Journal Article

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

Mann, Victoria H., Caldeira, Roberta Lima, dos Santos Carvalho, Omar, Miller, André, Ittiprasert, Wannaporn, Brindley, Paul J., Karinshak, Shannon E., Coelho, Fernanda Sales, de Moraes Mourão, Marina, Rodpai, Rutchanee

2020

Overview

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 -Bg AIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF ( Bg AIF) gene. Transcript levels for Cas9 and for Bg AIF 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 Bg AIF gene in the Bge population of cells were mutated. Transcript levels for Bg AIF were reduced by up to 73% (49.5 ± 20.2% SD, P  ≤ 0.05, n  = 12). Adherence by Bg AIF gene-edited (Δ Bg AIF) 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 Δ Bg AIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, Δ Bg AIF, 2.30 ± 0.22 ( P  ≤ 0.01). Conclusions The findings supported the hypothesis that Bg AIF 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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: adhesion / Allograft inflammatory factor / allografting / Animals / Bge / Biomedical and Life Sciences / Biomedicine / Biomphalaria - cytology / Biomphalaria - genetics / Biomphalaria - parasitology / Biomphalaria glabrata / Biomphalaria glabrata embryonic cell line / Biotechnology / Calcium-Binding Proteins - genetics / Cell activation / Cell adhesion / Cell adhesion & migration / Cell Adhesion - genetics / Cell culture / Cell Line - parasitology / cell lines / Cell morphology / Cell proliferation / cell structures / Cell-mediated immunity / Cells / coculture / CRISPR / CRISPR-Cas Systems / CRISPR/Cas9 / Cytology / Cytomegalovirus / Deoxyribonucleic acid / Developmental stages / DNA / E coli / Electroporation / Embryos / Entomology / exons / Gene editing / Gene Editing - methods / Gene Knockout Techniques / gene targeting / Genetic modification / Genetic testing / Genetic transcription / Genome editing / Genomes / genomics / Hemocytes / Hemocytes - immunology / Host-Parasite Interactions / host-parasite relationships / Humans / Immunity / In vitro methods and tests / Infections / Infectious Diseases / Inflammation / intermediate hosts / Invertebrates / Larval development / Larval stage / Leukocyte migration / macrophage activation / Macrophages / Marine molluscs / Microfilament Proteins / Morphology / Nuclease / Nucleic acids / Nucleotide sequence / Parasites / Parasitology / PCR / Phagocytes / phenotype / Phenotypes / Plasmids / Proliferation / Ribonucleic acid / RNA / Schistosoma mansoni / Schistosoma mansoni - parasitology / Schistosoma mansoni - pathogenicity / Schistosomiasis / Schistosomiasis - transmission / snails / Sporocysts / Transcription / Transplantation of organs, tissues, etc / Tropical Medicine / Veterinary Medicine/Veterinary Science / Virology