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

Abstract

Background Larval 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. Methods Bge 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. 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 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). Conclusions The 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.

PIWI silencing mechanism involving the retrotransposon nimbus orchestrates resistance to infection with Schistosoma mansoni in the snail vector, Biomphalaria glabrata

“…Toward this objective, molecular toolkits for gene editing in B. glabrata are being developed. CRISPR gene editing has been used to edit the allograft inflammatory factor gene in the Bge embryonic cell line from B. glabrata [53] and is finding utility in editing the schistosome genome [54,55]. Confirming earlier findings, we show that in intact juvenile resistant and susceptible snails, within a short period post-exposure to S. mansoni, the non-random movement of the PIWI locus within interphase nuclei in relation to its active transcription depending on the susceptibility phenotype of the snail [19].…”

Hit-and-Run Epigenetic Editing for Vectors of Snail-Borne Parasitic Diseases

“…Three gRNAs targeting DNA methylation canyon on the target locus: LGUN_random_Scaffold680: 40871-42922 on B. glabrata gene database ( https://legacy.www.vectorbase.org ) were elaborated by a software for designing CRISPR/Cas9 guide RNAs called CHOPCHOP ( Montague et al, 2014 ). The off-target was also predicted against B. glabrata genome by CHOPCHOP as previously described ( Coelho et al, 2020 ) and following considerations for dCas9-DNMT3 system described in ( Brezgin et al, 2019 ). The three sgRNAs were designed in the CUFF52452.…”

“…were elaborated by a software for designing CRISPR/Cas9 guide RNAs called CHOPCHOP (Montague et al, 2014). The off-target was also predicted against B. glabrata genome by CHOPCHOP as previously described (Coelho et al, 2020) and following considerations for dCas9-DNMT3 system described in (Brezgin et al, 2019). The three sgRNAs were designed in the CUFF52452.…”

Exploring the immune interactions between Oncomelania hupensis and Schistosoma japonicum, with a cross-comparison of immunological research progress in other intermediate host snails

“…Given the important role of haemocytes in the immune response against S. mansoni , it is crucial to study their proliferation and differentiation mechanisms. The known growth factor is granulocyte protein [ 27 ], and AIF is not only involved in the proliferation of haemocytes but also related to cell migration and encapsulation [ 52 ]. Of course, before regulating and controlling the signal pathway of immune response activation, immune recognition receptors involved in pathogen recognition include PRRs, integrin-related proteins, and growth factor/cytokine-like receptors.…”