Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway

Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.

Bacterial outer membrane vesicle-based cancer nanovaccines

Tumor vaccines,a type of personalized tumor immunotherapy,have developed rapidly in recent decades.These vaccines evoke tumor antigen-specific T cells to achieve immune recognition and killing of tumor cells.Because the immunogenicity of tumor antigens alone is insufficient,immune adjuvants and nanocarriers are often required to enhance anti-tumor immune responses.At present,vaccine carrier development often integrates nanocarriers and immune adjuvants.Among them,outer membrane vesicles(OMVs)are receiving increasing attention as a delivery platform for tumor vaccines.OMVs are natural nanovesicles derived from Gramnegative bacteria,which have adjuvant function because they contain pathogen associated molecular patterns.Importantly,OMVs can be functionally modified by genetic engineering of bacteria,thus laying a foundation for applications as a delivery platform for tumor nanovaccines.This review summarizes 5 aspects of recent progress in,and future development of,OMV-based tumor nanovaccines:strain selection,heterogeneity,tumor antigen loading,immunogenicity and safety,and mass production of OMVs.

Newly Detected Transmission of bla_(KPC-2) by Outer Membrane Vesicles in Klebsiella Pneumoniae

Objective The prevalence of carbapenem-resistant Klebsiella pneumoniae(CR-KP)is a global public health problem.It is mainly caused by the plasmid-carried carbapenemase gene.Outer membrane vesicles(OMVs)contain toxins and other factors involved in various biological processes,includingβ-lactamase and antibiotic-resistance genes.This study aimed to reveal the transmission mechanism of OMV-mediated drug resistance of Klebsiella(K.)pneumoniae.Methods We selected CR-KP producing K.pneumoniae carbapenemase-2(KPC-2)to study whether they can transfer resistance genes through OMVs.The OMVs of CR-KP were obtained by ultracentrifugation,and incubated with carbapenem-sensitive K.pneumoniae for 4 h.Finally,the carbapenem-sensitive K.pneumoniae was tested for the presence of bla_(KPC-2)resistance gene and its sensitivity to carbapenem antibiotics.Results The existence of OMVs was observed by the electron microscopy.The extracted OMVs had bla_(KPC-2)resistance gene.After incubation with OMVs,bla_(KPC-2)resistance gene was detected in sensitive K.pneumoniae,and it became resistant to imipenem and meropenem.Conclusion This study demonstrated that OMVs isolated from KPC-2-producing CR-KP could deliver bla_(KPC-2)to sensitive K.pneumoniae,allowing the bacteria to produce carbapenemase,which may provide a novel target for innovative therapies in combination with conventional antibiotics for treating carbapenem-resistant Enterobacteriaceae.

Proteomic and Functional Analyses of Outer Membrane Vesicles Secreted by Vibrio splendidus

Vibrio splendidus is an important opportunistic pathogen ubiquitously present in the marine environment,exhibiting virulence to a variety of cultured animals.The extracellular products secreted by V.splendidus are crucial to bacterial survival and virulence.In this study,the secretion of outer membrane vesicles(OMVs)by V.splendidus was determined,purified,and morphologically characterized.The protein composition of OMVs was analyzed by proteomic analysis.The results showed that approximately 120 proteins were contained in these OMVs,including outer membrane proteins,flagellins,ABC transporters,protease,and iron regulation proteins,etc.,which were involved in bacterial motility,formation of biofilms and the cell membrane components,and cellular localization based on their structural molecule activity,passive transmembrane transporter activity,channel activity,neurotransmitter receptor activity,extracellular ligand-gated ion channel activity,glutamate receptor activity,ligand-gated ion channel activity,and transmembrane signaling receptor activity.To explore the biological functions of OMVs in V.splendidus,the effects of OMVs on the bacterial adaption to iron limitation,antibiotic,and the coelomic fluid of the Apostichopus japonicus were confirmed.This study is the first time to show that V.splendidus secretes OMVs,and OMVs carry functional proteins that enhance bacterial survival under various stresses.

Dynamic shielding of bacterial outer membrane vesicles for safe and efficient chemo-immunotherapy against tumors

Bacterial outer membrane vesicles(OMVs)are potent immunostimulants of regulating the tumor microenvironment(TME)for immunotherapy,and can be used to deliver drugs.However,the severe systemic inflammatory response triggered by OMVs upon intravenous(i.v.)injection has limited their application.Here,we developed a safe and effective strategy by conjugating doxorubicin-loaded serum albumin(SA-DOX,AD)onto the surface of OMVs using a matrix metalloproteinase(MMP)-cleavable peptide linker(cL).This approach enabled the dynamic shielding of OMVs to reduce the systemic side effects while simultaneously enhancing the anti-tumor effects through chemo-immunotherapy.Specifically,the resulting OMV-cL-AD formulation exhibited significantly enhanced accumulation at the tumor site after i.v.administration,facilitated by the SA decoration on the OMVs surface.Subsequently,the shield on the OMV-cL-AD was cleaved by the over-expressed MMP in the TME,leading to the release of both OMVs and AD.This process provided OMV-induced immunotherapy and DOX-induced chemotherapy,resulting in synergistic tumor inhibition.In conclusion,our work demonstrated the potential of OMV-cL-AD as an effective immunochemotherapy strategy that can prolong the survival time of mice without inducing side effects.

Bacterial outer membrane vesicles in the fight against cancer

Bacterial outer membrane vesicles(OMVs)are diminutive vesicles naturally released by Gram-negative bacteria.These vesicles possess distinctive characteristics that attract attention for their potential use in drug administration and immunotherapy in cancer treatment.Therapeutic medicines may be delivered via OMVs directly to the tumor sites,thereby minimizing exposure to healthy cells and lowering the risk of systemic toxicity.Furthermore,the activation of the immune system by OMVs has been demonstrated to facilitate the recognition and elimination of cancer cells,which makes them a desirable tool for immunotherapy.They can also be genetically modified to carry specific antigens,immunomodulatory compounds,and small interfering RNAs,enhancing the immune response to cancerous cells and silencing genes associated with disease progression.Combining OMVs with other cancer treatments like chemotherapy and radiation has shown promising synergistic effects.This review highlights the crucial role of bacterial OMVs in cancer,emphasizing their potential as vectors for novel cancer targeted therapies.As researchers delve deeper into the complexities of these vesicles and their interactions with tumors,there is a growing sense of optimism that this avenue of study will bring positive outcomes and renewed hope to cancer patients in the foreseeable future.

Suppression of host plant defense by bacterial small RNAs packaged in outer membrane vesicles

Noncoding small RNAs(sRNAs)packaged in bacterial outer membrane vesicles(OMVs)function as novel mediators of interspecies communication.While the role of bacterial sRNAs in enhancing virulence is well established,the role of sRNAs in the interaction between OMVs from phytopathogenic bacteria and their host plants remains unclear.In this study,we employ RNA sequencing to characterize differentially pack-aged sRNAs in OMVs of the phytopathogen Xanthomonas oryzae pv.oryzicola(Xoc).Our candidate sRNA(Xosr001)was abundant in OMVs and involved in the regulation of OsJMT1 to impair host stomatal immu-nity.Xoc loads Xosr001 into OMVs,which are specifically ttransferred into the mechanical tissues of rice leaves.Xosr001 suppresses OsJMT1 transcript accumulation in vivo,leading to a reduction in MeJA accu-mulation in rice leaves.Furthermore,the application of synthesized Xosr001 sRNA to the leaves of OsJMT1-HA-OE transgenic line results in the suppression of OsJMT1 expression by Xosr001.Notably,the OsJMT1-HA-OE transgenic line exhibited attenuated stomatal immunity and disease susceptibility upon infection with DXosr001 compared to Xoc.These results suggest that Xosr001 packaged in Xoc OMVs functions to suppress stomatal immunity in rice.

Bacterial outer membrane vesicles-based therapeutic platform eradicates triple-negative breast tumor by combinational photodynamic/chemo-/immunotherapy

Bacterial outer membrane vesicles(OMVs)are potent immuno-stimulating agents and have the potentials to be bioengineered as platforms for antitumor nanomedicine.In this study,OMVs are demonstrated as promising antitumor therapeutics.OMVs can lead to beneficial M2-to-M1 polarization of macrophages and induce pyroptosis to enhance antitumor immunity,but the therapeutic window of OMVs is narrow for its toxicity.We propose a bioengineering strategy to enhance the tumor-targeting ability of OMVs by macrophage-mediated delivery and improve the antitumor efficacy by co-loading of photosensitizer chlorin e6(Ce6)and chemotherapeutic drug doxorubicin(DOX)into OMVs as a therapeutic platform.We demonstrate that systemic injection of the DOX/Ce6-OMVs@M therapeutic platform,providing combinational photodynamic/chemo-/immunotherapy,eradicates triple-negative breast tumors in mice without side effects.Importantly,this strategy also effectively prevents tumor metastasis to the lung.This OMVs-based strategy with bioengineering may serve as a powerful therapeutic platform for a synergic antitumor therapy.

Effects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigera

Brush border membrane vesicles （BBMV） isolated from insect midguts have been widely used to study CrylA binding proteins. Sample preparation is important in two- dimensional electrophoresis （2-DE）, so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）. All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2- DE analysis.

Personalized cancer vaccines from bacteria-derived outer membrane vesicles with antibody-mediated persistent uptake by dendritic cells

Nanocarriers with intrinsic immune adjuvant properties can activate the innate immune system while delivering tumor antigen,thus efficiently facilitating antitumor adaptive immunity.Bacteria-derived outer membrane vesicles(OMVs)are an excellent candidate due to their abundance of pathogen associated molecular patterns.However,during the uptake of OMVs by dendritic cells(DCs),the interaction between lipopolysaccharide and toll-like receptor 4 induces rapid DC maturation and uptake blockage,a phenomenon we refer to as“maturation-induced uptake obstruction"(MUO).Herein we decorated OMV with the DC-targeting aDEC205 antibody(OMV-DEC),which endowed the nanovaccine with an uptake mechanism termed as 4<not restricted to maturation via antibody modifying”(Normandy),thereby overcoming the MUO phenomenon.We also proved the applicability of this nanovaccine in identifying the human tumor neoantigens through rapid antigen display.In summary,this engineered OMV represents a powerful nanocarrier for personalized cancer vaccines,and this antibody modification strategy provides a reference to remodel the DC uptake pattern in nanocarrier design.

Decoration of bacterial outer membrane vesicles with liposomes

In this study, liposomes were used to decorate bacterial outer membrane vesicles(OMVs), and decorated OMVs were evaluated in vitro. The OMVs of Pseudomonas aeruginosa were extracted by pressure-induced ammonium sulfate precipitation,and their particle size, distribution, zeta potential, protein content and stability were determined. Several types of liposomes were prepared by thin film dispersion method, and the OMVs were decorated by vortexing, sonication and extrusion, respectively. The interaction between liposome and OMV was studied with fluorescence resonance energy transfer(FRET) method. The results showed that the OMVs were spherical in shape and negatively charged. The vortexing method exerted little effect on the particle size and distribution of the decorated OMVs. The sonication process reduced the particle size and distribution of OMVs. FRET experiment indicated that the OMVs were decorated through membrane fusion. The above-mentioned results indicated that liposomes could successfully decorate OMVs, and decorated OMVs certainly widened their applications.

Binding of Cry δ-endotoxins to brush border membrane vesicles of Helicoverpa armigera and Helicoverpa punctigera （Lepidoptera： Noctuidae）

The relatively low susceptibility ofHelicoverpa armigera to CrylAc, its history of resistance to chemical insecticides and the seasonal decline in expression of CrylAc in transgenic cotton necessitated the development of cotton expressing two insecticidal proteins to provide sustainable control of this multinational pest. To manage the resistance issue, it was essential that the second insecticidal protein have a significantly different mode of action to CrylAc. A common feature of resistance to CrylA proteins in several species as well as H. armigera has been a change in the binding site. A study of binding sites for some Cry proteins in the brush border membrane vesicles （BBMV） ofH. armigera and Helicoverpa punctigera was undertaken. The binding affinity for CrylAc was higher than for CrylAb, matching their relative toxicities, and CrylAc and CrylAb were found to share at least one binding site in both I-1. armigera and I-1. punctigera. However Cry2Aa did not compete with CrylAc for binding and so could be used in transgenic cotton in combination with CrylAc to control H. armigera and manage resistance. Variation in the susceptibilities of three different H. armigera strains to CrylAc correlated with the parameter Bmax/Kcom.

ACTIVE CALCIUM TRANSPORT IN PLASMA MEMBRANE VESICLES FROM DEVELOPING COTYLEDONS OF COMMON BEAN

Plasma membrane vesicles were prepared from the developing cotyledons of common beau (Phaseolus vulgaris L cv Diyundou) by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes.The putative plasma membrane fraction was minimally contaminated by membranes Other than plasma membrane and hence was of high purity. It exhibited a Ca2+dependent ATPase activity, which was inhibited by 1μ mol/L EB and promoted by calcium ionophore A23187. Such an activity was responsible for the observed ATPdependent 45Ca2+ uptake into inside-out plasma membrane vesicles. This process was stimulated by 0.6μmol/L CaM and 20μmol/L IAA but inhibited by 2μmol/L ABA and abolished by A23187. Possible role of cytoplasmic Ca2+ in mediating phytohormones activity is discussed.

An AIE-active probe for efficient detection and high-throughput identification of outer membrane vesicles

Rapid detection and quantification of outer membrane vesicle(OMV)are of both scientific value and clinical implications.However,limited tools are available for investigations of OMVs.Herein,we report a novel fluorescent probe with aggregation-induced emission(AIE)characteristics,namely,OEO-TPE-MEM(OTM),for OMV detection.OTM emits faintly in an aqueous medium,but its fluorescence could be effectively turned on upon interacting with bacteria bodies and OMVs produced by Gram-negative bacteria.Notably,OTM could provide quantitative information on bacterial membrane remodeling and OMV secretion and be applied to high-throughput screening of OMV-inducing agents.This study presents a powerful AIE probe for imaging and quantitative analysis of bacteria envelop and derived OMVs,which might be applied for evaluating research and clinical antimicrobial materials in future studies.

Role of Outer Membrane Vesicles in Bacterial Physiology and Host Cell Interactions

Outer membrane vesicles(OMVs)are spherical particles shed from the outer membrane of Gram-negative bacteria,which contain the typical components present in the outer membrane,although enrichment of specific molecules may occur,and furthermore a variety of periplasmic components and occasionally some inner membrane or cytoplasmic fractions.Although the detailed mechanisms of OMV biogenesis are not fully illuminated yet,several models have been proposed that demonstrate OMV biogenesis is an orchestrated well-regulated process.OMV secretion offers a way for both intra-and inter-species bacterial communication and for interaction or modulation of the bacterial environment.Therefore,OMVs have proven to be functionally versatile and important for bacterial physiology and survival of the host environment.In the host,OMVs are internalized via host cell endocytosis pathways,allowing them to subsequently trigger a variety of cellular responses.In this review,we discuss the recent advances in establishing the mechanisms involved in OMV biogenesis and the impact of OMVs on bacterial physiology and intracellular modulation of the host.

CD133 and membrane microdomains:Old facets for future hypotheses

Understanding all facets of membrane microdomains in normal and cancerous cells within the digestive tract is highly important,not only from a clinical point of view,but also in terms of our basic knowledge of cellular transformation.By studying the normal and cancer stem cell-associated molecule CD133 (prominin-1),novel aspects of the organization and dynamics of polarized epithelial cells have been revealed during the last decade.Its association with particular membrane microdomains is highly relevant in these contexts and might also offer new avenues in diagnosis and/or targeting of cancer stem cells.

A smart DNA hydrogel for isolation of bacterial outer membrane vesicles and localized cancer immunotherapy

Bacterial outer membrane vesicles(OMVs)have shown great potential in cancer immunotherapy.The isolation of OMVs from complex media with high purity and high bioactivity is the prerequisite of therapeutic applications,which remains highly challenging.Herein,we report a smart DNA hydrogel for the efficient isolation of OMVs from bacterial culture medium,which is further applied for localized cancer immunotherapy.The DNA hydrogel is constructed through the cross-linking of two ultralong DNA chains generated via rolling circle amplification(RCA).One chain contains polyvalent GN6 aptamer for the specific capture of OMVs,and the other contains polyvalent programmed death-1(PD-1)aptamer for the blocking of PD-1 immune checkpoint on the surface of T lymphocytes.The OMVs capsulated by DNA hydrogel maintain high immunostimulatory bioactivity.In the mouse model of melanoma,this OMVs-containing DNA hydrogel shows a remarkable tumor inhibition rate of∼95%.This smart DNA hydrogel represents a promising biomedical platform for the efficient isolation of bacterial-derived OMVs,and provides a powerful strategy for cancer immunotherapy.

Novel method for extracting exosomes of hepatocellular carcinoma cells

AIM: To develop a novel method for the rapid and efficient extraction of exosomes secreted by tumor cells.

Tailoring therapeutics via a systematic beneficial elements comparison between photosynthetic bacteria-derived OMVs and extruded nanovesicles

Photosynthetic bacteria(PSB)has shown significant potential as a drug or drug delivery system owing to their photothermal capabilities and antioxidant properties.Nevertheless,the actualization of their potential is impeded by inherent constraints,including their considerable size,heightened immunogenicity and compromised biosafety.Conquering these obstacles and pursuing more effective solutions remains a top priority.Similar to extracellular vesicles,bacterial outer membrane vesicles(OMVs)have demonstrated a great potential in biomedical applications.OMVs from PSB encapsulate a rich array of bioactive constituents,including proteins,nucleic acids,and lipids inherited from their parent cells.Consequently,they emerge as a promising and practical alternative.Unfortunately,OMVs have suffered from low yield and inconsistent particle sizes.In response,bacteria-derived nanovesicles(BNVs),created through controlled extrusion,adeptly overcome the challenges associated with OMVs.However,the differences,both in composition and subsequent biological effects,between OMVs and BNVs remain enigmatic.In a groundbreaking endeavor,our study meticulously cultivates PSB-derived OMVs and BNVs,dissecting their nuances.Despite minimal differences in morphology and size between PSB-derived OMVs and BNVs,the latter contains a higher concentration of active ingredients and metabolites.Particularly noteworthy is the elevated levels of lysophosphatidylcholine(LPC)found in BNVs,known for its ability to enhance cell proliferation and initiate downstream signaling pathways that promote angiogenesis and epithelialization.Importantly,our results indicate that BNVs can accelerate wound closure more effectively by orchestrating a harmonious balance of cell proliferation and migration within NIH-3T3 cells,while also activating the EGFR/AKT/PI3K pathway.In contrast,OMVs have a pronounced aptitude in anti-cancer efforts,driving macrophages toward the M1 phenotype and promoting the release of inflammatory cytokines.Thus,our findings not only provide a promising methodological framework but also establish a definitive criterion for discerning the optimal application of OMVs and BNVs in addressing a wide range of medical conditions.

SNARE complex in axonal guidance and neuroregeneration

Through complex mechanisms that guide axons to the appropriate routes towards their targets, axonal growth and guidance lead to neuronal system formation. These mechanisms establish the synaptic circuitry necessary for the optimal performance of the nervous system in all organisms. Damage to these networks can be repaired by neuroregenerative processes which in turn can re-establish synapses between injured axons and postsynaptic terminals. Both axonal growth and guidance and the neuroregenerative response rely on correct axonal growth and growth cone responses to guidance cues as well as correct synapses with appropriate targets. With this in mind, parallels can be drawn between axonal regeneration and processes occurring during embryonic nervous system development. However, when studying parallels between axonal development and regeneration many questions still arise; mainly, how do axons grow and synapse with their targets and how do they repair their membranes, grow and orchestrate regenerative responses after injury. Major players in the cellular and molecular processes that lead to growth cone development and movement during embryonic development are the Soluble N-ethylamaleimide Sensitive Factor （NSF） Attachment Protein Receptor （SNARE） proteins, which have been shown to be involved in axonal growth and guidance. Their involvement in axonal growth, guidance and neuroregeneration is of foremost importance, due to their roles in vesicle and membrane trafficking events. Here, we review the recent literature on the involvement of SNARE proteins in axonal growth and guidance during embryonic development and neuroregeneration.