A Novel Toll-Like Receptor (TLR) Influences Compatibility between the Gastropod Biomphalaria glabrata, and the Digenean Trematode Schistosoma mansoni.

Schistosomiasis, a devastating disease caused by parasitic flatworms of the genus Schistosoma, affects over 260 million people worldwide especially in tropical and sub-tropical regions. Schistosomes must undergo their larval development within specific species of snail intermediate hosts, a trait that is shared among almost all digenean trematodes. This unique and long-standing host-parasite relationship presents an opportunity to study both the importance of conserved immunological features in novel immunological roles, as well as new immunological adaptations that have arisen to combat a very specific type of immunological challenge. While it is well supported that the snail immune response is important for protecting against schistosome infection, very few specific snail immune factors have been identified and even fewer have been functionally characterized. Here, we provide the first functional report of a snail Toll-like receptor, which we demonstrate as playing an important role in the cellular immune response of the snail Biomphalaria glabrata following challenge with Schistosoma mansoni. This TLR (BgTLR) was identified as part of a peptide screen of snail immune cell surface proteins that differed in abundance between B. glabrata snails that differ in their compatibility phenotype to challenge by S. mansoni. The S. mansoni-resistant strain of B. glabrata (BS-90) displayed higher levels of BgTLR compared to the susceptible (M-line) strain. Transcript expression of BgTLR was found to be very responsive in BS-90 snails when challenged with S. mansoni, increasing 27 fold relative to β-actin (non-immune control gene); whereas expression in susceptible M-line snails was not significantly increased. Knockdown of BgTLR in BS-90 snails via targeted siRNA oligonucleotides was confirmed using a specific anti-BgTLR antibody and resulted in a significant alteration of the resistant phenotype, yielding patent infections in 43% of the normally resistant snails, which shed S. mansoni cercariae 1-week before the susceptible controls. Our results represent the first functional characterization of a gastropod TLR, and demonstrate that BgTLR is an important snail immune receptor that is capable of influencing infection outcome following S. mansoni challenge

siRNA-mediated knockdown of BgTLR transcripts.

<p>(A) Graphic view of BgTLR (not drawn to scale) showing the leucine-repeat motifs and Toll/IL-1 domain. Red bars indicate the approximate positions targeted by siRNA oligonucleotides used to inject the snails, while the blue bar indicates the targeted position of the antibody used. (B) Confirmation of BgTLR (~135 kDa) knockdown in BS-90 haemocytes and Bge cells respectively. Protein was detected via Western blot with primary antibody developed against the extracellular region of BgTLR. BgActin (~42 kDa) served as the loading control.</p

BgTLR displayed increased transcript expression in BS-90 snails following challenge with <i>S</i>. <i>mansoni</i>.

<p>(A) BgTLR transcript expression with and without <i>S</i>. <i>mansoni</i> challenge. Snails (BS-90 and M-line strains) were individually exposed to ~5 miracidia or left unexposed (control). Five snails were collected at indicated time points over the incubation period of the parasite. RNA was extracted from whole snails, converted to cDNA and BgTLR expression was measured by quantitative PCR. Expression was quantified in fold changes normalized to time 0-hour controls. Bars represent standard error (n = 5). Asterisk (*) indicates significant difference (P < 0.05) between experimental and control samples, while hash (#) indicates significant difference between BS-90 and M-line snails at the respective time points. (B) Knockdown of BgTLR or GFP (control) in BS-90 snails responding to <i>S</i>. <i>mansoni</i> parasite challenge. (C) Knockdown of BgTLR in unexposed M-line and BS-90 snails. Five snails were collected for each time point in B and C for RNA extraction and cDNA synthesis. Bars represent standard error (n = 5). Asterisks indicate significant difference (P < 0.05) from 0-hour time points.</p

siRNA oligonucleotide sequences.

<p>siRNA oligonucleotide sequences.</p

Knockdown of BgTLR in BS-90 snails decreases haemocyte phagocytic response.

<p>(A) Mean number of phagocytosed beads per haemocyte. Five BS-90 snails each were injected with siRNA targeting BgTLR or GFP (control) and 96 hours later, haemolymph was extracted from the snails and immediately mixed with ~1 x 10⁶ 1μM FITC-labelled streptavidin-coated beads pre-incubated with biotinylated <i>S</i>. <i>mansoni</i> excretory/secretory products and sporocyst. After 3 hours, haemocytes from each snail were counted from a random field of view on the slide, and 30 haemocytes for each snail were assessed for the number of beads within each cell from which mean number of beads per haemocyte was calculated. Asterisk (*) indicates significant reduction (P < 0.05) in the mean number of beads per haemocyte in BgTLR knockdown snails. (B) Frequency of number of beads observed in 30 haemocytes from one field of view (FOV). Asterisk (*) indicates significant difference (P < 0.05) in the average number of beads phagocytosed between BgTLR and GFP knockdown.</p

BgTLR immunolocalization in haemocytes and Bge cells.

<p>Haemocytes (A-C) and Bge cells (D-E) were labelled with DAPI and anti-BgTLR primary antibody. Control haemocyte (C) and Bge (E) samples were stained with DAPI but the primary antibody step omitted. Scale bars represent 20 μM.</p

Primers used in this study.

<p>Primers used in this study.</p