Internalization of extracellular vesicles from Lactobacillus johnsonii N6.2 elicit an RNA sensory response in human pancreatic cell lines

Abstract

Cells of all domains of life can secrete extracellular vesicles (EV). These secreted vesicles have been indicated as vehicles carrying molecules that facilitate intra‐ and inter‐species interaction. Lactobacillus johnsonii N6.2, a bacterium used in probiotic preparations, has been shown to produce nano‐sized EV. In the present work we used L. johnsonii N6.2 EV, concentrated from exosome‐depleted MRS supernatant, to identify the uptake mechanisms of EV and the impact of the RNA cargo in the EV on the upregulation of the cellular response of βlox5 human pancreatic cells. Using eukaryotic uptake inhibitors, it was found that EV are internalized by the clathrin/dynamin mediated endocytosis pathway. Further co‐localization experiments with the endosome markers RAB5, RAB7 and LAMP1 as well as calcein indicated that EV escape the endosome shortly after RAB7 fusion. Using the expression of the 2′,5′‐oligoadenylate synthetase (OAS) host pathway, previously identified as targeted by L. johnsonii EV, we found that the host cellular response to the EV are dependent on the integrity of the external components of the EV as well as on the RNA cargo. Global transcriptome analysis was performed on EV and the bacterial whole cell. It was found that the RNA transcripts found within the EV largely represent the most abundantly transcribed genes in the bacterial cells such as those associated with protein synthesis and glycolysis. Further analysis showed an enrichment of smaller size transcripts as well as those encoding for membrane bound or extracellular proteins in L. johnsonii’s EV.

Bile promotes Lactobacillus johnsonii N6.2 extracellular vesicle production with conserved immunomodulatory properties

“…It is currently unclear whether an active sorting mechanism is necessary for the inclusion of immunomodulatory components within EVs, or if these components are simply present due to their proximity to sites of EV release. Recently, L. johnsonii N6.2 EVs were shown to be enriched with RNA transcripts encoding membrane bound and extracellular proteins compared to total cell RNA 45 . These data suggest that transcription and translation coupling drives RNA packaging into EVs.…”

“…We reported that in human pancreatic βlox5 cells, the addition of EVs at 10 10 induce the expression of multiple genes involved in nucleic acid sensing and degradation, including the OAS genes, IFI44L, and MX2 5 , 45 . Next we evaluated whether EVs isolated from L. johnsonii N6.2 grown in the presence of bile (EV BILE ) would elicit a similar response in host cell lines to EVs obtained from standard conditions (EV CTRL ).…”