Abstract
BackgroundLarval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma 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, from Biomphalaria glabrata is 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.MethodsBge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed.ResultsGene 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 the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5 ± 20.2% SD, P ≤ 0.05, n = 12). Adherence by BgAIF gene-edited (ΔBgAIF) 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 ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01).ConclusionsThe findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes 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.
Highlights
Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni
We hypothesized that B. glabrata AIF (BgAIF) was involved in cell mediated immune response(s) by B. glabrata through activation of hemocyte cell adhesion and/or migration after the schistosome miracidium has penetrated into the tissues of the snail. We addressed this hypothesis by using Clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based programmed genome editing to interrupt the BgAIF gene of B. glabrata in the Biomphalaria glabrata embryonic cell line (Bge) cell line, following reports that indicated the utility of using CRISPR-based programmed gene knockout approach in other mollusks including the Pacific oyster, Crassostrea gigas and the slipper limpet, Crepidula fornicata and the gastropod, Lymnaea stagnalis [46,47,48]
Sequential isolation of total Ribonucleic acid (RNA) and genomic Deoxyribonucleic acid (DNA) To monitor the transfection of Bge cell by pCas9BgAIFx4, we investigated the expression of Cas9 in Bge cells by reverse transcription Polymerase chain reaction (PCR) (RT-PCR)
Summary
Larval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life-cycle of Schistosoma mansoni. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocytelike phenotype. Several species of the freshwater snail genus Biomphalaria are the intermediate host for Schistosoma mansoni. The Neotropical species, Biomphalaria glabrata has been studied extensively with respect to host-parasite relationship and coevolution with S. mansoni, especially on mechanisms of susceptibility and/or resistance to the compatible parasites [1, 2]. The resistance phenotype is underpinned by a complex genetic trait, where the schistosome larva fails to develop as the consequence of innate and cellular immune responses. Hemocytes of resistant snails encapsulate and destroy the sporocyst [11,12,13,14,15,16,17,18]
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