Fig 1.
Biomphalysin 1 distribution in Biomphalaria glabrata tissues assessed through Western blotting.
Tissues from five BgBRE2 strain individuals were dissected, pooled and lysed. A protein dosage was performed, ensuring a homogeneous deposition of the samples on gels. SDS-PAGE were performed and biomphalysin 1 was revealed through immunoblotting. Biomphalysin 1 has an approximative size of 54 kDa (monomeric form), there is a slight difference in size between several tissues. Some tissues (hemocytes, mantle edge, posterior part of the foot, kidney -including tegument of the paleal cavity-) exhibit high molecular weight bands. aF: anterior part of the Foot, AG: Albumin Gland, H: Heart, He: Hemocytes, HP: Hepatopancreas, K: Kidney, M: Mantle edge, OVO: Ovotestis, pF: posterior part of the Foot, PL: Plasma, STO: Stomach. A: migration on a 12% gel. B: migration on a gradient 4-20% gel.
Fig 2.
Biomphalysin 1/2 distribution in Biomphalaria glabrata tissues through immunohistology.
Cuts are shown by pairs (A to F), an immunolabeled cut with anti-B1/2 antibody revealed through an Alexa 594-labeled secondary antibody (above), and a HES-stained cut permitting the identification of the immunolabeled tissues (below). Asterisks indicate a similar position between the fluorescence labeled cut and the HES stained one. The cuts were 3µm thick. A: Anterior part of the foot, under the buccal mass. B: Superior part of the buccal mass, centered on the mantle edge. C: Posterior part of the foot. D: First part of the visceral mass, rich in serous gland, salivary glands. E: Visceral mass, focused on heart sinuses area. F: Magnification on serous gland of the first part of the visceral mass. G: Magnification on D, focused on the paleal cavity. Abbreviation: AG: Albumen Gland, APO: Amoebocyte Producing Organ, BM: Buccal Mass, CM: Columellar Muscle, E: Epithelium (floor of the paleal cavity), HV: Hemolymphatic Vessel, MC: Mantle Collar, MSL: Median Sensory Lobe, NG: Neural Ganglia, OSL: Outer Secretory Lobe, PC: Paleal Cavity, PG: Pedal Gland, S-S1-S2: Sinus, SG: Serous Gland, SV: Seminal Vesicle, V: Ventricle, VD: Vas Deferens.
Fig 3.
Biomphalysin 1/2 distribution in Biomphalaria glabrata hemocytes through immunocytology.
A to F: Immunolabeling of biomphalysin 1 in hemocytes of BgBS-90 strain was performed on circulating hemocytes from the hemolymph compartment. The labeling is specific to a subpopulation of blast-like cells, unbound by phalloidin. Green: Alexa488 coupled secondary antibody (B1/2), Red: Alexa594 coupled phalloidin, Blue: 4′,6-diamidino-2-phenylindole. The same immunolabeling procedure was performed on BgBRE2 strain (shown in S4 Fig). A: General view on several hemocytes, showing an overview of the proportion of the B1/2-positive population. B, C, D, E: Shape and size comparison between B1/2-positive cells and other cell types such as hyalinocytes. F: B1/2-positive cells aren’t stained with phalloidin and some cells, close in size, can be negative for B1/2. G to J: Immunolabeling of biomphalysin 1 and TEP1 in hemocytes of BgBS-90 strain. Immunolabeling of hemocytes by TEP1 and biomphalysin 1 antibodies shows a co-location in the same blast-like cells. Green: Alexa488 coupled secondary antibody (TEP1), Red: Alexa594 coupled secondary antibody (B1/2), Blue: 4′,6-diamidino-2-phenylindole. G: B1 labelling (same observation field than H). H: TEP1 labelling (same observation field than G). I and J: Magnification of B1/2/TEP1 positive cells.
Fig 4.
Lightsheet and confocal visualizations of biomphalysin 1/2 immunolabeling on tissue-cleared Biomphalaria glabrata snails.
Tissue clearing was performed using X-CLARITY system, after fixation snails are incubated in acryl solution and optically cleared through electrophoretic delipidation using SDS. Propidium iodide (red) allows to locate snail tissues by DNA staining around B1/2 labeling (green). A: Z-stack of the head-foot region of a tissue-cleared snail (ventral view using a lightsheet microscope) scale bar = 300µm, B: Z-stack facing the pedal sole of the foot, C: 90-degree horizontal rotation of stack B, highlighting the dorsal outlet of the pear-shaped structures labeled in the foot, D: Mantel edge of the snail, exhibiting similar biomphalysin 1/2-labeled structures. B, C and D pictures were taken using a confocal microscope. Red: propidium iodide, green: immunolabeling of B1/2. A short video showing the rotation between picture B and C is provided in S5 Fig.
Fig 5.
Biomphalysin genes expression in response to S. mansoni intrusion.
qRTPCR was performed on whole snail organisms exposed to S. mansoni either in the case of a compatible (BgBRE2 snail and SmBRE parasite strains) or an incompatible interaction (BgBRE2 snail and SmGH2 parasite strains). Expression was measured at six time points (3, 6, 12, 24, 48 and 96 hours) and was normalized to S19 housekeeping gene expression and compared with the expression obtained in non-exposed snails. Asterisks indicate a significant difference between non-exposed and exposed snails (p < 0.05). RQ: Relative Quantification (2-ΔΔCT).
Fig 6.
Biomphalysin 1/2 binding to several cell types.
A, B, C, D, E, F and G: Confocal observation of biomphalysin 1 binding to S. mansoni sporocyst (A to C) and HeLa cells (D to F). Z-stacks are shown here for sporocysts and one focal plane for HeLa cells. Unexposed sporocysts were used as control, maintained in CBSS (A). Sporocysts were incubated with plasma for one hour, washed with CBSS and immunolabeled using anti-B1/2 antibody (B and C). Scale bars are 20µm. Labeling is only present at the surface of the sporocysts. HeLa cells were exposed either to CBSS (D), or undiluted ultracentrifuged plasma (E and F) for 1 hour. HeLa cells are not lysed following CBSS exposure. Green: Alexa488-coupled secondary antibody (B1/2), Blue: 4′,6-diamidino-2-phenylindole. Sale bars are 20 µm. White arrowheads show some of the multiple vesicles from different sizes visible on and around the lysed HeLa cells. Those vesicles range in diameter from about 1 µm for the smallest to over 10 µm for the largest. Cells or microorganisms were incubated with ultracentrifuged plasma for two hours (plasma final dilution of 1/2), washed and mixed in reducing Laemmli buffer for gel migration (G). For each cell type, one well is dedicated to the plasma-exposed condition (e.g., HPL) and one well to an unexposed condition (e.g., H). C: C2C12 cells, Fh: Fasciola hepatica miracidia, H: HeLa cells, H2O: Water in which F. hepatica eggs hatched (control), J: BCL2 Jurkat cells, Pl: Plasma, Sm: Schistosoma mansoni mother sporocysts (48h transformation).
Fig 7.
New insights related to predicted structure of biomphalysin 1, based on alphafold2 aminoacyl pair representation and mass spectrometry data support.
A: Abundance of biomphalysin 1 individual amino acids, permitted by peptide coverage and determined through mass analysis of plasma compartment. Biomphalysin 1 reference sequence provided by NCBI, NP_001298219. Identified peptides for biomphalysin 1 and 2 following mass spectrometry analysis were aligned on the same sequence of biomphalysin 1. Identified amino acids are colored from white to dark orange depending on their relative representation among identified peptides. Unidentified amino acids are colored in gray (except for the signal peptide colored in yellow). B: Biomphalysin monomer structure prediction (left) and comparison to proaerolysin crystal structure (right). PDB accession number: 3C0N. By analogy with aerolysin structure: domain I (red), domain II (blue), domain III (purple), domain IV (cyan), transmembrane domain containing the insertion loop (yellow) and unfolded Cter residues (black) were defined. Small lobe = domain I (red), large lobe = domains II, III and IV. C: Ribbon representation of biomphalysin 1 domain I (including the Nter residues). Each face of the β-prism is colored in a different color. Cysteines are highlighted in orange. There is an accordance between the non-organized Cter sequence shape in structure prediction and the lack of detection of the Cter sequence shown in A. D: Topological representation of biomphalysin 1 domain I. β-strands are depicted with arrows and α-helices with cylinders. The boundaries of each secondary structure element are indicated, as well as the positions of cysteine residues, depicted with orange circles. The six disulfide bridges are indicated with capitalized identical bold letters. The repeated structural patterns are framed in cyan. E: Position and distance between close cysteines within domain I (small lobe) and domain II.