The Proteome of Extracellular Vesicles Produced by the Human Gut Bacteria Bacteroides thetaiotaomicron In Vivo Is Influenced by Environmental and Host-Derived Factors

Extracellular Vesicles
/References

Stentz, Régis, Emily Jones, Rokas Juodeikis, Udo Wegmann, Maria Guirro, Andrew J. Goldson, Arlaine Brion, et al. 2022. “The Proteome of Extracellular Vesicles Produced by the Human Gut Bacteria Bacteroides Thetaiotaomicron in Vivo Is Influenced by Environmental and Host-Derived Factors.” Edited by Knut Rudi. Applied and Environmental Microbiology 88 (16). https://doi.org/10.1128/aem.00533-22.

Bacterial extracellular vesicles (BEVs) released from both Gram-negative and Gram-positive bacteria provide an effective means of communication and trafficking of cell signaling molecules. In the gastrointestinal tract (GIT) BEVs produced by members of the intestinal microbiota can impact host health by mediating microbe-host cell interactions. A major unresolved question, however, is what factors influence the composition of BEV proteins and whether the host influences protein packaging into BEVs and secretion into the GIT. To address this, we have analyzed the proteome of BEVs produced by the major human gut symbiont Bacteroides thetaiotaomicron both in vitro and in vivo in the murine GIT in order to identify proteins specifically enriched in BEVs produced in vivo. We identified 113 proteins enriched in BEVs produced in vivo, the majority (62/113) of which accumulated in BEVs in the absence of any changes in their expression by the parental cells. Among these selectively enriched proteins, we identified dipeptidyl peptidases and an asparaginase and confirmed their increased activity in BEVs produced in vivo. We also showed that intact BEVs are capable of degrading bile acids via a bile salt hydrolase. Collectively these findings provide additional evidence for the dynamic interplay of host-microbe interactions in the GIT and the existence of an active mechanism to drive and enrich a selected group of proteins for secretion into BEVs in the GIT.

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Background Previous studies found that cigarette smoke (CS) exposure could induce NSCLC malignancy and miRNA dysregulation. Yet, the association of CS-induced miRNA dysregulation and NSCLC malignancy has not been clearly understood. This study aimed to evaluate the effect of CS exposure in smokers on the expression of miR-10b-5p and miR-320b in extracellular vesicles (EVs) from NSCLC patients. Material and methods Bioinformatic analysis was conducted to validate miRNA candidates. Blood and tissue samples were collected from NSCLC patients (n = 21) with smoking and non-smoking history. EVs were isolated from plasma and miRNAs were extracted from the isolated EVs. The miRNAs relative expression was analyzed and compared. Results In silico analysis identified miR-320b and miR-10b-5p as potential biomarkers for diagnosing NSCLC in smokers. Experimental analysis revealed differential expression of EVs-associated miRNAs in NSCLC patients with smoking and non-smoking histories. EVs-associated miR-10b-5p was significantly overexpressed in smoker NSCLC patients (p = 0.000), while miR-320b expression was significantly lower in this group (p = 0.018). Additionally, smoking intensity influenced miRNA expression, with higher smoking intensity correlating with increased miR-10b-5p expression and decreased miR-320b expression. ROC analysis demonstrated that EVs were a superior source of miRNAs compared to plasma for NSCLC diagnostics. miR-10b-5p and miR-320b in EVs showed higher diagnostic performance (AUC 0.878; 0.739) compared to plasma (AUC 0.628; 0.559). Conclusion CS exposure induces different expression of miR-10b-5p and miR-320b in EVs of NSCLC patients with smoking history. EV-related miR-10b-5p and miR-320b showed potential to be utilized as prognostic biomarker for smokers NSCLC patients.

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Extracellular vesicles (EVs) have emerged as promising therapeutics with broad clinical applications as diagnostic biomarkers and therapeutic drug delivery systems. Yet, these biopharmaceuticals pose a challenge in terms of manufacturing due to their complexity and heterogeneity. Despite advancements in the field, current purification technologies lack scalability and/or selectivity. Affinity chromatography (AC) − coupling unmatched specificity and scalability − could be used to simplify purification processing and generate clinical-grade EVs with higher titers and purity. In the present work, we report the implementation of an immuno-AC resin to capture and purify EVs directly from clarified cellular feedstocks. Firstly, to guide and support marker selection, vesicle phenotype characterization was conducted using single particle interferometric reflectance image sensing (SP-IRIS) coupled with immunofluorescence. CD81 was the marker which shown to be more present and more likely to have the other markers (CD63 and CD9). Thus, anti-CD81 VHH ligand was generated and evaluated towards recombinant CD81 protein and CD81 bearing EV particles using surface plasmon resonance (SPR). Different chromatographic studies with Anti-CD81 ligand immobilized onto agarose beads resin were conducted to optimize the process parameters (residence time, dynamic binding capacity and impurity clearance). At residence time of 2 min, on average 40 % of pure triple tetraspanin-positive EV fraction was recovered. The enrichment in EV particles herein obtained, based on scale-up calculations, it would be possible to produce 1 × 1013 EVs from a 1L cell culture, while meeting impurity requirements in a single-step purification process (impurity removal over 2 log reduction value). A single-step purification process is possible, enabling the successful isolation of homogeneous EVs population, counting with a final HCP titer of 60 ng/mL and 9 ng/mL of dsDNA impurities. EV’s morphological integrity and internalization ability were also demonstrated, showcasing elution’s efficiency under mild conditions. Overall, this work contributes to the development of a novel, highly specific, AC technology using a camelid-derived affinity ligand which, bridging the scalability requirements demanded of large-scale production, could potentiate the advent of EV-based therapies.

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Extracellular vesicles (EVs) have emerged as promising therapeutics with broad clinical applications as diagnostic biomarkers and therapeutic drug delivery systems. Yet, these biopharmaceuticals pose a challenge in terms of manufacturing due to their complexity and heterogeneity. Despite advancements in the field, current purification technologies lack scalability and/or selectivity. Affinity chromatography (AC) − coupling unmatched specificity and scalability − could be used to simplify purification processing and generate clinical-grade EVs with higher titers and purity. In the present work, we report the implementation of an immuno-AC resin to capture and purify EVs directly from clarified cellular feedstocks. Firstly, to guide and support marker selection, vesicle phenotype characterization was conducted using single particle interferometric reflectance image sensing (SP-IRIS) coupled with immunofluorescence. CD81 was the marker which shown to be more present and more likely to have the other markers (CD63 and CD9). Thus, anti-CD81 VHH ligand was generated and evaluated towards recombinant CD81 protein and CD81 bearing EV particles using surface plasmon resonance (SPR). Different chromatographic studies with Anti-CD81 ligand immobilized onto agarose beads resin were conducted to optimize the process parameters (residence time, dynamic binding capacity and impurity clearance). At residence time of 2 min, on average 40 % of pure triple tetraspanin-positive EV fraction was recovered. The enrichment in EV particles herein obtained, based on scale-up calculations, it would be possible to produce 1 × 1013 EVs from a 1L cell culture, while meeting impurity requirements in a single-step purification process (impurity removal over 2 log reduction value). A single-step purification process is possible, enabling the successful isolation of homogeneous EVs population, counting with a final HCP titer of 60 ng/mL and 9 ng/mL of dsDNA impurities. EV’s morphological integrity and internalization ability were also demonstrated, showcasing elution’s efficiency under mild conditions. Overall, this work contributes to the development of a novel, highly specific, AC technology using a camelid-derived affinity ligand which, bridging the scalability requirements demanded of large-scale production, could potentiate the advent of EV-based therapies.

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