Search Results (251)

Bacterial membrane vesicles (BMVs), encompassing outer membrane vesicles (OMVs) released from Gram-negative bacteria and extracellular vesicles (EVs) released from Gram-positive bacteria, have emerged as promising vaccine platforms owing to their intrinsic immunostimulatory properties and capacity to deliver a wide range of antigens. Although conventional vaccines effectively prevent infectious diseases, their long-term efficacy is often limited by antigenic variation and reliance on a restricted number of licensed adjuvants. BMVs, as self-adjuvanting systems, enable both antigen delivery and innate immune activation. BMVs are nanoscale lipid bilayer structures enriched with pathogen-associated molecular patterns (PAMPs), facilitating their recognition and uptake by antigen-presenting cells. This leads to the activation of pattern recognition receptors and the induction of pro-inflammatory cytokines, type I interferons, and adaptive immune responses, including antibody production and Th1- and Th17-biased cellular immunity. Recent studies highlight the versatility of BMVs as vaccine platforms across bacterial, fungal, and viral infection models. BMVs induce protective immunity by promoting both systemic and mucosal immune responses, thereby reducing bacterial burden and limiting pathogen colonization across diverse infection models. These properties have supported their application in viral vaccine development, including influenza and SARS-CoV-2, with the potential to enhance mucosal immunity. Despite these advantages, challenges remain in standardization, safety, and antigen-loading efficiency. Engineered BMVs incorporating protein or mRNA antigens may further enhance antigen presentation and CD8⁺ T cell responses. This review summarizes the biological features, immunological mechanisms, and future potential of BMVs in vaccine development. Full article

Melanoma is an aggressive skin cancer with rapid progression and high metastatic potential, and resistance to current therapies remains a major clinical challenge. In this study, Listeria monocytogenes-derived membrane vesicles (LM MVs) were isolated, characterized, and evaluated for their immunomodulatory and antitumor activities. LM MVs showed an average diameter of approximately 160 nm and contained multiple bacterial proteins, including listeriolysin O. In vitro, LM MVs promoted pro-inflammatory activation of RAW264.7 macrophages, as indicated by increased CD80/CD86 expression and enhanced transcription of inflammatory mediators. LM MV treatment was accompanied by IκB-α degradation and NF-κB p65 nuclear translocation, whereas pharmacological inhibition of NF-κB attenuated macrophage activation. In a macrophage–melanoma co-culture system, LM MVs-activated macrophages reduced the viability, migration, and invasion of B16 melanoma cells and increased tumor cell apoptosis. Additional inhibition and immunofluorescence analyses suggested that iNOS and TNF-α-associated mechanisms contributed to these tumor-suppressive effects. In a murine melanoma model, LM MVs significantly inhibited tumor growth without overt systemic toxicity, whereas macrophage depletion markedly weakened this effect. These findings indicate that LM MVs exert antitumor activity against melanoma, at least in part through macrophage activation involving NF-κB signaling. Full article

Background/Objectives: The escalating crisis of antibiotic resistance and the inherent limitations of conventional antibiotics necessitate the development of innovative therapeutic strategies. Targeted drug delivery (TDD) offers a powerful approach to enhance efficacy, minimize systemic toxicity, and circumvent bacterial resistance. This systematic review aims to evaluate the potential of unique bacterial transport systems (BTSs), surface specific receptors and intracellular enzymes as platforms for TDD via the “Trojan Horse” strategy (THS). Methods: A comprehensive literature review was conducted, focusing on studies that investigated the specificity and mechanisms of BTSs responsible for the uptake of metabolites that are essential for and unique to bacteria. This includes an analysis of transport systems for siderophores, bacteria-specific sugars, cell wall components, D-amino acids, and vitamins. We assessed preclinical and clinical examples of drug conjugates utilizing these pathways, as well as emerging platforms such as bacteriophage-derived proteins, antibody–antibiotic conjugates, and bacterial extracellular vesicles (EVs). Results: BTSs demonstrate high specificity for their cognate substrates, providing effective molecular gateways for TDD of drugs photosensitizers and diagnostic probes in form of conjugates. The siderophore–cephalosporin conjugate cefiderocol represents a clinically validated example, having received FDA approval. Preclinical studies further reveal that conjugates utilizing sugars (e.g., maltose, trehalose) and vitamins (e.g., B12) can significantly enhance antibiotic uptake and activity against both Gram-positive and Gram-negative pathogens, including drug-resistant strains. Emerging platforms like bacteriophage endolysins and engineered EVs show promise for overcoming biological barriers such as bacterial outer membranes and intracellular host niches. Conclusions: The THS leveraging BTSs represents a clinically viable and promising avenue for next-generation antibacterial therapies. Advantages of BTS include overcoming bacterial resistance, such as reduced membrane permeability and efflux pumps, enabling the “revival” of antibiotics that are poorly permeable or toxic, increasing their local concentration at the target site and reducing side effects on host cells. While significant progress has been made, a striking disconnect persists between the hundreds of conjugates demonstrating potent in vitro activity and the limited agent that has achieved clinical use. This in vitro–in vivo gap reflects, in large part, the early stage of this field rather than a fundamental failure. Further research is critically needed not only to identify novel BTSs and optimize drug-linker chemistry, but also to systematically address the translational barriers—including poor pharmacokinetics, immunogenicity, and unexpected toxicity—that have prevented most promising candidates from advancing beyond preclinical evaluation. Full article

Pertussis is a highly contagious respiratory infection caused by Bordetella pertussis and remains a persistent global health challenge despite widespread vaccination. This review aims to analyze the immune pathogenesis of B. pertussis infection and to identify key immunological limitations of current acellular pertussis vaccines that contribute to ongoing transmission. A narrative review of the literature was conducted, focusing on mechanisms of host–pathogen interaction, immune evasion, and vaccine-induced immunity. Evidence indicates that although acellular vaccines effectively reduce disease severity, they fail to prevent nasopharyngeal colonization and transmission, largely due to insufficient induction of mucosal immunity, T helper 1 (Th1) and T helper 17 (Th17) responses, and airway tissue-resident memory T cells. In contrast, natural infection induces broader immune responses, including secretory IgA production and robust cellular immunity, which are associated with improved bacterial clearance. Emerging next-generation vaccine strategies, including mucosal, outer membrane vesicle-based, and live-attenuated platforms, demonstrate enhanced ability to reduce bacterial colonization in preclinical and clinical models. In conclusion, effective control of pertussis transmission will require vaccine approaches that replicate infection-induced immunity at the respiratory mucosa, emphasizing the need for redesigned immunization strategies. Full article

Alzheimer’s disease is a complex neurodegenerative condition characterized by progressive cognitive decline, neuroinflammation, metabolic dysregulation, and abnormal protein deposition. While genetic factors and amyloid-beta-focused hypotheses have been extensively investigated, they fail to fully account for the prolonged prodromal phase or the early susceptibility of olfactory and limbic regions. Emerging evidence suggests chronic peripheral and mucosal infections may influence disease risk; however, mechanisms by which microbial activity outside the central nervous system contributes to persistent neuropathology remain poorly understood. This review explores the emerging concept that bacterial outer membrane vesicles act as mobile, lipid-rich vectors linking peripheral microbial reservoirs to neuroimmune and metabolic dysfunction in the aging brain. We discuss evidence suggesting vesicles originating from oral, olfactory, and upper airway niches can access the central nervous system via vascular routes and direct neural pathways, including olfactory and trigeminal nerves, where they influence glial and endothelial cell function. We also propose the Accumulative Vesicle Load Hypothesis, which describes how cumulative lifetime exposure to bacterial vesicles shapes disease onset, anatomical vulnerability, and progression, and incorporates components of other hypotheses proposed for Alzheimer’s disease. This offers a system-level perspective for early diagnosis and upstream therapeutic strategies, including minimally invasive vesicle profiling in nasal fluid, saliva, blood, and cerebrospinal fluid. This work is a conceptual review that summarizes current evidence in a hierarchically organized manner and proposes a testable model; it does not assert causality where direct human evidence is currently limited. Full article

Streptococcus mutans (S. mutans), one of the main etiological pathogens of dental caries, forms dental plaque biofilms that drive tooth decay. Although bacterial membrane vesicles (MVs) are increasingly recognized as modulators of biofilm biology, little is known about MVs generated by S. mutans. The objective of this study is to investigate the role of S. mutans-derived MVs in the development of S. mutans biofilms formed under static conditions in plates or confocal dishes. Transmission electron microscopy and nanoparticle tracking analysis revealed that the MVs were cup-shaped with bilayered membranes and averaged 80.49 $\pm$ 32.24 nm in diameter. The addition of ≥5 µg/mL MVs enhanced biofilm formation during the initial adhesion stage (0 to 6 h), as demonstrated by crystal violet staining and XTT assays. Confocal laser scanning microscopy and scanning electron microscopy confirmed the incorporation of PKH26-labeled MVs into S. mutans biofilms and showed that supplemental MVs increased bacterial viability and extracellular polysaccharide biomass. Furthermore, RT-qPCR analysis revealed upregulated expression of genes related to adhesion and quorum-sensing systems in MV-treated biofilms. In conclusion, these findings indicate that S. muants MVs are integral biofilm components that promote biofilm establishment at the early stage of biofilm formation. Full article

Complex I (NADH:quinone oxidoreductase, CI) is central to cellular aerobic energy metabolism. The L-shaped structure of CI is unique, where the hydrophilic arm is responsible for the electron transfer function and the membrane arm operates proton pumping. These two functional sites are spatially far apart yet functionally connected. This basic core subunit architecture is highly conserved from bacterial to mammalian CI. Here, to gain detailed mechanistic insight into the role of the membrane subunit ND2 in the coupling mechanism, we mutated several highly conserved residues in the middle of the membrane axis of NuoN, the E. coli CI homolog of ND2. To more precisely investigate the consequences of mutational effects on highly conserved residues, we purified each mutant CI and compared the mutational effects on electron transfer and proton pumping activity using our instant membrane reconstitution method with E. coli double knockout (DKO) membrane vesicles lacking both CI and alternative NADH dehydrogenase (NDH-2). Thre results were corroborated by conventional proteoliposome reconstitution experiments. We found that Lys247 and Lys395 are absolutely essential for both electron transfer and proton pumping activities, while about 50% reduction of NADH oxidase activity but no reduction in proton pumping activity was observed in Lys217, and no significant decrease was detected in Glu133. Furthermore, unexpectedly, we were able to purify an NuoN knockout (ΔNuoN) mutant, which contained stoichiometric peripheral subunits NuoB, NuoCD, NuoE, NuoF, NuoG, and NuoI; and a substoichiometric amount of NuoH and a reduced amount of quinone. However, surprisingly, this isolated ΔNuoN CI showed CI activities (~30% of the WT) after being reconstituted into DKO membranes but not into proteoliposomes. Later, we confirmed by blue native PAGE that the wild-type CI was partially formed from ΔNuoN CI by recruiting its missing membrane subunits that existed in DKO membranes. Our data strongly suggest that ND2/NuoN plays an essential role in the coupling mechanism in CI. CI is the entry respiratory chain enzyme and is central to cellular energy metabolism. Two highly conserved lysine residues in the center of the antiporter-like membrane subunit ND2 are essential for the coupling mechanism between electron transfer and proton translocation. Full article

Bacterial membrane vesicles (BMVs) are non-replicative, bilayered nanostructures secreted by both Gram-negative and Gram-positive bacteria. Rather than being passive byproducts of cell envelope turnover, BMVs are increasingly recognized as regulated particles that selectively package proteins, lipids, nucleic acids, and other bioactive molecules. Through these cargos, BMVs mediate a wide range of biological processes, including bacterial stress adaption, intercellular communication, virulence delivery, and host immune modulation. In this review, we integrate recent advancements in understanding the molecular mechanisms underlying BMV biogenesis and composition and discuss how their heterogeneity contributes to their functional diversity. Beyond their biological roles, we critically examine the translational potential of BMVs in vaccine development, targeted drug delivery, cancer therapy, diagnostic tools, and biotechnological applications. However, significant challenges related to their safety, efficacy, and large-scale production must be addressed to realize their full clinical potential. We review recent progress and ongoing obstacles in the use of BMVs across various biomedical applications and propose strategies for their clinical translation. Full article

The cause of chronic neurological effects associated with Lyme disease (LD) remains unclear. We propose that bacterial extracellular vesicles (BEVs) released by Borrelia burgdorferi, the causative agent of LD, exacerbate spirochete-induced damage and serve as a persistent source of antigenic stimulation. We showed that, over a 10-day period, in vitro cultures of B. burgdorferi B31 produced 38,000 BEVs per spirochete with a distinctive double-membrane structure and median diameter of 143.3 nm. BEVs contained known immunogenic and immunomodulatory molecules such as peptidoglycan, p66, flagellar filament protein (FlaB), basic membrane proteins A/B/D, BdrV, GroEL, CRASP-1, ErpA8, glycerophosphodiester phosphodiesterase, p37, OMS28, p13, OspA/B/C, VlsE, and outer membrane glycolipids (e.g., cholesteryl 6-O acyl beta D galactopyranoside). Chromosome-encoded 16S ribosomal RNA and cp32 plasmid-encoded OspE and terminase genes were also detected in the BEVs. Of the 45 Borrelia proteins identified in the urine of a C3H/HeJ murine model of Lyme disease, 14 were associated with BEVs. In human urine samples, 31 of 289 spirochete proteins detected in patients with either acute Lyme disease or persistent borreliosis post-treatment symptoms, including p66 and FlaB, were also BEV-associated. BEV treatment of HMC3 human microglial cells reduced phagocytic activity and triggered aberrant activation of inflammatory and immunometabolic pathways, including upregulation of interferon-alpha (IFN-α), aconitate decarboxylase 1 (Acod1), and Toll-like receptor 2 (TLR2) gene expression. BEVs also induced NRF2 nuclear translocation. In conclusion, these findings support that BEVs can amplify spirochete-induced damage and act as antigenic debris, driving dampened phagocytic activity and dysregulated inflammation, with implications for diagnostics and therapeutics targeting vesicle-mediated pathology. Full article

Colorectal cancer (CRC) cachexia is a multifactorial, treatment-limiting syndrome characterized by progressive loss of skeletal muscle with or without loss of fat mass, accompanied by systemic inflammation, anorexia, metabolic dysregulation, and impaired treatment tolerance. Despite decades of work, cachexia remains clinically underdiagnosed and therapeutically underserved, in part because canonical models treat tumor-derived factors and host inflammatory mediators as a largely ‘host-only’ network. In parallel, CRC is strongly linked to intestinal dysbiosis, barrier disruption, and microbial translocation. Extracellular vesicles (EVs)—host small EVs, tumor-derived EVs, and bacterial extracellular vesicles (including outer membrane vesicles)—may provide a mechanistically plausible, information-dense route by which these domains could be coupled. Here, we synthesize emerging evidence suggesting that cross-kingdom EV signaling may operate as a vesicular ecosystem spanning gut lumen, mucosa, circulation, and peripheral organs. We propose the “vesicular intersection layer” as a unifying framework for how heterogeneous EV cargos converge on shared host decoding hubs (e.g., pattern-recognition receptors and stress-response pathways) to potentially contribute to muscle catabolism. We critically evaluate what is known—and what remains unproven—about EV biogenesis, trafficking, and causal mechanisms in CRC cachexia, highlight methodological constraints in microbial EV isolation and attribution, and outline minimum evidentiary standards for cross-kingdom claims. Finally, we translate the framework into actionable hypotheses for EV-informed endotyping, biomarker development (including stool EV assays), and therapeutic strategies targeting shared signaling nodes (e.g., TLR4–p38) and endocrine mediators that are predominantly soluble but may be fractionally vesicle-associated (e.g., GDF15). By reframing CRC cachexia as an emergent property of tumor–host–microbiota vesicular communication, this review provides a roadmap for mechanistic studies and clinically tractable interventions. Full article

Bacterial extracellular vesicles (BEVs) are nanosized (10–400 nm), membrane-enclosed particles naturally secreted by both Gram-negative and Gram-positive bacteria. Initially characterized as virulence factors in pathogenic species, BEVs are now recognized as multifunctional entities with significant biotechnological potential. Their cargo—comprising proteins, lipids, nucleic acids, and metabolites—enables diverse biological activities, including immune modulation, epithelial barrier protection, stress tolerance, and intercellular communication. Recent studies have highlighted BEVs from biotechnologically relevant bacteria—such as plant growth-promoting rhizobacteria, lactic acid bacteria, bifidobacteria, cyanobacteria, bacilli, and streptomycetes—for their different roles in biological and ecological interactions. These properties underpin emerging applications in health, agriculture, and bioprocessing, including next-generation postbiotics, vaccine platforms, drug and RNA delivery systems, and novel plant biostimulants. However, major challenges persist, particularly low production yields, variability in cargo composition, and scalability. Addressing these limitations requires a deeper understanding of vesiculation mechanisms and the development of process-oriented strategies for BEV recovery and purification. This review synthesizes recent advances in genetic analysis, physiological modulation, physicochemical stimuli, and bioprocess optimization aimed at enhancing BEV production and stabilizing cargo profiles, providing a comprehensive overview of approaches to unlock the full potential of BEVs as versatile biotechnological tools. Full article

Background/Objectives: The gut microbiota is increasingly recognized as a key determinant of human health, playing a vital role in metabolism, immunity, and disease susceptibility. Dysbiosis, or microbial imbalance, is associated with gastrointestinal disorders such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and Clostridioides difficile infection (CDI), as well as extraintestinal conditions, including obesity, cardiovascular disease, and neuropsychiatric disorders. This review aims to provide an updated overview of emerging therapeutic strategies to modulate the gut microbiota to restore eubiosis and improve health outcomes. Methods: A narrative review of recent literature was conducted, focusing on preclinical and clinical studies investigating microbiota-targeted therapies. The review primarily covers innovative interventional approaches, including fecal microbiota transplantation (FMT), bacterial consortium transplantation (BCT), bacteriophage therapy and outer membrane vesicles (OMVs). Results: Evidence supports the role of probiotics, prebiotics, and synbiotics in remodeling microbial communities and improving host health, although their effects may be strain- and context-dependent. FMT has demonstrated high efficacy in the treatment of recurrent Clostridium difficile infections and is being studied for IBD, IBS and extraintestinal diseases, following the recent Food and Drug Administration approval of the first commercial FMT products. BCT offers a standardized alternative to donor-derived material, with early clinical successes such as FDA-approved SER-109. Phage therapy and OMVs represent promising frontiers, offering targeted microbial modulation and interactions with the immune system, although clinical data remain limited. Conclusions: Emerging gut microbiota modulation strategies offer new perspectives for precision medicine and could transform the prevention and treatment of many diseases, but further studies are needed to ensure their safety, standardization, and clinical application. Full article

Background: Porphyromonas gingivalis outer membrane vesicles (OMVs) are key mediators of host–pathogen interactions and have been implicated in both periodontal disease and systemic conditions, including pregnancy complications. Although OMV production and cargo are known to be influenced by environmental stress, how oxidative stress reshapes P. gingivalis OMVs and their functional impact on trophoblast cells remains poorly understood. Here, we investigated how exposure to hydrogen peroxide (H₂O₂) affects OMV biogenesis, composition, and their ability to modulate bacterial invasion in trophoblast cells. Methods: P. gingivalis was cultured anaerobically and exposed to 30 mM H₂O₂ during the final 24 h of growth. OMVs were isolated by differential ultracentrifugation and characterized by nanoparticle tracking analysis and transmission electron microscopy and OMV protein cargo was analyzed by proteomics. Functional effects were assessed using invasion and persistence assays in HTR-8/SVneo trophoblast cells pretreated with OMVs. Results: Oxidative stress did not significantly alter total OMV yield but resulted in smaller vesicles (control OMV 168.2 ± 8.7 nm vs. OMV from H₂O₂-treated cultures 130.0 ± 13.8 nm) with reduced negative surface charge and increased membrane-associated FM4-64 fluorescence. Proteomic analysis revealed a remodeling of the OMV protein cargo under oxidative stress, including the selective enrichment of a von Willebrand factor type A domain-containing protein. Functionally, OMVs from control cultures led to a 2.5-fold increase in P. gingivalis invasion and a 4-fold increase in intracellular persistence in trophoblast cells, whereas OMVs produced under oxidative stress failed to promote these processes. Conclusions: Together, these findings highlight oxidative stress as a key determinant of OMV-mediated host–pathogen interactions at the maternal–fetal interface. Full article

Background/Objectives: Bacterial outer membrane vesicles (OMVs) play a role in bacterial communication, virulence, antimicrobial resistance, and host–pathogen interaction. OMV isolation is a key step for studying these particles’ functions; nevertheless, isolation procedures can greatly influence the yield, purity, and structural integrity of OMVs, thereby affecting downstream biological analyses and functional interpretation. Methods: In this study, we compared the efficacy of two OMV isolation techniques, differential ultracentrifugation (dUC) and size-exclusion chromatography (SEC), in separating and concentrating vesicles produced by two Escherichia coli strains belonging to uropathogenic (UPEC) and Shiga toxin-producing (STEC) pathotypes. The isolated OMVs were characterized using a multi-analytical approach including transmission and scanning electron microscopy (TEM, SEM), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), ζ-potential measurement, and protein quantification to assess the purity of the preparations. Results: Samples obtained by dUC exhibited higher total protein content, broader particle size distributions, and more pronounced contamination by non-vesicular material. In contrast, SEC yielded morphologically homogeneous and structurally well-preserved vesicles, higher particle-to-protein ratios, and lower total protein content, reflecting reduced co-isolation of protein aggregates. NTA and DLS analyses revealed polydisperse populations in samples obtained with both isolation methods, with DLS measurements highlighting the contribution of larger or transient aggregates. ζ-potential values were close to neutrality for all samples, consistent with limited electrostatic repulsion and with the aggregation tendencies observed in some preparations. Conclusions: This study describes features of OMV produced by two relevant E. coli strains considering two isolation strategies which exert method- and strain-dependent effects on vesicle properties, including size distribution and surface charge, and emphasizes the trade-offs between yield, purity, and vesicle integrity. Full article

Helicobacter pylori is a prevalent gastric pathogen that establishes chronic infection and contributes to gastritis, peptic ulcer disease, and gastric cancer. Its persistence depends on immune evasion strategies that promote sustained low-grade inflammation in the gastric mucosa. Nucleotide-binding oligomerization domain-like receptors (NLRs) are cytosolic pattern recognition receptors that play key roles in innate immune responses against H. pylori. Nod1 and Nod2 detect bacterial peptidoglycan delivered via the type IV secretion system or outer membrane vesicles, activating NF-κB, MAPK, and interferon signaling pathways that regulate inflammatory cytokine production, epithelial barrier function, autophagy, and antimicrobial defense. The NLRP3 inflammasome mediates the maturation of IL-1β and IL-18 primarily in myeloid cells, thereby shaping inflammatory and immunoregulatory responses during infection. In contrast, NLRC4 functions in a context-dependent manner in epithelial cells and is largely dispensable for myeloid IL-1β production. Emerging evidence also implicates noncanonical NLRs, including NLRP6, NLRP9, NLRP12, NLRX1, and NLRC5, in regulating inflammation, epithelial homeostasis, and gastric tumorigenesis. In addition, genetic polymorphisms in NLR genes influence host susceptibility to H. pylori-associated diseases. This review highlights the interplay between NLR signaling, bacterial virulence, and host immunity and identifies potential therapeutic targets. Full article