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Bacterial extracellular vesicles (BEVs) are nano-size vesicles containing a cargo of bioactive molecules that can play key roles in microbe-microbe and microbe-host interactions. In tracking their biodistribution in vivo, BEVs can cross several physical host barriers including the intestinal epithelium, vascular endothelium, and blood-brain-barrier (BBB) to ultimately accumulate in tissues such as the liver, lungs, spleen, and the brain. This tissue-specific dissemination has been exploited for the delivery of biomolecules such as vaccines for mucosal delivery. Although numerous strategies for labeling and tracking BEVs have been described, most have constraints that impact on interpreting in vivo bioimaging patterns. Here, we describe a general method for labeling BEVs using lipophilic fluorescent membrane stains which can be adopted by non-expert users. We also describe how the procedure can be used to overcome potential limitations. Furthermore, we outline methods of quantitative ex vivo tissue imaging that can be used to evaluate BEV organ trafficking., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

TenA thiamin-degrading enzymes are commonly found in prokaryotes, plants, fungi and algae and are involved in the thiamin salvage pathway. The gut symbiont Bacteroides thetaiotaomicron (Bt) produces a TenA protein (BtTenA) which is packaged into its extracellular vesicles. An alignment of BtTenA protein sequence with proteins from different databases using the basic local alignment search tool (BLAST) and the generation of a phylogenetic tree revealed that BtTenA is related to TenA-like proteins not only found in a small number of intestinal bacterial species but also in some aquatic bacteria, aquatic invertebrates, and freshwater fish. This is, to our knowledge, the first report describing the presence of TenA-encoding genes in the genome of members of the animal kingdom. By searching metagenomic databases of diverse host-associated microbial communities, we found that BtTenA homologues were mostly represented in biofilms present on the surface of macroalgae found in Australian coral reefs. We also confirmed the ability of a recombinant BtTenA to degrade thiamin. Our study shows that BttenA-like genes which encode a novel sub-class of TenA proteins are sparingly distributed across two kingdoms of life, a feature of accessory genes known for their ability to spread between species through horizontal gene transfer., (© 2023. The Author(s).)

Bacterial extracellular vesicles (BEVs) produced by gut commensal bacteria have been proposed to play an important role in maintaining host homeostasis via interactions with the immune system. Details of the mediators and pathways of BEV-immune cell interactions are however incomplete. In this study, we provide evidence for the anti-inflammatory and immunomodulatory properties of extracellular vesicles produced by the prominent human gut commensal bacterium Bacteroides thetaiotaomicron (Bt BEVs) and identify the molecular mechanisms underlying their interaction with innate immune cells. Administration of Bt BEVs to mice treated with colitis-inducing dextran sodium sulfate (DSS) ameliorates the symptoms of intestinal inflammation, improving survival rate and reducing weight loss and disease activity index scores, in association with upregulation of IL-10 production in colonic tissue and in splenocytes. Pre-treatment (conditioning) of murine bone marrow derived monocytes (BMDM) with Bt BEVs resulted in higher ratio of IL-10/TNFα production after an LPS challenge when compared to LPS pre-conditioned or non-conditioned BMDM. Using the THP-1 monocytic cell line the interactions between Bt BEVs and monocytes/macrophages were shown to be mediated primarily by TLR2. Histone (H3K4me1) methylation analysis showed that Bt BEVs induced epigenetic reprogramming which persisted after infectious challenge, as revealed by increased levels of H3K4me1 in Bt BEV-conditioned LPS-challenged BMDM. Collectively, our findings highlight the important role of Bt BEVs in maintaining host immune homeostasis and raise the promising possibility of considering their use in immune therapies., Competing Interests: 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., (Copyright © 2022 Fonseca, Carvalho, Miquel-Clopés, Jones, Juodeikis, Stentz and Carding.)

The processes by which bacteria proactively scavenge essential nutrients in crowded environments such as the gastrointestinal tract are not fully understood. In this context, we observed that bacterial extracellular vesicles (BEVs) produced by the human commensal gut microbe Bacteroides thetaiotaomicron contain multiple high-affinity vitamin B 12 binding proteins suggesting that the vesicles play a role in micronutrient scavenging. Vitamin B 12 belongs to the cobamide family of cofactors that regulate microbial communities through their limited bioavailability. We show that B. thetaiotaomicron derived BEVs bind a variety of cobamides and not only deliver them back to the parental bacterium but also sequester the micronutrient from competing bacteria. Additionally, Caco-2 cells, representing a model intestinal epithelial barrier, acquire cobamide-bound vesicles and traffic them to lysosomes, thereby mimicking the physiological cobalamin-specific intrinsic factor-mediated uptake process. Our findings identify a novel cobamide binding activity associated with BEVs with far-reaching implications for microbiota and host health., Competing Interests: The authors declare that there is no conflict of interest., (© 2022 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

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. IMPORTANCE The gastrointestinal tract (GIT) harbors a complex community of microbes termed the microbiota that plays a role in maintaining the host's health and wellbeing. How this comes about and the nature of microbe-host cell interactions in the GIT is still unclear. Recently, nanosized vesicles naturally produced by bacterial constituents of the microbiota have been shown to influence responses of different host cells although the molecular basis and identity of vesicle-born bacterial proteins that mediate these interactions is unclear. We show here that bacterial extracellular vesicles (BEVs) produced by the human symbiont Bacteroides thetaiotaomicron in the GIT are enriched in a set of proteins and enzymes, including dipeptidyl peptidases, an asparaginase and a bile salt hydrolase that can influence host cell biosynthetic pathways. Our results provide new insights into the molecular basis of microbiota-host interactions that are central to maintaining GIT homeostasis and health.

Bacterial extracellular vesicles (BEVs) possess features that make them well suited for the delivery of therapeutics and vaccines. This chapter describes methods for engineering the commensal human intestinal bacterium Bacteroides thetaiotaomicron (Bt) to produce BEVs carrying vaccine antigens and accompanying methods for isolating and purifying BEVs for mucosal vaccination regimens., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The gastrointestinal (GI) tract harbours a complex microbial community, which contributes to its homeostasis. A disrupted microbiome can cause GI-related diseases, including inflammatory bowel disease (IBD), therefore identifying host-microbe interactions is crucial for better understanding gut health. Bacterial extracellular vesicles (BEVs), released into the gut lumen, can cross the mucus layer and access underlying immune cells. To study BEV-host interactions, we examined the influence of BEVs generated by the gut commensal bacterium, Bacteroides thetaiotaomicron, on host immune cells. Single-cell RNA sequencing data and host-microbe protein-protein interaction networks were used to predict the effect of BEVs on dendritic cells, macrophages and monocytes focusing on the Toll-like receptor (TLR) pathway. We identified biological processes affected in each immune cell type and cell-type specific processes including myeloid cell differentiation. TLR pathway analysis highlighted that BEV targets differ among cells and between the same cells in healthy versus disease (ulcerative colitis) conditions. The in silico findings were validated in BEV-monocyte co-cultures demonstrating the requirement for TLR4 and Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) in BEV-elicited NF-kB activation. This study demonstrates that both cell-type and health status influence BEV-host communication. The results and the pipeline could facilitate BEV-based therapies for the treatment of IBD., (© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

The gastrointestinal tract harbors the gut microbiota, structural alterations of which (dysbiosis) are linked with an increase in gut permeability ("leaky gut"), enabling luminal antigens and bacterial products such as nanosized bacterial extracellular vesicles (BEVs) to access the circulatory system. Blood-derived BEVs contain various cargoes and may be useful biomarkers for diagnosis and monitoring of disease status and relapse in conditions such as inflammatory bowel disease (IBD). To progress this concept, we developed a rapid, cost-effective protocol to isolate BEV-associated DNA and used 16S rRNA gene sequencing to identify bacterial origins of the blood microbiome of healthy individuals and patients with Crohn's disease and ulcerative colitis. The 16S rRNA gene sequencing successfully identified the origin of plasma-derived BEV DNA. The analysis showed that the blood microbiota richness, diversity, or composition in IBD, healthy control, and protocol control groups were not significantly distinct, highlighting the issue of 'kit-ome' contamination in low-biomass studies. Our pilot study provides the basis for undertaking larger studies to determine the potential use of blood microbiota profiling as a diagnostic aid in IBD.

Prophages are widely present in Lactococcus lactis, a lactic acid bacterium (LAB) that plays a key role in dairy fermentations. L. lactis MG1363 is a laboratory strain used worldwide as a model LAB. Initially regarded as plasmid and prophage free, MG1363 carries two complete prophages, TP712 and MG-3. Only TP712 seems to be inducible but unable to lyse the host. Several so-called TP712 lysogens able to lyse upon prophage induction were reported in the past, but the reason for their lytic phenotype remained unknown. In this work, we describe CAP, a new P335 prophage detected in the "lytic TP712 lysogens" which had remained unnoticed. CAP is able to be excised after mitomycin C treatment, along with TP712, and able to infect L. lactis MG1363-like strains but not the lytic TP712 lysogens. Both phages cooperate for efficient host lysis. While the expression in trans of the CAP lytic genes was sufficient to trigger cell lysis, this process was boosted when the resident TP712 prophage was concomitantly induced. Introduction of mutations into the TP712 lytic genes revealed that its holin but not its endolysin plays a major role. Accordingly, it is shown that the lytic activity of the recombinant CAP endolysin relies on membrane depolarization. Revisiting the seminal work that generated the extensively used L. lactis MG1363 strain led us to conclude that the CAP phage was originally present in its ancestor, L. lactis NCDO712, and our results solved long-standing mysteries around the MG1363 resident prophage TP712 reported in the "presequencing" era. IMPORTANCE Prophages are bacterial viruses that integrate into the chromosomes of bacteria until an environmental trigger induces their lytic cycle, ending with lysis of the host. Prophages present in dairy starters can compromise milk fermentation and represent a serious threat in dairy plants. In this work, we discovered that two temperate phages, TP712 and CAP, infecting the laboratory strain Lactococcus lactis MG1363 join forces to lyse the host. Based on the in vitro lytic activity of the LysCAP endolysin, in combination with mutated versions of TP712 lacking either its holin or endolysin, we conclude that this cooperation relies on the combined activity of the holins of both phages that boost the activity of LysCAP. The presence of an additional prophage explains the lytic phenotype of the lysogens formerly thought to be single TP712 lysogens that had remained a mystery for many years.

Gut microbes have critical roles in maintaining host physiology, but their effects on epithelial chemosensory enteroendocrine cells (EEC) remain unclear. We investigated the role that the ubiquitous commensal gut bacterium Bacteriodes thetaiotaomicron (Bt) and its major fermentation products, acetate, propionate, and succinate (APS) have in shaping EEC networks in the murine gastrointestinal tract (GIT). The distribution and numbers of EEC populations were assessed in tissues along the GIT by fluorescent immunohistochemistry in specific pathogen free (SPF), germfree (GF) mice, GF mice conventionalized by Bt or Lactobacillus reuteri (Lr), and GF mice administered APS. In parallel, we also assessed the suitability of using intestinal crypt-derived epithelial monolayer cultures for these studies. GF mice up-regulated their EEC network, in terms of a general EEC marker chromogranin A (ChrA) expression, numbers of serotonin-producing enterochromaffin cells, and both hormone-producing K- and L-cells, with a corresponding increase in serum glucagon-like peptide-1 (GLP-1) levels. Bt conventionalization restored EEC numbers to levels in SPF mice with regional specificity; the effects on ChrA and L-cells were mainly in the small intestine, the effects on K-cells and EC cells were most apparent in the colon. By contrast, Lr did not restore EEC networks in conventionalized GF mice. Analysis of secretory epithelial cell monolayer cultures from whole small intestine showed that intestinal monolayers are variable and with the possible exclusion of GIP expressing cells, did not accurately reflect the EEC cell makeup seen in vivo . Regarding the mechanism of action of Bt on EECs, colonization of GF mice with Bt led to the production and accumulation of acetate, propionate and succinate (APS) in the caecum and colon, which when administered at physiological concentrations to GF mice via their drinking water for 10 days mimicked to a large extent the effects of Bt in GF mice. After withdrawal of APS, the changes in some EEC were maintained and, in some cases, were greater than during APS treatment. This data provides evidence of microbiota influences on regulating EEC networks in different regions of the GIT, with a single microbe, Bt, recapitulating its role in a process that may be dependent upon its fermentation products., (Copyright © 2020 Modasia, Parker, Jones, Stentz, Brion, Goldson, Defernez, Wileman, Ashley Blackshaw and Carding.)

Background and Aims: As the importance of gut-brain interactions increases, understanding how specific gut microbes interact with the enteric nervous system (ENS), which is the first point of neuronal exposure becomes critical. Our aim was to understand how the dominant human gut bacterium Bacteroides thetaiotaomicron (Bt) regulates anatomical and functional characteristics of the ENS., Methods: Neuronal cell populations, as well as enteroendocrine cells, were assessed in proximal colonic sections using fluorescent immunohistochemistry in specific pathogen-free (SPF), germ-free (GF) and Bt conventionalized-germ-free mice (Bt-CONV). RNA expression of tight junction proteins and toll-like receptors (TLR) were measured using qPCR. Colonic motility was analyzed using in vitro colonic manometry., Results: Decreased neuronal and vagal afferent innervation observed in GF mice was normalized by Bt-CONV with increased neuronal staining in mucosa and myenteric plexus. Bt-CONV also restored expression of nitric oxide synthase expressing inhibitory neurons and of choline acetyltransferase and substance P expressing excitatory motor neurons comparable to those of SPF mice. Neurite outgrowth and glial cells were upregulated by Bt-CONV. RNA expression of tight junction protein claudin 3 was downregulated while TLR2 was upregulated by Bt-CONV. The enteroendocrine cell subtypes L-cells and enterochromaffin cells were reduced in GF mice, with Bt-CONV restoring L-cell numbers. Motility as measured by colonic migrating motor complexes (CMMCs) increased in GF and Bt-CONV., Conclusion: Bt, common gut bacteria, is critical in regulating enteric neuronal and enteroendocrine cell populations, and neurogenic colonic activity. This highlights the potential use of this resident gut bacteria for maintaining healthy gut function.

Background: Bacteroides thetaiotaomicron (Bt) is a prominent member of the human intestinal microbiota that, like all gram-negative bacteria, naturally generates nanosized outer membrane vesicles (OMVs) which bud off from the cell surface. Importantly, OMVs can cross the intestinal epithelial barrier to mediate microbe-host cell crosstalk involving both epithelial and immune cells to help maintain intestinal homeostasis. Here, we have examined the interaction between Bt OMVs and blood or colonic mucosa-derived dendritic cells (DC) from healthy individuals and patients with Crohn's disease (CD) or ulcerative colitis (UC)., Results: In healthy individuals, Bt OMVs stimulated significant (p < 0.05) IL-10 expression by colonic DC, whereas in peripheral blood-derived DC they also stimulated significant (p < 0.001 and p < 0.01, respectively) expression of IL-6 and the activation marker CD80. Conversely, in UC Bt OMVs were unable to elicit IL-10 expression by colonic DC. There were also reduced numbers of CD103 + DC in the colon of both UC and CD patients compared to controls, supporting a loss of regulatory DC in both diseases. Furthermore, in CD and UC, Bt OMVs elicited a significantly lower proportion of DC which expressed IL-10 (p < 0.01 and p < 0.001, respectively) in blood compared to controls. These alterations in DC responses to Bt OMVs were seen in patients with inactive disease, and thus are indicative of intrinsic defects in immune responses to this commensal in inflammatory bowel disease (IBD)., Conclusions: Overall, our findings suggest a key role for OMVs generated by the commensal gut bacterium Bt in directing a balanced immune response to constituents of the microbiota locally and systemically during health which is altered in IBD patients. Video Abstract.

Gram-negative bacteria ubiquitously produce and release nano-size, non-replicative outer membrane vesicles (OMVs). In the gastrointestinal (GI-) tract, OMVs generated by members of the intestinal microbiota are believed to contribute to maintaining the intestinal microbial ecosystem and mediating bacteria-host interactions, including the delivery of bacterial effector molecules to host cells to modulate their physiology. Bacterial OMVs have also been found in the bloodstream although their origin and fate are unclear. Here we have investigated the interactions between OMVs produced by the major human gut commensal bacterium, Bacteroides thetaiotaomicron (Bt), with cells of the GI-tract. Using a combination of in vitro culture systems including intestinal epithelial organoids and in vivo imaging we show that intestinal epithelial cells principally acquire Bt OMVs via dynamin-dependent endocytosis followed by intracellular trafficking to LAMP-1 expressing endo-lysosomal vesicles and co-localization with the perinuclear membrane. We observed that Bt OMVs can also transmigrate through epithelial cells via a paracellular route with in vivo imaging demonstrating that within hours of oral administration Bt OMVs can be detected in systemic tissues and in particular, the liver. Our findings raise the intriguing possibility that OMVs may act as a long-distance microbiota-host communication system., (Copyright © 2020 Jones, Booth, Fonseca, Parker, Cross, Miquel-Clopés, Hautefort, Mayer, Wileman, Stentz and Carding.)

Gram-negative bacteria naturally produce and secrete nanosized outer membrane vesicles (OMVs). In the human gastrointestinal tract, OMVs produced by commensal Gram-negative bacteria can mediate interactions amongst host cells (including between epithelial cells and immune cells) and maintain microbial homeostasis. This OMV-mediated pathway for host-microbe interactions could be exploited to deliver biologically active proteins to the body. To test this we engineered the Gram-negative bacterium Bacteroides thetaiotaomicron (Bt), a prominent member of the intestinal microbiota of all animals, to incorporate bacteria-, virus- and human-derived proteins into its OMVs. We then used the engineered Bt OMVs to deliver these proteins to the respiratory and gastrointestinal (GI)-tract to protect against infection, tissue inflammation and injury. Our findings demonstrate the ability to express and package both Salmonella enterica ser. Typhimurium-derived vaccine antigens and influenza A virus (IAV)-derived vaccine antigens within or on the outer membrane of Bt OMVs. These antigens were in a form capable of eliciting antigen-specific immune and antibody responses in both mucosal tissues and systemically. Furthermore, immunisation with OMVs containing the core stalk region of the IAV H5 hemagglutinin from an H5N1 strain induced heterotypic protection in mice to a 10-fold lethal dose of an unrelated subtype (H1N1) of IAV. We also showed that OMVs could express the human therapeutic protein, keratinocyte growth factor-2 (KGF-2), in a stable form that, when delivered orally, reduced disease severity and promoted intestinal epithelial repair and recovery in animals administered colitis-inducing dextran sodium sulfate. Collectively, our data demonstrates the utility and effectiveness of using Bt OMVs as a mucosal biologics and drug delivery platform technology.

As part of their life cycle, Gram-negative bacteria produce and release microvesicles (outer membrane vesicles, OMVs) consisting of spherical protrusions of the outer membrane that encapsulate periplasmic contents. OMVs produced by commensal bacteria in the gastrointestinal (GI) tract of animals are dispersed within the gut lumen with their cargo and enzymes being distributed across and throughout the GI tract. Their ultimate destination and fate is unclear although they can interact with and cross the intestinal epithelium using different entry pathways and access underlying immune cells in the lamina propria. OMVs have also been found in the bloodstream from which they can access various tissues and possibly the brain. The nanosize and non-replicative status of OMVs together with their resistance to enzyme degradation and low pH, alongside their ability to interact with the host, make them ideal candidates for delivering biologics to mucosal sites, such as the GI and the respiratory tract. In this mini-review, we discuss the fate of OMVs produced in the GI tract of animals with a focus on vesicles released by Bacteroides species and the use of OMVs as vaccine delivery vehicles and other potential applications., (© 2018 The Author(s).)

There is considerable interest in studying the function of Bacteroides species resident in the human gastrointestinal (GI)-tract and the contribution they make to host health. Reverse genetics and protein expression techniques, such as those developed for well-characterized Escherichia coli cannot be applied to Bacteroides species as they and other members of the Bacteriodetes phylum have unique promoter structures. The availability of useful Bacteroides-specific genetic tools is therefore limited. Here we describe the development of an effective mannan-controlled gene expression system for Bacteroides thetaiotaomicron containing the mannan-inducible promoter-region of an α-1,2-mannosidase gene (BT&#95;3784), a ribosomal binding site designed to modulate expression, a multiple cloning site to facilitate the cloning of genes of interest, and a transcriptional terminator. Using the Lactobacillus pepI as a reporter gene, mannan induction resulted in an increase of reporter activity in a time- and concentration-dependent manner with a wide range of activity. The endogenous BtcepA cephalosporinase gene was used to demonstrate the suitability of this novel expression system, enabling the isolation of a His-tagged version of BtCepA. We have also shown with experiments performed in mice that the system can be induced in vivo in the presence of an exogenous source of mannan. By enabling the controlled expression of endogenous and exogenous genes in B. thetaiotaomicron this novel inducer-dependent expression system will aid in defining the physiological role of individual genes and the functional analyses of their products.