Outer membrane vesicles from Bacteroides fragilis contain coding and non-coding small RNA species that modulate inflammatory signaling in intestinal epithelial cells

Abstract

Alterations to the community structure and function of the microbiome are associated with changes to host physiology, including immune responses. However, the contribution of microbe-derived RNAs carried by outer membrane vesicles (OMVs) to host immune responses remain unclear. This study investigated the role of OMVs and OMV-associated small RNA (sRNA) species from pathogenic and commensal Bacteroides fragilis (ETBF and NTBF respectively) in eliciting different immune responses from intestinal epithelial cells. To distinguish the differences in the sRNA profiles of the two strains and their OMVs, RNA-seq, qRT-PCR, and northern blotting were conducted to identify enrichment of discrete sRNA species in OMVs, which were also differentially expressed between the two strains. Specifically, both coding and non-coding RNAs were enriched in OMVs from NTBF and ETBF, with BF9343_RS22680 and BF9343_RS17870 being significantly enriched in ETBF OMVs compared to NTBF. To understand the effects of OMVs on pattern recognition receptors, reporter cells of Toll-like receptor (TLR) activation were treated with OMVs, demonstrating activation of TLRs 2, 3, and 7. Treatment of Caco-2 and HT29-MTX cells with OMVs demonstrated increased expression of IL-8. Surprisingly, we discovered that degradation of RNase-accessible RNAs within ETBF OMVs, but not NTBF OMVs, resulted in vesicles with enhanced capacity to stimulate IL-8 expression, indicating that these extravesicular RNAs exert an immunosuppressive effect. This suggests a dual role for OMV-associated RNAs in modulating host immune responses, with implications for both bacterial pathogenesis and therapeutic applications.

Keywords: Bacteroides fragilis; outer membrane vesicles; small RNA.

Figure 1:. RNA sub-type enrichment is significantly different between ETBF and NTBF OMVs and whole cells.

(A/B) the average proportion of sequencing reads mapped to the NTBF (A) and ETBF (B) genomes and represented by RNA class. Data are the mean of n = 3 biological replicates. (C/D) Unsupervised hierarchical clustering of DESeq2 analysis of gene coding regions using DESeq2 reveals enrichment of RNA sequences in OMVs and whole cell lysate (WC) samples when they are aligned to the NTBF (C) and ETBF (D) genomes. (E/F) Unsupervised hierarchical clustering of DESeq2 analysis of intergenic, non-coding regions aligned to the NTBF (E) and ETBF (F) genomes. E = ETBF and N = NTBF. (G/H) Proportion of transcript types represented in data aligned to NTBF genome (G) or ETBF genome (H). Statistical significance was determined using a paired t-test, p-value <0.05 significant.

Figure 2:. B. fragilis OMV RNA transcripts show differential abundance between ETBF and NTBF.

(A/B) Enrichment of transcripts from established gene coding regions for combined OMV and whole cell lysate (WC) samples when they are aligned to the NTBF (A) and ETBF (B) genomes. (C/D) Enrichment of transcripts from intergenic regions for combined OMV and WC samples when they are aligned to the NTBF (C) and ETBF (D) genomes. (E/F) Enrichment of transcripts from established gene coding regions for OMV samples from ETBF and NTBF when they are aligned to the NTBF (E) and ETBF (F) genomes. All panels are derived from the same RNA-seq dataset; RNA transcript IDs are in the supplemental datasets. Limits of biological and statistical significance are log₂FC was set at 5, and the adjusted p value was set to 10⁻⁴. (G) qRT-PCR for indicated known transcripts and intergenic regions. Normalized to rubredoxin and referenced to NTBF WC. n = 3 n.d. = not detected. (H/I) To confirm the size of tRNA (H) and CoA Ligase (I) transcripts, northern blot probes complimentary to the RNAseq reads were used and successfully detected transcripts in OMVs at lower length than in WC samples (denoted by arrows).

Figure 3:. RNA cargo from ETBF and NTBF OMVs is taken up and localized to the endoplasmic reticulum in colonic epithelial cells.

(A) Confocal microscopy images showing co-staining of RNA and OMVs using SytoRNASelect (green) and Vybrant DiD (red) in OMVs isolated from NTBF. The merged image indicates colocalization (yellow) of RNA and OMVs. Scale bar: 10 μm. (B) Confocal microscopy images of Caco2 cells incubated for 30 min with OMVs from ETBF. The images show the nuclei stained with DAPI (blue), RNA stained with SytoRNASelect (green), OMVs stained with Vybrant DiD (red), and the merged image showing colocalization (yellow) of RNA and OMVs within the cells. Scale bar: 10 μm. (C) Confocal microscopy images of Caco2 cells treated with pre-stained OMVs containing RNA, showing nuclei stained with DAPI (blue), OMV RNA stained with SytoRNASelect (green), OMVs stained with Vybrant DiD (magenta), endoplasmic reticulum (ER) stained with protein disulfide isomerase (PDI) (red), and the merged image showing colocalization of OMV RNA with the ER (yellow). Scale bar: 5 μm. (D) Boxplot depicting the normalized Mander’s split colocalization coefficients for images in (C) NTBF OMV and RNA cargo colocalization within colonic epithelial cells. The coefficients for Red-Green and Green-Red channels show significant colocalization in both channels.

Figure 4:. Specific OMV-associated RNAs are protected from RNase degradation.

(A) Quantification of RNA recovered from equivalent amounts of OMVs with and without RNase treatment; individual points represent biological replicates (n=4) and data is presented as mean ± S.E.M.; statistical significance was determined using a Welch’s test. * p<0.05; ** p<0.01 (B/C). To confirm the size of tRNA-Phe (4B) and CoA Ligase (4C), northern blot probes complimentary to the most abundant RNAseq reads in the genes were used and successfully detected transcript fragments in RNase-treated OMVs (denoted by arrows). Probes bound to OMV-associated RNAs at shorter length in the in the blot compared to the whole cell samples, suggesting that the sequences contained in the OMVs are fragments of the larger RNA transcript.

Figure 5:. NTBF and ETBF OMVs activate TLRs in a dose-dependent manner.

(A-D) OMVs from ETBF and NTBF were administered to HEK-293T cells expressing the indicated TLR proteins and an NFκB-responsive SEAP reporter gene. TLR activity was calculated by comparing vesicle and positive control treatments (gray columns) to the corresponding TLR cells treated with PBS (PBS treatment activity is represented by the dashed lines at y = 1). Points on the graph represent biological replicates (n=3) and data is presented as mean ± S.E.M. Statistical significance was found using Dunnett’s multiple comparison test. Stars represent significantly different from PBS control. ** p < 0.01, **** p < 0.0001. LPS = TLR4 agonist, Poly I:C = TLR3 agonist, CL307 = TLR7 agonist.

Figure 6:. RNase-treated OMVs from ETBF amplify the IL-8 response to TLR2 and TLR4 agonists in colorectal adenocarcinoma cells.

(A) Caco-2 cells treated with PBS, LPS, RNase-naïve or RNase-treated OMVs from NTBF and ETBF were assayed by qRT-PCR for IL8 expression. (B-C) Caco-2 cells treated with PBS, RNase-naïve or RNase-treated OMVs from NTBF and ETBF for 6h prior to withdrawal of pre-treatment and addition of LPS (TLR4 agonist) at 100 ng/mL (B) or Plasmotest (TLR2 agonist) (C). Signal normalized to vehicle only control. Points on the graph represent biological replicates and data is presented as mean ± st. dev. Statistical significance was found using Dunnett’s multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 7:. Protein components of B. fragilis OMVs differ between ETBF and NTBF.

(A) Silver staining of OMV samples of ETBF and NTBF show different protein profiles. ETBF and NTBF OMVs at different concentrations were run on a 10% SDS-PAGE gel followed by silver staining. (B) Protein IDs detected for ETBF and NTBF with >1 peptide match and which possess a molecular weight within the range of excised gel band.