A Somatically Diversified Defense Factor, FREP3, Is a Determinant of Snail Resistance to Schistosome Infection

Schistosomiasis, a neglected tropical disease, owes its continued success to freshwater snails that support production of prolific numbers of human-infective cercariae. Encounters between schistosomes and snails do not always result in the snail becoming infected, in part because snails can mount immune responses that prevent schistosome development. Fibrinogen-related protein 3 (FREP3) has been previously associated with snail defense against digenetic trematode infection. It is a member of a large family of immune molecules with a unique structure consisting of one or two immunoglobulin superfamily domains connected to a fibrinogen domain; to date fibrinogen containing proteins with this arrangement are found only in gastropod molluscs. Furthermore, specific gastropod FREPs have been shown to undergo somatic diversification. Here we demonstrate that siRNA mediated knockdown of FREP3 results in a phenotypic loss of resistance to Schistosoma mansoni infection in 15 of 70 (21.4%) snails of the resistant BS-90 strain of Biomphalaria glabrata. In contrast, none of the 64 control BS-90 snails receiving a GFP siRNA construct and then exposed to S. mansoni became infected. Furthermore, resistance to S. mansoni was overcome in 22 of 48 snails (46%) by pre-exposure to another digenetic trematode, Echinostoma paraensei. Loss of resistance in this case was shown by microarray analysis to be associated with strong down-regulation of FREP3, and other candidate immune molecules. Although many factors are certainly involved in snail defense from trematode infection, this study identifies for the first time the involvement of a specific snail gene, FREP3, in the phenotype of resistance to the medically important parasite, S. mansoni. The results have implications for revealing the underlying mechanisms involved in dictating the range of snail strains used by S. mansoni, and, more generally, for better understanding the phenomena of host specificity and host switching. It also highlights the role of a diversified invertebrate immune molecule in defense against a human pathogen. It suggests new lines of investigation for understanding how susceptibility of snails in areas endemic for S. mansoni could be manipulated and diminished

Histological sections of BS-90 snails made susceptible to <i>S. mansoni</i> infection by previous infection with irradiation-attenuated <i>E. paraensei</i>.

<p>A. Control snail not exposed to <i>S. mansoni</i> infection showing normal architecture of digestive gland. B–D. BS-90 snails with disseminated <i>S. mansoni</i> infections (arrows) following exposure to irradiated <i>E. paraensei</i>, including in the digestive gland. E. Exposure to irradiated <i>E. paraensei</i> did not result in disseminated infection or shedding of <i>E. paraensei</i> cercariae, however degenerating irradiated <i>E. paraensei</i> sporocysts were observed in the heart, as expected. Two degenerating sporocysts (arrows) of <i>E. paraensei</i> in the heart of a sensitized snail (28 dpe to <i>S. mansoni</i>). Scale bar = 10 um.</p

Knockdown of FREP3 reduces resistance to <i>S. mansoni</i> infection in BS-90 strain <i>B. glabrata</i>.

<p>A) RNAi knockdown of FREP3 in BS-90 <i>B. glabrata</i> snails confirmed at the transcriptional level by RT-PCR (25 cycles) over the course of 96 hours post injection. Shown are representative results from injection of FREP3-specific siRNA oligos and control GFP-specific siRNA oligos on FREP3 transcript expression. Experimental values are compared to the endogenous control elongation factor 1-α (EF-1α). B) Confirmation of protein-level knockdown of FREP3 in 4 individual BS-90 snails (A–D) before, and 4 days after injection of FREP3-specific siRNA oligos. FREP3 was visualized using a specific anti-FREP3 antibody. As a control for protein loading 100 µg of cell-free plasma was loaded into each well, and the same samples were probed for FREP4, using an anti-FREP4 antibody. FREP4 is a different FREP family member, related to FREP3, and was detectable in all individuals both before and after FREP3 knockdown. C) Percentage of BS-90 snails shedding <i>S. mansoni</i> cercariae (21%) after knockdown of FREP3, as compared to controls which were either susceptible M-line snails exposed to <i>S. mansoni</i> (85%) or BS-90 snails injected with the GFP siRNA constructs and challenged with <i>S. mansoni</i> (0%).</p

A. Total number of transcripts exhibiting increased (above zero line) or decreased (below zero line) expression in BS-90 snails immunocompromised by irradiated <i>E. paraensei</i> before challenge with <i>S. mansoni</i> (see key for bar colors on figure).

<p>Analysis compared experimental snails at 2 or 4 days post <i>S. mansoni</i> challenge to time and size matched control BS-90 snails exposed to irradiated <i>E. paraensei</i> only. Of the 6 individual snails analyzed at each time point, 3 were successfully infected with <i>S. mansoni</i>, and 3 remained resistant. B) Expression profiles of transcripts deemed important to <i>S. mansoni</i> resistance in snails. Fold-changes in expression of snails suppressed by irradiated <i>E. paraensei</i> before <i>S. mansoni</i> challenge are compared to snails exposed only to irradiated <i>E. paraensei</i>. Bars represent standard error (n = 3).</p

Percentage of BS-90 snails shedding <i>S. mansoni</i> cercariae after attenuation with irradiated <i>E. paraensei</i> miracidia.

<p>Experimental snails (46% infection rate) were compared to <i>S. mansoni</i>-susceptible M-line <i>B. glabrata</i> (82% infected), and to BS-90 snails challenged with <i>S. mansoni</i> only (0% infected).</p