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.

Keywords: 2’−5’-oligoadenylate synthetase; Lactobacillus johnsonii N6.2; endosome; extracellular vesicles; human beta cell line; nanovesicles; probiotic; βlox5.

FIGURE 1

βLox5 cells in an energy depleted state (4°C) shows impairment in the internalization of L. johnsonii N6.2 labelled EV compared to βLox5 cells standard energy state (37°C). (a) βLox5 cells were treated with AlexaFluor 594 labelled EV for 0, 15, 30 and 45 min and incubated at 37°C or 4°C, as indicated on the side of each panel, and visualized by fluorescent confocal microscopy. Cell nucleus was stained with DAPI in cyan, the cell membrane was stained with AlexaFluor 488 WGA in green and the EV were stained with AlexaFluor 594 in red. Z‐stacks were made with a composite of 15 images. (b) Quantification of the AlexaFluor 594 labelled EV signal per cell area, different letters on top of the corresponding bar indicated statistical significance of p ≤ 0.05 from ANOVA analysis and post‐hoc Tukey test performed on 10 individual cells.

FIGURE 2

L. johnsonii N6.2 EV are internalized by a clathrin/dynamin dependent endocytic pathway. βLox5 cells were pretreated with 1.61 μL/mL of DMSO (Control), 80 μM of dynasore (Dyna), 0.5 μM of chlorpromazine (Chlor), 50 μM of methyl‐β‐cyclodextrin (MBC), 4 nM of 5(N‐ethyl‐N‐isopropyl)‐amiloride (5N) or 1 nM of cytochalasin D (Cyto), incubated with AlexaFluor 594 labelled EV and incubated at 37°C for 45 min, and visualized by fluorescent confocal microscopy. (a) Cell nucleus was stained with DAPI in cyan, the cell membrane was stained with AlexaFluor 488 WGA in green and the EV were stained with AlexaFluor 594 in red. Z‐stacks were made with a composite of 15 images. (b) Quantification of the AlexaFluor 594 labelled EV signal per cell area, different letters on top of the corresponding bar indicated statistical significance of p ≤ 0.05 from ANOVA analysis and post‐hoc Tukey test performed on 10 individual cells.

FIGURE 3

Inhibition of clathrin endocytic pathway hinders induction of the host RNA sensing genes. Cells were treated with L. johnsonii N6.2 EV and incubated at 37°C for 1 (a–c) or 5 h (d–f). βLox5 EV interaction was quantified using qRT‐PCR and relative expression is shown as fold change for (a, d). OAS2, (b, e) MX2 and (c, f) IFI44L when cells are pretreated with 1.61 μL/mL of DMSO (Control), 80 μM of dynasore (Dyna), 0.5 μM of chlorpromazine (Chlor), 50 μM of methyl‐β‐cyclodextrin (MBC), 4 nM of 5(N‐ethyl‐N‐isopropyl)‐amiloride (5N) or 1 nM of cytochalasin D (Cyto). Different letters on top of each bar indicates statistical significance of p ≤ 0.001 from ANOVA analysis and post‐hoc Tukey test performed on three biological replicates (with two qRT‐PCR technical replicates each).

FIGURE 4

Colocalization of fluorescently labelled L. johnsonii N6.2 EV and early, late endosomal markers. Representative colocalization immunofluorescent images of (a) RAB5 and (b) RAB7 as shown in green and AlexaFluor 594 labelled EV in red. Colocalization coefficients of the EV and (c) RAB5 or (d) RAB7 signal. Different letters on top of the corresponding bar indicates statistical significance of p ≤ 0.05 from ANOVA analysis and post‐hoc Tukey test performed on 5 individual images.

FIGURE 5

L. johnsonii N6.2 EV escape the late endosome. Representative images of the timepoint analysis of βLox5 incubated with media containing 100 μg/mL of calcein in the (a) absence and (b) presence of native L. johnsonii N6.2 EV or (c) Rhodamine B‐labelled L. johnsonii N6.2 EV visualized by fluorescent confocal microscopy. Endosomal escape is evidenced by a cellular distribution of the calcein dye in green while endosomal retention shows a punctuated green phenotype. (d) Colocalization coefficient of the EV and calcein, different letters on top of the corresponding bar indicated statistical significance of p ≤ 0.05 from ANOVA analysis and post‐hoc Tukey test performed on 5 individual images.

FIGURE 6

L. johnsonii N6.2 EV require a protein component to initiate cellular internalization while internalized RNA alone is sufficient to trigger a host RNA sensing response. (a–c) βLox5 cells treated with lipofectamine alone (Lpf), RNA alone and Lpf containing L. johnsonii N6.2 cellular RNA (Lpf + Cell RNA) or EV RNA (Lpf + EV RNA). (d–f) βLox5 cells were treated with protein‐free liposomes (Lipo) made from total lipid extract from L. johnsonii N6.2 and Lipo containing cellular RNA (Lipo + Cell RNA) or EV RNA (Lipo + EV RNA). Relative gene expression shown as fold change for (a, d) OAS2, (b, e) MX2 and (c, f) IFI44L. Different letters on top of each bar indicates statistical significance of p ≤ 0.001 from ANOVA analysis and post‐hoc Tukey test performed on three biological replicates (with two qRT‐PCR technical replicates each).

FIGURE 7

L. johnsonii N6.2 EV structure is critical in protecting the intravesicular cargo and in mediating the internalization into the host cell. (a–c) RNase protection assay was performed by treating EV with RNAse as described in the methods section. (d–i) EV were thermally disrupted (tdEV, d–f) by boiling for 10 min at 100°C or chemically disrupted (cdEV, g–i) by incubating the EV with 0.05% Triton X‐100 for 10 min. βLox5 cells were incubated with the treated EV as indicated earlier. The impact of the treatments was followed by determining the relative expression of (a, d, g) OAS2, (b, e, h) MX2 and (c, f, i) IFI44L genes. Different letters on top of each bar indicates statistical significance of p ≤ 0.001 from ANOVA analysis and post‐hoc Tukey test performed on three biological replicates (with two qRT‐PCR technical replicates each).

FIGURE 8

Global comparative transcriptomic analysis of the RNA in L. johnsonii N6.2 and its EV. (a) Principal component (PC) plot comparing the RNA content of the cellular and EV fractions, EV RNA samples are in red and cellular RNA samples are in blue. (b) Size analysis of the enriched transcripts within the cellular and EV fractions showing both the density distribution and count distribution according to gene length (dashed vertical lines represent average size). (c) Volcano plot of the differentially enriched transcripts within the cellular and EV fractions (red represents EV enrichment and blue represents cellular enrichment). (d) Comparison of the average normalized transcript count of the genes encoding for membrane and extracellular localized proteins. EV enriched RNA fraction (red) and the cellular enriched RNA fraction (blue). Different letters on top of each bar indicates statistical significance of p ≤ 0.05 from student's t‐test. (e) KEGG pathway analysis of the enriched transcript found within the EV compared against the cellular fraction (* indicates a statistical significance of p ≤ 0.05).