Conforti, J. M. | bioRxiv Science

bioRxiv2025

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

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. Graphical Abstract

molecular biology↗

bioRxiv2024

Differences in Protein Capture by SP3 and SP4 Demonstrate Mechanistic Insights of Proteomics Clean-up Techniques

The goal of proteomics experiments is to identify proteins to observe changes in cellular processes and diseases. One challenge in proteomics is the removal of contaminants following protein extraction, which can limit protein identification. Single-pot, solid-phase-enhanced sample preparation (SP3) is a clean-up technique in which proteins are captured on carboxylate-modified particles through a proposed hydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism. However, recent results have suggested that proteins are captured in SP3 due to a protein-aggregation mechanism. Thus, solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) is a newer clean-up technique that employs protein-aggregation to capture proteins without modified particles. SP4 has previously enriched low-solubility proteins, though differences in protein capture could affect which proteins are detected and identified. We hypothesize that the mechanisms of capture for SP3 and SP4 are distinct. Herein, we assess the proteins identified and enriched using SP3 versus SP4 for MCF7 subcellular fractions and correlate protein capture in each method to protein hydrophobicity. Our results indicate that SP3 captures more hydrophilic proteins through a combination of HILIC-like and protein-aggregation mechanisms, while SP4 captures more hydrophobic proteins through a protein-aggregation mechanism. From these results, we recommend clean-up techniques based on protein-sample hydrophobicity to yield high proteome coverage in biological samples.

biochemistry↗