Schistosome infectivity in the snail, Biomphalaria glabrata, is partially dependent on the expression of Grctm6, a Guadeloupe Resistance Complex protein

Abstract

Schistosomiasis is one of the most important neglected tropical diseases. Despite effective chemotherapeutic treatments, this disease continues to afflict hundreds of millions of people. Understanding the natural intermediate snail hosts of schistosome parasites is vital to the suppression of this disease. A recently identified genomic region in Caribbean Biomphalaria glabrata snails strongly influences their resistance to infection by Schistosoma mansoni. This region contains novel genes having structural similarity to known pathogen recognition proteins. Here we elaborate on the probable structure and role of one of these genes, grctm6. We characterised the expression of Grctm6 in a population of Caribbean snails, and performed a siRNA knockdown of Grctm6. We show that this protein is not only expressed in B. glabrata hemolymph, but that it also has a role in modulating the number of S. mansoni cercariae released by infected snails, making it a possible target for the biological control of schistosomiasis.

Fig 1. grctm6 encodes a single-pass transmembrane protein with an extraordinarily variable extracellular domain.

Alignment of the protein products of the three alleles of grctm6 found in BgGUA. Residues identical to those in the R allele are indicated by dots. The putative signal peptide is shown with a dark blue outlined box. The putative transmembrane domain is shown with an orange box, while the extracellular and intracellular domains are flanked by green and purple bars, respectively. Highly variable regions, defined by 20aa windows containing at least 10 sequence differences, have a yellow background. Cysteines are shown in red. Asparagines predicted to be glycosylated are shown in blue/green.

Fig 2. Susceptibility of BgGUA lines is not explained by grctm6 mRNA levels.

(A) The susceptibility of the three different homozygous BgGUA genotypes (S1S1, S2S2, RR) after challenge with 20 miracidia (n = 64, 68, 86 snails respectively). Susceptibility data are presented as the proportion of infected snails +/- standard error of proportions. (B) Constitutive grctm6 mRNA levels (whole snail) of these same homozygous lines (n = 17, 21, 17 snails respectively). Note that the susceptibility differences among the three genotypes does not correlate with expression levels of grctm6. mRNA levels are normalized to β-actin and presented as mean +/- SD relative to levels in S2S2. Significant differences among lines (Susceptibility: Z score of proportion; mRNA levels: ln transformed for equal variance, ANOVA, p<0.05) are denoted by asterisks (*).

Fig 3. grctm6 mRNA expression and protein detection.

(A) Constitutive grctm6 mRNA levels in resistant whole snail (WS), head-foot (HF), albumen gland (ABG), and hemolymph (HL) lysates (n = 4 samples). HL has significantly elevated levels compared to HF only. (B) Western blot analysis of constitutive Grctm6 protein levels in resistant whole snail after hemolymph was removed (WS- HL), head-foot (HF), albumen gland (ABG), hemolymph with cells removed (HL—cells), whole hemolymph (HL), hemocyte lysates, sample buffer containing no snail tissue, negative control peptide, and Grctm6 peptide (provided by genscript). Equivalent total protein was loaded into each well for experimental samples (500 ng/sample). The band shown is at the ~68 kDa size for Grctm6 (~42 for BgActin loading control), Grctm6 peptides appear lower down on the gel as they are not the full length protein. HL- cells shows lower levels of BgActin protein because most of the actin producing cells were removed by centrifugation. Additionally, hemolymph samples with differing total protein concentrations are shown below. mRNA levels are normalized to β-actin and presented as mean +/- SD relative to WS samples. Significant differences (ln transformed for equal variance, ANOVA, p<0.05) are denoted by asterisks (*).

Fig 4. siRNA mediated knockdown of Grctm6.

(A) Mortality of resistant BgGUA snails injected with siRNA taken on 4 separate days post injection (n = 3–6 independent experiments per day). (B) The kinetics of siRNA mediated knockdown of grctm6 mRNA in whole resistant snails after injection compared to GFP siRNA oligos during the first 4 days post-injection (n = 8–10 snails/treatment/day). A reduction of 60% below control was evident by day 3. (C) Western blot analysis of Grctm6 protein levels in resistant hemolymph 2–4 days after injecting snails with grctm6 or GFP siRNA. Equivalent total protein was loaded into each well (750 ng). The band shown in these representative blots is at the ~68 kDa size (~42 for BgActin loading control). Densitometry was preformed using Image J software (Days 2 and 4: n = 3 samples/treatment/day, n = hemolymph pooled from 4–5 individuals; Day 3: n = 5 samples/treatment, n = hemolymph pooled from 6–10 individuals, averaged from three independent Western blots and presented relative to the BgActin loading control for each individual day). A ~30% reduction, below control relative to BgActin, was found in samples taken on Day 3. mRNA levels are normalized to β-actin and relative to GFP oligo injected individuals taken on each individual day (no comparison between days was made). All data are presented as mean +/- SD; significant differences (student t-test, p<0.05) from the GFP control are denoted by asterisks (*).

Fig 5. siRNA knockdown of Grctm6 increases the number of cercariae released by shedding snails but does not modify susceptibility.

Resistant BgGUA snails were treated with GFP or grctm6 oligos and exposed to 10 or 20 miracidia 3 days after injection of oligos. (A-B) Susceptibility over weeks 5–10 post challenge with (A) 10 (n = 65 GFP, 63 snails grctm6) or (B) 20 miracidia (n = 55 GFP, 37 grctm6 snails). (C-D) The total number of cercariae released over a 15 h period (a 3 h period, every week, from weeks 5–10 post exposure) by shedding snails exposed to (C) 10 (n = 6 GFP, 5 grctm6 snails) or (D) 20 miracidia (n = 12 GFP, 11 grctm6 snails). Susceptibility data are presented as the proportion of infected snails +/- standard error of proportions. Cercariae counts are presented as mean +/- SD. Significant differences (Susceptibility: Z score of proportion; cercariae counts: ln transformed for equal variance, student t-test, p<0.05) from the GFP control are denoted by asterisks (*). Note the different scales on the Y-axes in figures for 10-miracidial versus 20-miracidial challenges.