of the Bge cDNA as encoding an authentic HSP70. Results of a Northern blot analysis, using Bge HSP70 clone-specific probes, indicated that gene expression was heat inducible and not constitutively expressed. This is the first reported sequence of an inducible HSP70 from cells originating from a freshwater gastropod and provides a first step in the development of a genetic transformation system for molluscs of medical importance.

Schistosoma japonicum: in vitro cultivation of miracidium to daughter sporocyst using a Biomphalaria glabrata embryonic cell line.

In vitro cultivation of Schistosoma japonicum miracidia to the mother sporocyst (MS) and then to the daughter sporocyst (DS) stage was achieved using the Biomphalaria glabrata embryonic (Bge) cell line as a coculture system. When comparing the effect of Bge cell and MS density on MS development, it was apparent that Bge cell density had a highly significant effect on both MS viability and growth. Viability and growth rate of MS cultured under high cell density conditions (350 cells/mm2) were almost 2 times greater than those of MS cultured under conditions of low cell density (60 cells/mm2). Growth under high cell density conditions corresponded to a 20 to 30 times increase in MS estimated volume within the first 9 weeks of cultivation. Emergence of fully formed motile DS was first observed after 11 weeks of cocultivation. A few DS lived for 14 weeks after emergence and attained a size of 770 +/- 100 microns in length and 48 +/- 13 microns in width. In contrast to what was observed in Bge cell/Schistosoma mansoni cocultures, Bge cells did not encapsulate S. japonicum MS. Our results show that, although the cellular interactions between Bge cells and schistosomes MS display some level of specificity, Bge cells apparently secrete soluble factors that permit excellent survival and can trigger advanced in vitro development of S. japonicum.