Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor

Background: Larval development in an intermediate host gastropod snail of the genusBiomphalariais an obligatory component of the life-cycle ofSchistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, fromBiomphalaria glabratais a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro. Methods: Bge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of theB. glabrataAIF (BgAIF) gene. Transcript levels for Cas9 and forBgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. Results: 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 the copies of theBgAIF gene in the Bge population of cells were mutated. Transcript levels forBgAIF were reduced by up to 73% (49.5 +/- 20.2% SD,P <= 0.05,n = 12). Adherence byBgAIF gene-edited (Delta BgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer Delta BgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 +/- 0.10, Delta BgAIF, 2.30 +/- 0.22 (P <= 0.01). Conclusions: The findings supported the hypothesis thatBgAIF plays a role in the adherence ofB. glabratahemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.