Diminished Adherence of Biomphalaria Glabrata Embryonic Cell Line to Sporocysts of Schistosoma Mansoni Following Programmed Knockout of the Allograft Inflammatory Factor

Abstract

The development of the larval stage of Schistosoma mansoni in an intermediate host snail of the genus Biomphalaria is an obligatory component of the life cycle. Enhanced understanding of the mechanism(s) of host defense in the snail may hasten the development of tools that block transmission of schistosomiasis. The B. glabrata embryonic cell line, termed the Bge line, is a versatile resource for investigation of the snail-schistosome relationship. A key attribute of the Bge cell is the hemocyte-like phenotype, given the central role of the snail hemocyte in innate and cellular immunity. The allograft inflammatory factor 1, AIF, is evolutionarily conserved, typically is expressed in phagocytes and granular leukocytes, is a marker of macrophage activation in mammals and in invertebrates, and enhances cell proliferation and migration. AIF is highly expressed in strains of B. glabrata resistant S. mansoni infection in comparison with susceptible strains of the snail. We hypothesized that BgAIF may play a role in hemocyte proliferation, adhesion and/or migration after exposure of the snail to schistosomes. CRISPR/Cas gene knockout of the BgAIF gene in Bge cells was undertaken to investigate the hypothesis. Gene knockout manipulation induced gene-disrupting indels, frequently 1–2 bp insertions and/or 8–30 bp deletions, at the programmed target site; a range from 9 to 17% of BgAIF genes were mutated during 12 replicate experiments, and transcript levels for BgAIF were significantly reduced by up to 73% (range, 26 to 73%, mean 49.5 ± 20.2% S.D, n = 12) when monitored for up to nine days following the gene-editing manipulation. The adherence to sporocyst of BgAIF gene-edited (ΔBgAIF) Bge cells was significantly diminished in comparison to wild type cells, even though cell morphology did not differ between ΔBgAIF treatment and control groups of Bge cells. A Bge cell adherence index (CAI) to individual sporocysts was observed at 2.66 ± 0.10 for control and 2.30 ± 0.22 in ΔBgAIF cells (P < 0.05), revealing that ΔBgAIF cells were significantly less adherent than wild type Bge cells to primary sporocysts. The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection.