Allelic variation partially regulates galactose-dependent hydrogen peroxide release from circulating hemocytes of the snail Biomphalaria glabrata

Abstract

Freshwater snails are the intermediate hosts for numerous parasitic worms that are detrimental to human and agricultural health. Understanding the immune responses of these snails could be vital for finding ways to block transmission of those parasites. Allelic variation in a recently discovered genomic region in the snail, Biomphalaria glabrata, influences their susceptibility to schistosomes. Here we tested whether genes in that region, termed the Guadeloupe Resistance Complex (GRC), are involved in recognition of common pathogen-associated molecules that have been shown to be stimulants of the hydrogen peroxide defense pathway. We show that hemocytes extracted from individuals with one of the three GRC genotypes released less hydrogen peroxide than the other two genotypes, after stimulation with galactose. This difference was not observed after stimulation with several other microbial-associated carbohydrates, despite those ligands sharing the same putative pathway for hydrogen peroxide release. Therefore, we conclude that allelic variation in the GRC region may influence the recognition of galactose, rather than the conserved downstream steps in the hydrogen peroxide pathway. These results thus are consistent with the hypothesis that proteins produced by this region are involved in pathogen recognition.

Keywords: Biomphalaria glabrata; Carbohydrate; Hemocyte; Hydrogen peroxide; Innate immunity.

Fig 1. A simplified diagram outlining the known canonical hydrogen peroxide pathway in B. glabrata

Both PMA, and ligands that require a receptor on the cell surface, activate intracellular kinases before NOX2 is recruited and activated. NOX2 produces superoxide (O₂⁻), the substrate for SOD, which is then converted to hydrogen peroxide. Hydrogen peroxide acts as the final anti-microbial product in this pathway in hemocytes. The kinases and receptors which lead to the activation of NOX2 are not conclusively characterized in B. glabrata, though the downstream NOX2-SOD pathway is believed to be conserved with that of mammals. The receptor initiating the hemocyte response is likely to be the primary difference between stimulation with the ligands used in this study (other than PMA).

Fig 2. BgGUA hemocytes produce hydrogen peroxide in response to PMA, LPS, and microbial-associated BSA-conjugated sugars

(A) Stimulation of BgGUA hemocytes with PMA (500 nM), PMA (500 nM) + DPI (0.5 μM), or CBSS carrier alone (n=4). (B) A dose response of BgGUA hemocytes to sugars commonly found on microbes, and microbial products isolated directly from microbes. Doses 0–4 correspond to: LPS 0, 10, 50, 100, 150 μg/ml; BSA-sugars 0, 50, 100, 200, 300 nM (n=1 per treatment). All data are shown as RFU/5000 cells relative to the PMA + control. Data in (A) are presented as mean +/− SD relative to PMA stimulation. Significant differences (ANOVA p<0.05) in (A) are denoted by an asterisks (*).

Fig 3. Allelic variation at the GRC locus does not modify hydrogen peroxide stimulation induced by PMA or LPS

Stimulation of RR, S1S1, or S2S2 BgGUA hemocytes with (A) PMA (500 nM), or (B) LPS (100 μg/ml). All data are shown as mean +/− SD of RFU/5000 cells. PMA data (A) are presented relative to the S1S1 genotype (n=4). Data in (B) are normalized to independent PMA + controls (500 nM) within an individual genotype and presented relative to PMA (n=4). Significant differences between genotypes (ANOVA p<0.05) are denoted by an asterisks (*).

Fig 4. S2S2 hemocytes release less hydrogen peroxide in response to BSA-galactose, but not in response to other common microbial carbohydrates

Stimulation of RR, S1S1, or S2S2 BgGUA hemocytes with (A) BSA-mannose (200 nM), (B) BSA-fructose (200 nM), (C) BSA-arabanose (200 nM), (D) BSA-sialic acid (300 nM), (E) BSA-galactose (300 nM) (n=4). (F) Additionally, three individual families each of RR, S1S1, or S2S2 were independently tested with BSA-galactose (300 nM) (n=4–5). All experiments contained internal PMA + controls (500 nM) within an individual genotype or line. All data are shown as mean +/− SD of RFU/5000 cells, normalized to PMA + controls within an individual genotype, and presented relative to PMA stimulation. Significant differences (ANOVA p<0.05) are denoted by an asterisks (*).