Tanno, W. I. | bioRxiv Science

bioRxiv2020

Infection with CagA+ Helicobacter pylori induces epithelial to mesenchymal transition in human cholangiocytes

Recent reports suggest that the East Asian liver fluke, Opisthorchis viverrini, infection with which is implicated in opisthorchiasis-associated cholangiocarcinoma, serves as a reservoir of Helicobacter pylori. The opisthorchiasis-affected cholangiocytes that line the intrahepatic biliary tract are considered to be the cell of origin of this malignancy. Here, we investigated interactions in vitro among human cholangiocytes, a CagA-positive strain of Helicobacter pylori, and the related bacillus, Helicobacter bilis. Exposure to increasing numbers of H. pylori at 0, 1, 10, 100 bacilli per cholangiocyte induced phenotypic changes including the profusion of thread-like filopodia and a loss of cell-cell contact, in a dose-dependent fashion. In parallel, following exposure to H. pylori, changes were evident in levels of mRNA expression of epithelial to mesenchymal transition (EMT)-encoding factors including snail, slug, vimentin, matrix metalloprotease, zinc finger E-box-binding homeobox, and the cancer stem cell marker CD44. Transcription levels encoding the cell adhesion marker CD24 decreased. Analysis to quantify cellular proliferation, migration and invasion in real time using the xCELLigence approach revealed that exposure to [≥]10 H. pylori stimulated migration and invasion by the cholangiocytes through an extracellular matrix. In addition, 10 bacilli of CagA-positive H. pylori stimulated contact-independent colony establishment in soft agar. These findings support the hypothesis that infection with H. pylori contributes to the malignant transformation of the biliary epithelium.

molecular biology↗

bioRxiv2020

Diminished adherence of snail hemocytes to schistosome sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor of Biomphalaria glabrata

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 quantitative 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% S.D, P [≤] 0.05, n =12). Adherence by BgAIF gene-edited ({Delta}BgAIF) 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 {Delta}BgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66{+/-}0.10, {Delta}BgAIF, 2.30{+/-}0.22 (P [≤] 0.01). ConclusionThe 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.

molecular biology↗