HRP-labeling of bacterial extracellular vesicles for transmission electron microscopy imaging

Abstract. Extracellular vesicles (EVs) are small, membrane-bound carriers secreted by all cells. In humans, in addition to body’s own cells, also the bacteria of the microbiota releases bacterial EVs (BEVs). BEVs in human body can be examined from body fluids. For studying BEVs derived from gut microbiota, fecal samples are used. The isolation protocol of BEVs includes ultrafiltration, size-exclusion chromatography, and density gradient ultracentrifugation. For characterization, transmission electron microscopy and nanoparticle tracking analysis are commonly used. Several methods for labeling and imaging EVs have been demonstrated. Yet, none of them are suitable tracking EVs with high resolution imaging. In this thesis, a novel horseradish peroxidase (HRP) -labeling for BEVs for transmission electron microscopy (TEM) is established. For visualizing the HRP, carrying out a diaminobenzidine (DAB) -reaction had to be optimized. With multiple trials of in vitro experiments, a suitable protocol for labeling BEVs was established. As a result of successful HRP-labeling followed by DAB-reaction, BEVs appeared sharply stained on the edges. Further trials in ex vivo conditions were performed by injecting HRP-labeled BEVs to a muscle of a euthanized mouse. Thin sections of mouse muscle tissue in TEM revealed dark-stained clusters of structures resembling BEVs. The outcomes of the ex vivo mouse experiment were promising but will require optimizing in future studies.HRP-leimaus bakteeriperäisille solunulkoisille vesikkeleille läpäisyelektronimikroskooppikuvantamiseen. Tiivistelmä. Kaikki solut erittävät ulkopuolelleen kuljetukseen erikoistuneita pieniä kalvopeitteisiä rakkuloita, solunulkoisia vesikkeleitä. Solunulkoisia vesikkeleitä tuottavat paitsi elimistön omat solut, myös elimistössä elävän mikrobiomin solut. Ihmiselimistön suolistobakteerien tuottamia bakteeriperäisiä solunulkoisia vesikkeleitä tutkitaan ulosteesta, joiden eristämiseksi määrittämiseksi ja tutkimiseksi yhdistellään useita eri menetelmiä. Myös vesikkeleiden leimaamiseen ja kuvaamiseen on kehitetty useita menetelmiä, mutta mikään niistä ei sovellu solunulkoisten vesikkeleiden seurantaan läpäisyelektronimikroskoopilla. Tässä työssä testattiin menetelmää bakteeriperäisten solunulkoisten vesikkeleiden leimaamiseen piparjuuriperoksidaasilla (horseradish peroxidase, HRP). HRP:n havaitsemiseksi menetelmää varten optimoitiin myös diaminobentsidiinireaktio (DAB). Useiden kokeilujen jälkeen sopiva protokolla bakteeriperäisten solunulkoisten vesikkeleiden leimaamiseksi saavutettiin in vitro -olosuhteissa. Onnistuneen leimaamisen seurauksena läpäisyelektronimikroskoopissa nähtiin terävästi reunoiltaan värjäytyneitä bakteeriperäisiä solunulkoisia vesikkeleitä. In vitro -osuuden jälkeen kokeiluja jatkettiin ex vivo -olosuhteissa injektoimalla HRP-leimattuja vesikkeleitä lopetetun hiiren lihakseen. Läpäisyelektronimikroskoopissa lihaksesta tehdyissä ohutleikenäytteissä nähtiin rykelmiä tummaksi värjäytyneitä, vesikkeleitä muistuttavia rakenteita. Tulokset ex vivo -osuudesta olivat lupaavia, mutta vaativat vielä lisätutkimusta tulevaisuudessa

Microbiota and Extracellular Vesicles in Anti-PD-1/PD-L1 Therapy

Cancer is a deadly disease worldwide. In light of the requisite of convincing therapeutic methods for cancer, immune checkpoint inhibition methods such as anti-PD-1/PD-L1 therapy appear promising. Human microbiota have been exhibited to regulate susceptibility to cancer as well as the response to anti-PD-1/PD-L1 therapy. However, the probable contribution of bacterial extracellular vesicles (bEVs) in cancer pathophysiology and treatment has not been investigated much. bEVs illustrate the ability to cross physiological barriers, assemble around the tumor cells, and likely modify the tumor microenvironment (EVs). This systematic review emphasizes the correlation between cancer-associated extracellular vesicles, particularly bEVs and the efficacy of anti-PD-1/PD-L1 therapy. The clinical and pharmacological prospective of bEVs in revamping the contemporary treatments for cancer has been further discussed

Impact of extracellular vesicles of Staphylococcus aureus N305 on the immune response of the host

Impact of extracellular vesicles of Staphylococcus aureus N305 on the immune response of the host. STLOpenday

Outer membrane vesicles from probiotic and commensal Escherichia coli activate NOD1-mediated immune responses in intestinal epithelial cells

Gut microbiota plays a critical role in maintaining human intestinal homeostasis and host health. Bacterial extracellular vesicles are key players in bacteria-host communication, as they allow delivery of effector molecules into the host cells. Outer membrane vesicles (OMVs) released by Gram-negative bacteria carry many ligands of pattern recognition receptors that are key components of innate immunity. NOD1 and NOD2 cytosolic receptors specifically recognize peptidoglycans present within the bacterial cell wall. These intracellular immune receptors are essential in host defense against bacterial infections and in the regulation of inflammatory responses. Recent contributions show that NODs are also fundamental to maintain intestinal homeostasis and microbiota balance. Peptidoglycan from non-invasive pathogens is delivered to cytosolic NODs through OMVs, which are internalized via endocytosis. Whether this pathway could be used by microbiota to activate NOD receptors remains unexplored. Here, we report that OMVs isolated from the probiotic Escherichia coli Nissle 1917 and the commensal ECOR12 activate NOD1 signaling pathways in intestinal epithelial cells. NOD1 silencing and RIP2 inhibition significantly abolished OMV-mediated activation of NF-κB and subsequent IL-6 and IL-8 expression. Confocal fluorescence microscopy analysis confirmed that endocytosed OMVs colocalize with NOD1, trigger the formation of NOD1 aggregates, and promote NOD1 association with early endosomes. This study shows for the first time the activation of NOD1-signaling pathways by extracellular vesicles released by gut microbiota. Keywords: gut microbiota, Escherichia coli Nissle 1917, NF-κB activation, bacterial extracellular vesicles, NOD

Bacterial extracellular vesicles – brain invaders? A systematic review

IntroductionKnowledge on the human gut microbiota in health and disease continues to rapidly expand. In recent years, changes in the gut microbiota composition have been reported as a part of the pathology in numerous neurodegenerative diseases. Bacterial extracellular vesicles (EVs) have been suggested as a novel mechanism for the crosstalk between the brain and gut microbiota, physiologically connecting the observed changes in the brain to gut microbiota dysbiosis.MethodsPublications reporting findings on bacterial EVs passage through the blood–brain barrier were identified in PubMed and Scopus databases.ResultsThe literature search yielded 138 non-duplicate publications, from which 113 records were excluded in title and abstract screening step. From 25 publications subjected to full-text screening, 8 were excluded. The resulting 17 publications were considered for the review.DiscussionBacterial EVs have been described with capability to cross the blood–brain barrier, but the mechanisms behind the crossing remain largely unknown. Importantly, very little data exists in this context on EVs secreted by the human gut microbiota. This systematic review summarizes the present evidence of bacterial EVs crossing the blood–brain barrier and highlights the importance of future research on gut microbiota-derived EVs in the context of gut-brain communication across the blood–brain barrier

Roles of bacterial extracellular vesicles in systemic diseases

Accumulating evidence suggests that in various systems, not all bidirectional microbiota–host interactions involve direct cell contact. Bacterial extracellular vesicles (BEVs) may be key participants in this interkingdom crosstalk. BEVs mediate microbiota functions by delivering effector molecules that modulate host signaling pathways, thereby facilitating host–microbe interactions. BEV production during infections by both pathogens and probiotics has been observed in various host tissues. Therefore, these vesicles released by microbiota may have the ability to drive or inhibit disease pathogenesis in different systems within the host. Here, we review the current knowledge of BEVs and particularly emphasize their interactions with the host and the pathogenesis of systemic diseases

Isolation and Analysis of Extracellular Vesicles from Lactic Acid Bacteria

Extracellular Vesicles, also referred to as EVs, are spherical lipid membrane-bound vesicles produced by both Gram positive and Gram negative bacteria. These vesicles are secreted into the extracellular space and play important functions in cellular and host communication, elimination of competitors, virulence, detoxification of environmental stress, and nutrition sensing. They are often packed with proteins, enzymes, lipids, and nucleic acids like DNA or RNA molecules among other biological entities. Streptococcus thermophilus is a lactic acid bacterium (LAB), inhabiting the human digestive tract, that has been shown to produce EVs. The bacterial flora has a great impact on the host immune system, metabolism, and neurological processes, however, not a lot is known about the biochemical pathways behind this impact. Since extracellular vesicles are involved in host communication, they play a key role in the impact that bacterial flora has on the biochemical processes of a host. Therefore S. Thermophilus was grown aerobically at 37° C in M17 media, two other LABs were grown including Lactobacillus acidophilus, and Lactobacillus bulgaricus. The extracellular vesicles will then be isolated through centrifugation, then the EVs content will be analyzed further. Size comparison can be conducted using gel electrophoresis, on various RNA molecules hypothesized to be held within the membranes of EVs. A previous study in this lab isolated the AsdS sRNA molecule, that is 152 base pairs in length, and is involved in quorum sensing. This gene is conserved among Streptococcus species and can be observed in S. pyogenes as the MarS. Since S. thermophilus is a non-pathogenic species the Asd gene cannot be involved in virulence as MarS is responsible for virulence in S. pyogenes. Based on functional predictions, AsdS is responsible for intraspecies communication, biofilm formation, and transport

Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction

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Proteomic profile of extracellular vesicles released by Lactiplantibacillus plantarum BGAN8 and their internalization by non-polarized HT29 cell line

In recent years the role of extracellular vesicles (EVs) of Gram-positive bacteria in host-microbe cross-talk has become increasingly appreciated, although the knowledge of their biogenesis, release and host-uptake is still limited. The aim of this study was to characterize the EVs released by the dairy isolate Lactiplantibacillus plantarum BGAN8 and to gain an insight into the putative mechanism of EVs uptake by intestinal epithelial cells. The cryo-TEM observation undoubtedly demonstrated the release of EVs (20 to 140 nm) from the surface of BGAN8, with exopolysaccharides seems to be part of EVs surface. The proteomic analysis revealed that the EVs are enriched in enzymes involved in central metabolic pathways, such as glycolysis, and in membrane components with the most abundant proteins belonging to amino acid/peptide ABC transporters. Putative internalization pathways were evaluated in time-course internalization experiments with non-polarized HT29 cells in the presence of inhibitors of endocytic pathways: chlorpromazine and dynasore (inhibitors of clathrin-mediated endocytosis-CME) and filipin III and nystatin (disrupting lipid rafts). For the first time, our results revealed that the internalization was specifically inhibited by dynasore and chlorpromazine but not by filipin III and nystatin implying that one of the entries of L. plantarum vesicles was through CME pathway