Bacterial Outer Membrane Vesicles Research Articles

Microbial Trojan Horses: Virulence Factors as Key Players in Neurodegenerative Diseases

Changes in population demographics indicate that the elderly population will reach 2.1 billion worldwide by 2050. In parallel, there will be an increase in neurodegenerative diseases such as Alzheimer’s and Parkinson’s. This review explores dysbiosis occurring in these pathologies and how virulence factors contribute to the worsening or development of clinical conditions, and it summarizes existing and potential ways to combat microorganisms related to these diseases. Microbiota imbalances can contribute to the progression of neurodegenerative diseases by increasing intestinal permeability, exchanging information through innervation, and even acting as a Trojan horse affecting immune cells. The microorganisms of the microbiota produce virulence factors to protect themselves from host defenses, many of which contribute to neurodegenerative diseases. These virulence factors are expressed according to the genetic composition of each microorganism, leading to a wide range of factors to be considered. Among the main virulence factors are LPS, urease, curli proteins, amyloidogenic proteins, VacA, and CagA. These factors can also be packed into bacterial outer membrane vesicles, which transport proteins, RNA, and DNA, enabling distal communication that impacts various diseases, including Alzheimer’s and Parkinson’s.

A "plug-and-display" nanoparticle based on attenuated outer membrane vesicles enhances the immunogenicity of protein antigens.

As natural nanoparticle, the bacterial outer membrane vesicles (OMV) hold great potential in protein vaccines because of its self-adjuvant properties and good biocompatibility. However, the inherent immunotoxicity seriously hampers the application of OMV as protein antigens delivery carrier. Here, an attenuated OMV was constructed by elimination of the flagella protein from its surface and removal of the phosphate group of LPS at position one via gene-editing strategy. The gene-edited outer membrane vesicles (EMV) effectively reduced the levels of pro-inflammatory factors TNF-α and IL-6 in mouse blood by at least 10-fold and 15-fold respectively, compared to wild type OMV (WT-OMV). Importantly, protein antigens are conveniently displayed on EMV by employing a plug-and-display procedure, whereby the exterior of biotinylated EMV can be readily decorated with a synthetic protein comprised of target antigen fused to a biotin-binding protein. EMV greatly increased the uptake of antigen by dendritic cells (DCs) and promoted their maturation. EMV-antigen complex induces a robust antigen-specific antibody responses and cellular immune responses. We propose that EMV have great potential as protein antigens delivery vehicle for preventing different infectious diseases.

An OMV-Based Nanovaccine as Antigen Presentation Signal Enhancer for Cancer Immunotherapy.

Antigen-presenting cells (APCs) process tumor vaccines and present tumor antigens as the first signals to T cells to activate anti-tumor immunity, which process requires the assistance of co-stimulatory second signals on APCs. The immune checkpoint programmed death ligand 1 (PD-L1) not only mediates the immune escape of tumor cells but also acts as a co-inhibitory second signal on APCs. The serious dysfunction of second signals due to the high expression of PD-L1 on APCs in the tumor body results in the inefficiency of tumor vaccines. To overcome this challenge, a previously established Plug-and-Display tumor vaccine platform based on bacterial outer membrane vesicles (OMVs) is developed into an "Antigen Presentation Signal Enhancer" (APSE) by surface-modifying PD-L1 antibodies (αPD-L1). While delivering tumor antigens, APSE can activate the expression of co-stimulatory second signals in APCs due to the high immunogenicity of OMVs. More importantly, the surface-modified αPD-L1 binds to the co-inhibitory signals PD-L1, potentially restoring CD80 function and ensuring efficient co-stimulatory second signals and activation of anti-tumor immunity. The results reveal the importance of PD-L1 blockage in the initiation process of anti-tumor immunity, and the second signal modulation capability of APSE can expand the application potential of cancer vaccines to less immunogenic malignancies.

Endotoxin-Free Outer Membrane Vesicles for Safe and Modular Anticancer Immunotherapy.

Bacterial outer membrane vesicles (OMVs) have emerged as promising vehicles for anticancer drug delivery due to their inherent tumor tropism, immune-stimulatory properties, and potential for functionalization with therapeutic proteins. Despite their advantages, the high lipopolysaccharide (LPS) endotoxin content in the OMVs raises significant safety and regulatory challenges. In this work, we produce LPS-attenuated and LPS-free OMVs and systematically assess the effects of LPS modification on OMVs' physicochemical characteristics, membrane protein content, immune-stimulatory capacity, tolerability, and anticancer efficacy. Our findings reveal that LPS removal increased the maximal tolerated dose of the OMVs by over 25-fold. When adjusted for comparable safety profiles, LPS-free OMVs exhibit superior anticancer effects compared with wild-type OMVs. Mechanistic investigations indicate that the LPS removal obviates immune cell death caused by LPS and reduces the negatory effects of wild type of OMVs on tumor immune cell infiltrates. We further show the functionality of the LPS-free OMV through the incorporation of an IL-2 variant protein (Neo-2/15). This functionalization augments OMV's ability of the OMV to inhibit tumor growth and promote lymphocyte infiltration into the tumor microenvironment. This study presents a safe and functionalizable OMV with improved translational prospect.

Platelet backpacking nanoparticles based on bacterial outer membrane vesicles enhanced photothermal-immune anti-tumor therapy.

Bacterial outer membrane vesicles (OMVs), produced by Gram-negative bacteria, retain the immunostimulatory capacity of parental bacteria. OMVs have been recognized as potent natural immune adjuvants and drug delivery vehicles. Photothermal therapy that triggers immunogenic cell death further stimulates the immune system by releasing damage-associated molecular patterns. This therapeutic effect can be synergized with OMVs to achieve enhanced anti-tumor outcomes. We also observed that tumors can recruit platelets. Leveraging the phenomenon, we have innovatively employed platelets as "couriers" to boost the tumor-targeting delivery efficiency of both OMVs and photothermal agents. In detail, based on OMVs, we meticulously engineered nanoparticles (IR780-SLN@O-P) with platelet-binding capacity. These "courier" platelets carry "cargo" IR780-SLN@O-P NPs to tumor sites via P-selectin, ensuring targeted delivery. Under laser irradiation, the photothermal agents generate significant photothermal effects, which combined with the immune-stimulating properties of OMVs, creating a robust anti-tumor immune response. For "cold" tumors such as triple-negative breast cancer (TNBC), our IR780-SLN@O-P NPs not only prolonged the survival of mice bearing 4T1 orthotopic tumors, but also significantly suppressed tumor growth. Moreover, they facilitated dendritic cell maturation and the infiltration of CD8+ T cells to ameliorate the immunosuppressive tumor environment. Our research aims to highlight the unique advantages of OMVs and explore the potential of the tumor-targeting strategy that synergizes photothermal therapy with immunotherapy. We hope that our findings can offer insights into TNBC clinical treatments.

Bacterial Outer Membrane Vesicle (OMV)-Encapsulated TiO2 Nanoparticles: A Dual-Action Strategy for Enhanced Radiotherapy and Immunomodulation in Oral Cancer Treatment.

Oral squamous-cell carcinoma (OSCC) poses significant treatment challenges due to its high recurrence rates and the limitations of current therapies. Titanium dioxide (TiO2) nanoparticles are promising radiosensitizers, while bacterial outer membrane vesicles (OMVs) are known for their immunomodulatory properties. This study investigates the potential of OMV-encapsulated TiO2 nanoparticles (TiO2@OMV) to combine these effects for improved OSCC treatment. TiO2 nanoparticles were synthesized using a hydrothermal method and encapsulated within OMVs derived from Escherichia coli. The TiO2@OMV carriers were evaluated for their ability to enhance radiosensitivity and stimulate immune responses in OSCC cell lines. Reactive oxygen species (ROS) production, macrophage recruitment, and selective cytotoxicity toward cancer cells were assessed. TiO2@OMV demonstrated significant radiosensitization and immune activation compared to unencapsulated TiO2 nanoparticles. The system selectively induced cytotoxicity in OSCC cells, sparing normal cells, and enhanced ROS generation and macrophage-mediated antitumor responses. This study highlights TiO2@OMV as a dual-action therapeutic platform that synergizes radiotherapy and immunomodulation, offering a targeted and effective strategy for OSCC treatment. The approach could improve therapeutic outcomes and reduce the adverse effects associated with conventional therapies.

An Oral Nanovaccine Secreted by Genetically Engineered and Ultrasound‐Responsive Bacteria for Colon Cancer Immunotherapy

AbstractColorectal cancers represent a major global morbidity and mortality burden, neccessitating improved treatment paradigms. In this work, an ingestible, genetically engineered Escherichia coli (E. coli) 1917 termed “E. coli (AH1‐CDA‐Co1)” is designed that upon ultrasound exposure secretes bacterial outer membrane vesicles (OMV) incorporating the AH1 tumor rejection epitope, an enzyme producing the stimulator of interferon genes (STING) agonist CDA, and the microfold cell‐targeting peptide Co1. For oral administration, a polydopamine system (iPDA) coating on bacteria is exploited to resist the acidic condition in stomach, increase the bacterial survival, and prolong the intestinal transit time. Upon harmless ultrasound exposure, sustained secretion of engineered OMV vaccines is triggered that efficiently cross the intestinal epithelium. Both cyclic GMP–AMP synthase (cGAS)‐STING and TLR4 innate immune signaling pathways are activated, triggering long‐term antigen‐specific immune responses that overcome the immunosuppressive tumor microenvironment. In subcutaneous and orthotopic murine colorectal tumor models, the E. coli (AH1‐CDA‐Co1)@iPDA system inhibits tumor growth and prolongs survival without recurrence. E. coli (AH1‐CDA‐Co1)@iPDA also inhibits tumor growth and recurrence in a postoperative orthotopic colonrectal tumor model of lymph node metastases. Taken together, E. coli (AH1‐CDA‐Co1)@iPDA demonstrates a potent oral vaccine system for improved colon cancer immunotherapy.

Antimonene and bacterial outer membrane vesicle modification nanoplatform enhanced photothermal immunotherapy

Antimonene and bacterial outer membrane vesicle modification nanoplatform enhanced photothermal immunotherapy

Bioorthogonal Engineering of Bacterial Outer Membrane Vesicles for NIR-II Fluorescence Imaging-Guided Synergistic Enhanced Immunotherapy.

The efficacy of immunotherapy in treating triple-negative breast cancer (TNBC) has been restricted due to its low immunogenicity and suppressive immune microenvironment. Bacterial outer membrane vesicles (OMVs) have emerged as innovative immunotherapeutic agents in antitumor therapy by stimulating the innate immune system, but intricate modifications and undesirable multiple dose administration severely hinder their utility. Herein, a two-step bacterial metabolic labeling technique was utilized for the bioorthogonal engineering of OMVs. At first, d-propargylglycine (DPG, an alkyne-containing d-amino acid) was introduced into the incubation process of probiotic Escherichia coli 1917 (Ecn) to produce DPG-functionalized OMVs, which were subsequently conjugated with azide-functionalized new indocyanine green (IR820) to yield OMV-DPG-IR820. The combination of phototherapy and immunostimulation of OMV-DPG-IR820 effectively arouses adaptive immune responses, causing maturation of dendritic cells, infiltration of T cells, repolarization of the M2 macrophage to the M1 macrophage, and upregulation of inflammatory factors. Remarkably, OMV-DPG-IR820 demonstrated tumor-targeting capabilities with guidance provided by near-infrared II (NIR-II) fluorescence imaging, leading to remarkable inhibition on both primary and distant tumors and preventing metastasis without causing noticeable adverse reactions. This study elucidates a sophisticated bioorthogonal engineering strategy for the design and production of functionalized OMVs and provides novel perspectives on the microbiome-mediated reversal of TNBC through a precise and efficient immunotherapy.

Pseudomonas aeruginosa- mediated cardiac dysfunction is driven by extracellular vesicles released during infection.

Pseudomonas aeruginosa (P.a.) is a gram-negative, opportunistic bacterium abundantly present in the environment. Often P.a. infections cause severe pneumonia, if left untreated. Surprisingly, up to 30% of patients admitted to the hospital for community- acquired pneumonia develop adverse cardiovascular complications such as myocardial infarction, arrhythmia, left ventricular dysfunction, and heart failure. However, the underlying mechanism of infection-mediated cardiac dysfunction is not yet known. Recently, we demonstrated that P.a. infection of the lungs led to severe cardiac electrical abnormalities and left ventricular dysfunction with limited P.a. dissemination to the heart tissue. To understand the mechanism of cardiac dysfunction during P.a. infection, we utilized both in vitro and in vivo models. Our results revealed that inflammatory cytokines contribute but are not solely responsible for severe contractile dysfunction in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Instead, exposure of hiPSC-CMs with conditioned media from P.a. infected human monocyte-derived macrophages (hMDMs) was sufficient to cause severe contractile dysfunction and arrhythmia in hiPSC-CMs. Specifically, exosomes released from infected hMDMs and bacterial outer membrane vesicles (OMVs) are the major drivers of cardiomyocyte contractile dysfunction. By using LC-MS/MS, we identified bacterial proteins, including toxins that are packaged in the exosomes and OMVs, which are responsible for contractile dysfunction. Furthermore, we demonstrated that systemic delivery of bacterial OMVs to mice caused severe cardiac dysfunction, mimicking the natural bacterial infection. In summary, we conclude that OMVs released during infection enter circulation and drive cardiac dysfunction.

Outer Membrane Vesicle-Cancer Hybrid Membrane Coating Indocyanine Green Nanoparticles for Enhancing Photothermal Therapy Efficacy in Tumors.

Cell membrane-coated nanomaterials are increasingly recognized as effective in cancer treatment due to their unique benefits. This study introduces a novel hybrid membrane coating nanoparticle, termed cancer cell membrane (CCM)-outer membrane vesicle (OMV)@Lip-indocyanine green (ICG), which combines CCMs with bacterial OMV to encapsulate ICG-loaded liposomes. Comprehensive analyses were conducted to assess its physical and chemical properties as well as its functionality. Demonstrating targeted delivery capabilities and good biocompatibility, CCM-OMV@Lip-ICG nanoparticles showed promising photothermal and immunotherapeutic effects in tumor models. By inducing hyperthermia-induced tumor therapy and bolstering antitumor immunity, CCM-OMV@Lip-ICG nanoparticles exhibit a synergistic therapeutic effect, providing a new perspective for the management of cancer.

Lipopolysaccharide Imprinted Polymers for Specific Recognition of Bacterial Outer Membrane Vesicles.

Outer membrane vesicles (OMVs) secreted by bacteria are emerging diagnostic markers for bacterial infection or disease detection due to their carriage of various signaling molecules. However, actual biological samples of patients are extremely complex, and applying OMVs to clinical diagnosis remains a major challenge. One of the major challenges is that there are still great difficulties in the enrichment of OMVs including tedious steps and lower concentration. And some commonly used exosome enrichment methods, such as ultracentrifugation, still have some shortcomings. Herein, we introduce lipopolysaccharide (LPS) molecularly imprinted polymer (MIP) for efficient capturing and analyzing OMVs, enabling a novel approach to bacterial disease diagnosis based on biorecognition materials. LPS, as a unique structure of Gram-negative bacteria, also widely expressed on the surface of OMVs, which will form cyclic hydrogen bonds with functional monomers of MIP with affinity interactions. The prepared MIP efficiently can isolate OMVs from 100 μL of culture broth via specific affinity LPS in less than 40 min with a recovery rate of over 95%. Moreover, MIP exhibits good reusability, with almost identical enrichment performance after 5 repeated cycles, contributing to reducing experimental costs in both time and economy. The captured OMVs can be detected using Western blotting with target protein antibodies or in combination with proteomic analysis, providing a proteomic biomarker platform for early bacteria disease diagnosis.

Clinical efficacy of washed microbiota transplantation on metabolic syndrome and metabolic profile of donor outer membrane vesicles.

To clarify the clinical efficacy of washed microbiota transplantation (WMT) for metabolic syndrome (MetS), and explore the differences in the metabolic profile of bacterial outer membrane vesicles (OMVs) in donor fecal bacteria suspension received by MetS patients with good and poor outcomes, and to construct a predictive model for the efficacy of WMT for MetS using differential metabolites. Medical data 65 MetS patients who had completed at least 2 courses of WMT from 2017.05 to 2023.07 were collected. Fecal bacteria suspension of WMT donors were collected, and the clinical data of MetS patients treated with WMT during this period were collected as well. The changes of BMI, blood glucose, blood lipids, blood pressure and other indicators before and after WMT were compared. OMVs were isolated from donor fecal bacteria suspension and off-target metabolomic sequencing was performed by Liquid Chromatograph Mass Spectrometer (LC-MS). Compared with baseline, Body mass index (BMI), Systolic blood pressure (SBP) and Diastolic blood pressure (DBP) of MetS patients showed significant decreases after the 1st (short-term) and 2nd (medium-term) courses, and fasting blood glucose (FBG) also showed significant decreases after the 1st session. There was a significant difference between the Marked Response OMVs and the Moderate Response OMVs. It was showed that 960 metabolites were significantly up-regulated in Marked Response OMVs and 439 metabolites that were significantly down-regulated. The ROC model suggested that 9-carboxymethoxymethylguanine, AUC = 0.8127, 95% CI [0.6885, 0.9369], was the most potent metabolite predicting the most available metabolite for efficacy. WMT had significant short-term and medium-term clinical efficacy in MetS. There were differences in the structure of metabolites between Marked Response OMVs and Moderate Response OMVs. The level of 9-Carboxy methoxy methylguanine in Marked Response OMVs can be a good predictor of the efficacy of WMT in the treatment of MetS.

Biodegradable Acid‐Responsive Nanocarrier for Enhanced Antibiotic Therapy Against Drug‐Resistant Helicobacter Pylori via Urease Inhibition

AbstractMetal ion‐based inhibition of urease activity is a promising strategy for treating Helicobacter pylori (H. pylori) infections. However, the challenges of safe delivery and reducing cytotoxicity persist. In this study, an innovative nanocarrier capable of acid‐responsive release of Ag+ and antibiotics is developed, with complete degradation after treatment. Mesoporous organosilica nanoparticle (MON) is encapsulated with hyaluronic acid (HA) to prevent drug leakage and further coated with bacterial outer membrane vesicle (OMV) from Escherichia coli Nissle 1917, creating a nanocarrier with cell‐protective capabilities. Ag+ and antibiotic clarithromycin (CLR) are incorporated into the nanocarrier to form CLR‐Ag+@MON@HA@OMV (CAMO), designed for the targeted treatment of gastric H. pylori infection. The HA encapsulation ensures acid‐responsive release of CLR and Ag+ in the stomach, preventing premature release at non‐inflammatory sites. There is a potential for Ag⁺ in CAMO to replace Ni2⁺ at the active site of urease, enhancing the bactericidal effect of CLR through urease inhibition. Furthermore, the OMV provides additional cytoprotection, mitigating cell damage and inflammation response induced by the H. pylori infection. This study introduces a safe and effective nanocarrier that eradicates H. pylori and alleviates gastric inflammation.

Dual-drug loaded manganese dioxide nanoparticles coated with bacterial outer-membrane vesicles for chemo-immunotherapy in lung cancer

Dual-drug loaded manganese dioxide nanoparticles coated with bacterial outer-membrane vesicles for chemo-immunotherapy in lung cancer

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

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

Determination of particle number concentration for biological particles using AF4-MALS: Dependencies on light scattering model and refractive index

Determining accurate counts and size distributions for biological particles (bioparticles) is crucial in wide-ranging fields, but current methods to this end are susceptible to bias from polydispersity in size. This bias can be mitigated by incorporating a separation step prior to characterization. For this reason, asymmetrical flow field-flow fractionation (AF4) with on-line multiangle light scattering (MALS) has become an important platform for determining particle size. AF4-MALS has also been increasingly used to report particle concentration, particularly for complex biological particles, yet the impact of light scattering models and particle refractive indices (RI) have not been quantitatively evaluated. Here, we develop an analysis workflow using AF4-MALS to simultaneously separate and determine particles sizes and concentrations. The impacts of the MALS particle counting model used to process data and the chosen RI value(s) on particle counts are systematically assessed for polystyrene latex (PSL) particles and bacterial outer membrane vesicles (OMVs) in the 20-500 nm size range. Across spherical models, PSL and OMV particle counts varied up to 13 % or 200 %, respectively. For the coated-sphere model used in the analysis of OMV samples, the sphere RI value greatly impacts particle counts. As the sphere RI value approaches the RI of the suspending medium, the model becomes increasingly sensitive to the light scattering signal-to-noise ratio ultimately causing erroneous particle counts. Overall, this work establishes the importance of selecting appropriate MALS models and RI values for bioparticles to obtain accurate counts and provides an AF4-MALS method to separate, enumerate, and size polydisperse bioparticles.

A novel outer membrane vesicle adjuvant improves vaccine protection against Bordetella pertussis

Pertussis is a vaccine-preventable respiratory disease caused by the Gram negative coccobacillus Bordetella pertussis. The licensed acellular pertussis (aP) vaccines protect against disease but do not prevent bacterial colonization and transmission. Here, we developed and tested an intranasal vaccine composed of aP antigens combined with T-vant, a novel adjuvant derived from bacterial outer membrane vesicles, that elicits both mucosal and systemic immune responses. We hypothesized that immunization of mice with aP-T-vant would enhance mucosal immunity and eliminate B. pertussis in the respiratory tract. In contrast to mice immunized intramuscularly with the licensed aP vaccine, intranasal immunization with aP-T-vant eliminated bacteria in both the lung and nasopharynx. Protection was associated with IFN-gamma and IL-17-producing, non-circulating CD4 + T cells in the lung and nasopharynx, and sterilizing immunity in the nasopharynx was dependent on IL-17. Novel mucosal adjuvants, such as T-vant, warrant further investigation to enhance the efficacy of next generation pertussis vaccines.

Quantitative Biodistribution of OMVs Using SPECT/CT Imaging with HYNIC-Duramycin Radiolabeling.

Introduction: Bacterial outer membrane vesicles (OMVs) are emerging as important players in the host-microbiome interaction, while also proving to be a promising platform for vaccine development and targeted drug delivery. The available methods for measuring their biodistribution, however, are limited. We aimed to establish a high-efficiency radiolabeling method for the treatment of OMVs. Methods: 99mTc-HYNIC-duramycin was incubated with OMVs isolated from E. coli BL21(DE3) ΔnlpI ΔlpxM. Radiolabeling efficiency (RLE) and radiochemical purity (RCP) were measured with size-exclusion high-performance liquid chromatography. The biodistribution was quantitatively measured in mice using SPECT/CT imaging. Results: RLE was 81.84 ± 2.03% for undiluted OMV suspension and 56.17 ± 2.29% for 100× dilution. Postlabeling purification with a spin-desalting column results in 100% radioactivity in the OMV fraction according to HPLC, indicating 100% RCP of the final product. The biodistribution was found to be in line with previous data reported in the literature using other OMV tracking attempts. Conclusions: Our findings illustrate that using HYNIC-duramycin for labeling of the OMVs enhances efficiency and is easily implementable for in vivo imaging studies, significantly improving upon earlier methods.

Bacterial outer membrane vesicles-clothed nanoparticles delivered by live macrophages for potentiating antitumor photoimmunotherapy

Bacterial outer membrane vesicles-clothed nanoparticles delivered by live macrophages for potentiating antitumor photoimmunotherapy