Allograft Inflammatory Factor 1 as an Immunohistochemical Marker for Macrophages in Multiple Tissues and Laboratory Animal Species

et al. 2020

Preprint

The development of the larval stage of Schistosoma mansoni in an intermediate host snail of the genus Biomphalaria is an obligatory component of the life cycle. Enhanced understanding of the mechanism(s) of host defense in the snail may hasten the development of tools that block transmission of schistosomiasis. The B. glabrata embryonic cell line, termed the Bge line, is a versatile resource for investigation of the snail-schistosome relationship. A key attribute of the Bge cell is the hemocyte-like phenotype, given the central role of the snail hemocyte in innate and cellular immunity. The allograft inflammatory factor 1, AIF, is evolutionarily conserved, typically is expressed in phagocytes and granular leukocytes, is a marker of macrophage activation in mammals and in invertebrates, and enhances cell proliferation and migration. AIF is highly expressed in strains of B. glabrata resistant S. mansoni infection in comparison with susceptible strains of the snail. We hypothesized that BgAIF may play a role in hemocyte proliferation, adhesion and/or migration after exposure of the snail to schistosomes. CRISPR/Cas gene knockout of the BgAIF gene in Bge cells was undertaken to investigate the hypothesis. 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 BgAIF genes were mutated during 12 replicate experiments, and transcript levels for BgAIF were significantly reduced by up to 73% (range, 26 to 73%, mean 49.5 ± 20.2% S.D, n = 12) when monitored for up to nine days following the gene-editing manipulation. The adherence to sporocyst of BgAIF gene-edited (ΔBgAIF) Bge cells was significantly diminished in comparison to wild type cells, even though cell morphology did not differ between ΔBgAIF treatment and control groups of Bge cells. A Bge cell adherence index (CAI) to individual sporocysts was observed at 2.66 ± 0.10 for control and 2.30 ± 0.22 in ΔBgAIF cells (P < 0.05), revealing that ΔBgAIF cells were significantly less adherent than wild type Bge cells to primary sporocysts. The findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection.

Section: Discussionmentioning

confidence: 99%

Diminished Adherence of Biomphalaria Glabrata Embryonic Cell Line to Sporocysts of Schistosoma Mansoni Following Programmed Knockout of the Allograft Inflammatory Factor

Coelho

et al. 2020

Preprint

“…However, even though Bge cells adhere to the schistosome, the parasite is not killed by these cells in vitro. The allograft in ammatory factor 1 (AIF) is a conserved calcium-binding protein typically expressed in phagocytic and granular leukocytes and is a marker of macrophage activation (38,41,45,(62)(63)(64)(65). An orthologue, termed BgAIF, is highly expressed in isolates of B. glabrata that are resistant to infection with S. mansoni and this gene may be linked to hemocyte activation (8,9).…”

Section: Discussionmentioning

confidence: 99%

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

Coelho¹,

et al. 2020

Preprint

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 quantitative 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% S.D, 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).Conclusion: 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.

“…Last, morphological changes were not apparent between the However, even though Bge cells adhere to the schistosome, the parasite is not killed by these cells in vitro. The allograft inflammatory factor 1 (AIF) is a conserved calcium-binding protein typically expressed in phagocytic and granular leukocytes and is a marker of macrophage activation (38,41,45,(62)(63)(64)(65). An orthologue, termed BgAIF, is highly expressed in isolates of B. glabrata that are resistant to infection with S. mansoni and this gene may be linked to hemocyte activation (8,9).…”

Section: Programmed Mutation Of Bgaif Confirmed Functional Crispr/casmentioning

confidence: 99%

Diminished adherence ofBiomphalaria glabrataembryonic cell line to sporocysts ofSchistosoma mansonifollowing programmed knockout of the allograft inflammatory factor

Coelho

et al. 2020

Preprint

Schistosomiasis mansoni is a debilitating neglected tropical disease caused by infection with the blood fluke, Schistosoma mansoni. Development of the larval stage of the parasite in an intermediate host snail of the genus Biomphalaria is an obligatory component of the life cycle. Enhanced understanding of the mechanism(s) of host defense in the intermediate host snail may hasten the development of new tools that block transmission of schistosomiasis. The embryonic cell line from B. glabrata, termed the Bge line, is a versatile resource in the investigation of snail-schistosome interactions. A key attribute of the Bge cell is its hemocyte-like phenotype, given the central role of the snail hemocyte in innate and cellular immunity. The allograft inflammatory factor 1 (AIF, aif) is a conserved antigen typically expressed in phagocytic and granular leukocytes and is a marker of macrophage activation. AIF is highly expressed in isolates of B. glabrata that are resistant to infection with S. mansoni. We targeted the aif gene of B. glabrata (BgAIF) for programmed gene knockout using the CRISPR/Cas9 approach, to investigate the activity of CRISPR/Cas9 gene editing in this gastropod species and, in turn, to investigate the loss of AIF on Bge cell-mediated adherence to schistosome sporocysts. Bge cells were transformed with gene editing plasmid encoding the Cas9 nuclease driven by the CMV promoter and a guide RNA specific for exon 4 of the BgAIF by square wave electroporation. Twelve independent transformations were carried out. Transcript levels of BgAIF were significantly reduced by up to 73% (range, 26 to 73%; mean 49.5±20.2% S.D; n = 12) from two to nine days following transformation. Sanger direct sequencing and bioinformatic analysis of the sequence reads revealed on-target programmed mutation on the BgAIF gene, including insertions of 1-2 bp and deletion of 8-30 bp, (range, 9 to 17% of BgAIF gene; n =12). In addition, the adherence of gene-edited Bge cells to sporocysts was significantly impeded in comparison to control cells, as ascertained using the cell adherence index (CAI); 2.66±0.10 (control) vs. 2.30±0.22 (ΔBgAIF cells), P < 0.05. This is the first report of gene editing in this medically important taxon of freshwater gastropods that are necessary for the transmission of schistosomiasis.