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. 2022 Sep 20:13:970725.
doi: 10.3389/fimmu.2022.970725. eCollection 2022.

Bacteroides fragilis outer membrane vesicles preferentially activate innate immune receptors compared to their parent bacteria

William J Gilmore  1   2 , Ella L Johnston  1   2 , Natalie J Bitto  1   2 , Lauren Zavan  1   2 , Neil O'Brien-Simpson  3 , Andrew F Hill  2   4   5 , Maria Kaparakis-Liaskos  1   2
Affiliations

Bacteroides fragilis outer membrane vesicles preferentially activate innate immune receptors compared to their parent bacteria

William J Gilmore et al. Front Immunol. .

Abstract

The release of bacterial membrane vesicles (BMVs) has become recognized as a key mechanism used by both pathogenic and commensal bacteria to activate innate immune responses in the host and mediate immunity. Outer membrane vesicles (OMVs) produced by Gram-negative bacteria can harbor various immunogenic cargo that includes proteins, nucleic acids and peptidoglycan, and the composition of OMVs strongly influences their ability to activate host innate immune receptors. Although various Gram-negative pathogens can produce OMVs that are enriched in immunogenic cargo compared to their parent bacteria, the ability of OMVs produced by commensal organisms to be enriched with immunostimulatory contents is only recently becoming known. In this study, we investigated the cargo associated with OMVs produced by the intestinal commensal Bacteroides fragilis and determined their ability to activate host innate immune receptors. Analysis of B. fragilis OMVs revealed that they packaged various biological cargo including proteins, DNA, RNA, lipopolysaccharides (LPS) and peptidoglycan, and that this cargo could be enriched in OMVs compared to their parent bacteria. We visualized the entry of B. fragilis OMVs into intestinal epithelial cells, in addition to the ability of B. fragilis OMVs to transport bacterial RNA and peptidoglycan cargo into Caco-2 epithelial cells. Using HEK-Blue reporter cell lines, we identified that B. fragilis OMVs could activate host Toll-like receptors (TLR)-2, TLR4, TLR7 and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), whereas B. fragilis bacteria could only induce the activation of TLR2. Overall, our data demonstrates that B. fragilis OMVs activate a broader range of host innate immune receptors compared to their parent bacteria due to their enrichment of biological cargo and their ability to transport this cargo directly into host epithelial cells. These findings indicate that the secretion of OMVs by B. fragilis may facilitate immune crosstalk with host epithelial cells at the gastrointestinal surface and suggests that OMVs produced by commensal bacteria may preferentially activate host innate immune receptors at the mucosal gastrointestinal tract.

Keywords: Bacteroides fragilis; Commensals; NOD1; TLRs; bacterial membrane vesicles; epithelial cells; innate immunity; outer membrane vesicles (OMVs).

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
B fragilis OMVs are heterogenous in size and morphology, and harbour protein, DNA, RNA, peptidoglycan and LPS cargo. (A) Purified B. fragilis OMVs were visualized using transmission electron microscopy (TEM). OMVs are indicated by black arrows. Images are representative of three biological replicates (Scale bar = 100 nm). (B) The size distribution of B. fragilis OMVs was determined using ZetaView Nanoparticle Tracking Analysis. Data shows the mean (black line) ± SEM (red error bars) of three biological replicates. (C) Quantification of OMV-associated protein, per 109 B. fragilis OMVs, using Qubit fluorometric analysis. Data shows the mean ± SEM of three biological replicates. (D) Quantification of DNA associated with DNase-treated (+) or non-treated (-) B. fragilis OMVs using Qubit fluorometric analysis. B. fragilis genomic DNA (Genomic DNA control), either treated with DNase (+) or non-treated (-), was used as a control for DNase activity. Shown is the mean ± SEM of three biological replicates. ns, not significant, ***p < 0.001 (unpaired t-test). (E) Quantification of RNA associated with RNase-treated (+) or non-treated (-) B. fragilis OMVs using Qubit fluorometric analysis. B. fragilis bacterial RNA (Bacterial RNA control), either treated with RNase (+) or not-treated (-), was used as a control for RNase activity. Shown is the mean ± SEM of three biological replicates. ns, not significant, ***p < 0.001 (unpaired t-test). (F) Quantification of the peptidoglycan cargo associated with 109 B. fragilis OMVs. Shown is the mean ± SEM of three biological replicates. (G) LPS associated with 109 B. fragilis OMVs was quantified using the Pierce™ Chromogenic Endotoxin Quant kit. Data represents the mean ± SEM of three biological replicates.
Figure 2
Figure 2
B. fragilis OMVs are enriched in protein, peptidoglycan and LPS cargo compared to their parent bacteria. (A) B. fragilis OMVs (10 μg) and B. fragilis bacteria (10 μg) were separated using SDS-PAGE and their protein cargo was stained using Sypro Ruby. Data is representative of three biological replicates. (B) The presence of peptidoglycan cargo associated with B. fragilis OMVs (10 μg) and B. fragilis bacteria (10 μg) was detected using Western immunoblot using an anti-peptidoglycan antibody. Peptidoglycan was used as a positive control (Pos control). Data shows three biological samples of B. fragilis OMVs and B. fragilis bacteria and is representative of three experiments. (C) B. fragilis OMVs and B. fragilis bacteria were treated with Proteinase K (P.K) (+) or not treated as controls (-), and their LPS cargo was detected by staining with ProQ Emerald 300 LPS stain. (D) ProQ Emerald-stained SDS-PAGE gels were counterstained with the protein-specific stain Sypro Ruby. Data in panel C and D represents three independent biological samples of B. fragilis OMVs and B. fragilis bacteria and is representative of three independent experiments. In all panels (A–D), black arrows represent cargo that is enriched in OMVs compared to bacteria, stars represent cargo that is less abundant in OMVs compared to bacteria.
Figure 3
Figure 3
B. fragilis OMVs activate TLR2 and TLR4 compared to their parent bacteria which only activate TLR2. (A, B) TLR2 and (C, D) TLR4 expressing HEK-Blue cells were stimulated with either B. fragilis OMVs (A, C, squares) or B fragilis bacteria (B, D, circles) at an increasing multiplicity of infection (MOI) for 18 hours. In all panels, open triangles indicate stimulation of the HEK-Blue null cell line with either OMVs (A, C) or bacteria (B, D) in each assay, filled triangles indicate positive controls for each respective cell line. Data represents mean ± SEM of three biological replicates. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (One-way ANOVA with Dunnett’s multiple comparisons test, compared to non-stimulated controls).
Figure 4
Figure 4
B fragilis OMVs enter host intestinal epithelial cells and transport their peptidoglycan and RNA cargo intracellularly. The (A) lipid (DiI; red), (B) peptidoglycan (BODIPY-FL vancomycin; green) and (C) RNA (Syto RNASelect; green) cargo associated with B. fragilis OMVs was fluorescently labelled. Fluorescently labelled B. fragilis OMVs were then incubated with Caco-2 cells for 4 hours, and OMV entry was visualized by confocal microscopy. Cell nuclei and cellular actin were visualized by staining with DAPI (blue) or Phalloidin (magenta). Intracellular B. fragilis OMVs are indicated by white arrows. DPBS containing each respective stain (Control) were incubated with Caco-2 cells to control for the non-specific uptake of each fluorescent stain by host cells. Images are representative of three biological replicates of Caco-2 cells stimulated with each type of fluorescently-labelled B. fragilis OMVs. Scale bar = 10 μm.
Figure 5
Figure 5
B fragilis OMVs activate NOD1 and TLR7 whereas B fragilis bacteria cannot. (A, B) NOD1, (C, D) NOD2, (E, F) TLR7, (G, H) TLR8 and (I, J) TLR9 expressing HEK-Blue cells were stimulated with an increasing MOI of either B. fragilis OMVs (A, C, E, G, I, squares) or B. fragilis bacteria (B, D, F, H, J, circles) for 18 hours. Open triangles indicate stimulation of the HEK-Blue null cell line with either B. fragilis OMVs or B. fragilis bacteria as a negative control in each assay. Filled triangles indicate positive controls for each respective cell line. Data represents mean ± SEM of three biological replicates. ns=not significant, *p < 0.05, **p < 0.01. (One-way ANOVA with Dunnett’s multiple comparisons test, compared to non-stimulated controls).
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