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

Parasites & Vectors

Rutchanee Rodpai¹  Wannaporn Ittiprasert²  Paul J. Brindley²  Shannon E. Karinshak²  Victoria H. Mann²  Roberta Lima Caldeira³  Marina de Moraes Mourão³  Omar dos Santos Carvalho³  Fernanda Sales Coelho³  André Miller⁴ 

[1] Department of Microbiology, Immunology and Tropical Medicine, School of Medicine and Health Sciences, George Washington University, Washington, D.C., USA;Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen province, Thailand;Department of Microbiology, Immunology and Tropical Medicine, School of Medicine and Health Sciences, George Washington University, Washington, D.C., USA;Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, George Washington University, Washington, D.C., USA;Grupo de Pesquisa Em Helmintologia E Malacologia Médica, Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil;Schistosomiasis Resource Center, Biomedical Research Institute, Rockville, MD, USA;

关键词: Biomphalaria glabrata;    Bge;    CRISPR/Cas9;    Gene editing;    Allograft inflammatory factor;    Cell adhesion;   

DOI  :  10.1186/s13071-020-04384-9

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY