Biomphalaria Glabrata Embryonic Cell Research Articles

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

BackgroundLarval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro.MethodsBge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed.ResultsGene knockout manipulation induced gene-disrupting indels, frequently 1–2 bp insertions and/or 8–30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n = 12). Adherence by BgAIF gene-edited (ΔBgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01).ConclusionsThe findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

Differential spatial repositioning of activated genes in Biomphalaria glabrata snails infected with Schistosoma mansoni.

Schistosomiasis is an infectious disease infecting mammals as the definitive host and fresh water snails as the intermediate host. Understanding the molecular and biochemical relationship between the causative schistosome parasite and its hosts will be key to understanding and ultimately treating and/or eradicating the disease. There is increasing evidence that pathogens that have co-evolved with their hosts can manipulate their hosts' behaviour at various levels to augment an infection. Bacteria, for example, can induce beneficial chromatin remodelling of the host genome. We have previously shown in vitro that Biomphalaria glabrata embryonic cells co-cultured with schistosome miracidia display genes changing their nuclear location and becoming up-regulated. This also happens in vivo in live intact snails, where early exposure to miracidia also elicits non-random repositioning of genes. We reveal differences in the nuclear repositioning between the response of parasite susceptible snails as compared to resistant snails and with normal or live, attenuated parasites. Interestingly, the stress response gene heat shock protein (Hsp) 70 is only repositioned and then up-regulated in susceptible snails with the normal parasite. This movement and change in gene expression seems to be controlled by the parasite. Other differences in the behaviour of genes support the view that some genes are responding to tissue damage, for example the ferritin genes move and are up-regulated whether the snails are either susceptible or resistant and upon exposure to either normal or attenuated parasite. This is the first time host genome reorganisation has been seen in a parasitic host and only the second time for any pathogen. We believe that the parasite elicits a spatio-epigenetic reorganisation of the host genome to induce favourable gene expression for itself and this might represent a fundamental mechanism present in the human host infected with schistosome cercariae as well as in other host-pathogen relationships.

Excreted/secreted Schistosoma mansoni venom allergen-like 9 (SmVAL9) modulates host extracellular matrix remodelling gene expression

The Schistosoma mansoni venom allergen-like (SmVAL) protein family consists of 29 members, each possessing a conserved α-β-α sandwich tertiary feature called the Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain. While the SmVALs have been found in both excretory/secretory (E/S) products and in intra/sub-tegumental (non-E/S) fractions, the role(s) of this family in host/parasite relationships or schistosome developmental processes remains poorly resolved. In order to begin quantifying SmVAL functional diversity or redundancy, dissecting the specific activity (ies) of individual family members is necessary. Towards this end, we present the characterisation of SmVAL9; a protein previously found enriched in both miracidia/sporocyst larval transformation proteins and in egg secretions. While our study confirms that SmVAL9 is indeed found in soluble egg products and miracidia/sporocyst larval transformation proteins, we find it to be maximally transcribed/translated in miracidia and subsequently down-regulated during in vitro sporocyst development. SmVAL9 localisation within sporocysts appears concentrated in parenchymal cells/vesicles as well as associated with larval germinal cells. Furthermore, we demonstrate that egg-derived SmVAL9 carries an N-linked glycan containing a schistosome-specific difucosyl element and is an immunogenic target during chronic murine schistosomiasis. Finally, we demonstrate that recombinant SmVAL9 affects the expression of extracellular matrix, remodelling matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) gene products in both Biomphalaria glabrata embryonic cell (BgMMP1) and Mus musculus bone marrow-derived macrophage (MmMMP2, MmMMP9, MmMMP12, MmMMP13, MmMMP14, MmMMP28, TIMP1 and TIMP2) in vitro cultures. These findings importantly suggest that excreted/secreted SmVAL9 participates in tissue reorganisation/extracellular matrix remodelling during intra-mammalian egg translocation, miracidia infection and intra-molluscan sporocyst development/migration.

Biomphalaria glabrata embryonic cells as a model system for toxicological and molecular studies of stress-induced expression of metallothionen genes

Biomphalaria glabrata embryonic cells as a model system for toxicological and molecular studies of stress-induced expression of metallothionen genes

Chemokinetic Effect of Interleukin-1β on Cultured Biomphalaria glabrata Embryonic Cells

Previous studies have indicated that a molecule with cytokine activity, possibly an interleukin-1-like (IL-1) molecule, plays a role in the killing of larval stages of the blood fluke Schistosoma mansoni in the snail host Biomphalaria glabrata. The purpose of the present experiment was to test the effects of recombinant-human IL-1beta (rhIL-1beta) on embryonic B. glabrata (Bge) cell motility to determine whether the cells respond to the cytokine. Response was measured using a variation of a chemokinetic assay in which cells in culture were separated from variable concentrations of rhIL-1beta by a semi-permeable membrane containing pores to allow migration. A double staining technique was developed to ascertain cell movement across the membrane. The number of cells moving across the membrane significantly increased in a concentration-dependent manner relative to the presence of increasing amounts of rhIL-1beta below the membrane. The number of cells that moved across the membrane increased until a threshold was reached, after which migration decreased. Further, the rhIL-1beta-mediated increase in Bge cell migration across the membrane was abrogated by the addition of IL-1 receptor antagonist protein. These data indicate that Bge cells respond specifically to rhIL-1beta. As such, these data also indicate that Bge cells may serve as a useful model for elucidation of the role of cytokines or cytokine-like molecules in the snail/schistosome relationship.

In vitro and in vivo encystment of the cercariae of Echinostoma caproni.

In vitro and in vivo studies were conducted on the cercariae of Echinostoma caproni. Of the 15 media tried, 2 resulted in effective in vitro encystment in petri dish cultures maintained at 23 +/- 1 C. They were a Locke's--artificial springwater (ASW) (1:1) medium (67% encystment) and a Biomphalaria glabrata embryonic cell line medium (23% encystment). To obtain large numbers of in vitro--formed cysts, finger bowl cultures containing 40 ml of the Locke's-ASW (1:1) medium were used at 23 +/- 1 C. Of 3,000 cercariae tested, 1,890 (63%) were encysted in this medium by 48 hr. Most of these cysts looked similar to those formed in vivo, although some showed abnormalities in the outer cyst wall and other malformations. A total of 200 in vitro-formed cysts treated in an alkaline trypsin-bile salts (TB) medium for 2 hr at 41 C showed 94% excystation. In vitro-formed cysts fed to mice produced ovigerous adults within 2 wk postinfection (PI). Eggs from these worms gave rise to miracidia that produced patent intramolluscan infections in B. glabrata snails. In vivo encystment was studied in lab-raised juvenile Helisoma trivolvis (Colorado strain) snails, 1-3 mm in shell diameter. From 6 to 24 hr PI, 93-100% of the cercariae were recovered as metacercarial cysts in the snail tissue. Treatment of these cysts in the TB medium resulted in 96% excystation within 2 hr at 41 C.

Gene expression changes in Schistosoma mansoni sporocysts induced by Biomphalaria glabrata embryonic cells.

Biomphalaria glabrataembryonic (Bge) cells have been shown to provide favourable environmental conditions for the development of Schistosoma mansoni sporocysts. We investigated the effect of Bge excretory-secretory products on metabolic activity and gene transcription in S. mansoni mother sporocysts. Using the differential-display technique, we identified several sporocyst transcripts regulated by exposure to Bge soluble components. Research in databases indicated that six of the eight differential products analysed were homologous to sequences already present in databases. Two transcripts appeared of interest for schistosome development since they could be associated with cell division and protein synthesis in developing sporocysts. Their up-regulation following contact with cell products was confirmed by semi-quantitative RT-PCR. The first fragment coded for a part of the chaperonin containing T-complex protein gamma subunit-like protein of S. mansoni (SmTCP 1-C). The second one represented a new S. mansoni expressed sequence tag encoding a protein homologous to various glutaminyl-tRNA synthetases (GlnRS). The full-length sequence of SmGlnRS was cloned from adult schistosomes and its primary sequence was compared to other GlnRS. The overexpression of SmTCP-1 and SmGlnRS could be correlated with the metabolic changes observed in Bge-exposed sporocysts.

Characterization of an insulin receptor-related receptor in Biomphalaria glabrata embryonic cells

Tyrosine kinase receptors play a key role in the communication of cells with their environment. Growth hormone receptors, such as insulin receptors, are involved in the regulation of cell growth, differentiation and metabolism in multicellular organisms. Insulin-related peptides and members of the insulin receptor subfamily have been described in a wide variety of invertebrates, including freshwater molluscs. In this paper, we describe the metabolic effect of insulin on a mollusc cell line (Bge) derived from embryos of the snail Biomphalaria glabrata. Using a PCR strategy, we have cloned from Bge cells a cDNA encoding a protein (BgIR) homologous to, and exhibiting all of the typical features of insulin receptors. Northern blot analysis confirmed the expression of BgIR in B. glabrata snails and suggested its wide distribution in the snail body. Bge cells have been shown to provide the environmental conditions necessary for the in vitro development of the sporocysts of Schistosoma mansoni, a trematode parasite that uses B. glabrata as an intermediate host. The possible implication of BgIR in the activating and proliferating processes observed in Bge cells during their coculture with S. mansoni larvae is discussed.

Biomphalaria glabrata embryonic cells express a protein with a domain homologous to the lectin domain of mammalian selectins.

We have cloned from Biomphalaria glabrata, the intermediate host of the helminth parasite Schistosoma mansoni, a 36-kDa apparent-molecular-mass molecule (BgSel) that shares sequence identity with selectins of the cell-adhesion-molecule superfamily. BgSel exhibited in its C-terminal part a putative C-type lectin domain similar to the selectin lectin domain. Using antibodies to the recombinant BgSel protein, we demonstrated the presence of BgSel in snail hemocytes as well as in the cell line derived from B. glabrata embryos (Bge). Anti-BgSel antibodies specifically recognized a 79-kDa component in Bge-cell-secreted products that was supposed to represent the native form of BgSel, as was confirmed after glycosidase treatment. Lectins are known to be implicated in recognition mechanisms participating in humoral and cellular immunity in molluscs. The possible role of BgSel in the interaction between sporocysts and Bge cells, particularly in the in vitro model of sporocyst development dependent on Bge cell factors, remains to be determined.

DNA transfer in a Biomphalaria glabrata embryonic cell line by DOTAP lipofection.

DNA transfer in a Biomphalaria glabrata embryonic cell line by DOTAP lipofection.