Outer Membrane Vesicle Production by Helicobacter pylori Represents an Approach for the Delivery of Virulence Factors CagA,VacA and UreA into Human Gastric Adenocarcinoma (AGS) Cells

Helicobacter pylori infection is the etiology of several gastric-related diseases including gastric cancer. Cytotoxin associated gene A (CagA), vacuolating cytotoxin A (VacA) and α-subunit of urease (UreA) are three major virulence factors of H. pylori, and each of them has a distinct entry pathway and pathogenic mechanism during bacterial infection. H. pylori can shed outer membrane vesicles (OMVs). Therefore, it would be interesting to explore the production kinetics of H. pylori OMVs and its connection with the entry of key virulence factors into host cells. Here, we isolated OMVs from H. pylori 26,695 strain and characterized their properties and interaction kinetics with human gastric adenocarcinoma (AGS) cells. We found that the generation of OMVs and the presence of CagA, VacA and UreA in OMVs were a lasting event throughout different phases of bacterial growth. H. pylori OMVs entered AGS cells mainly through macropinocytosis/phagocytosis. Furthermore, CagA, VacA and UreA could enter AGS cells via OMVs and the treatment with H. pylori OMVs would cause cell death. Comparison of H. pylori 26,695 and clinical strains suggested that the production and characteristics of OMVs are not only limited to laboratory strains commonly in use, but a general phenomenon to most H. pylori strains.     

Salivary Outer Membrane Vesicles and DNA Methylation of Small Extracellular Vesicles as Biomarkers for Periodontal Status: A Pilot Study

Periodontitis is an inflammatory disease, associated with a microbial dysbiosis. Early detection using salivary small extracellular vesicles (sEVs) biomarkers may facilitate timely prevention. sEVs derived from different species (i.e., humans, bacteria) are expected to circulate in saliva. This pilot study recruited 22 participants (seven periodontal healthy, seven gingivitis and eight periodontitis) and salivary sEVs were isolated using the size-exclusion chromatography (SEC) method. The healthy, gingivitis and periodontitis groups were compared in terms of salivary sEVs in the CD9+ sEV subpopulation, Gram-negative bacteria-enriched lipopolysaccharide (LPS+) outer membrane vesicles (OMVs) and global DNA methylation pattern of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and N6-Methyladenosine (m6dA). It was found that LPS+ OMVs, global 5mC methylation and four periodontal pathogens (T. denticola, E. corrodens, P. gingivalis and F. nucleatum) that secreted OMVs were significantly increased in periodontitis sEVs compared to those from healthy groups. These differences were more pronounced in sEVs than the whole saliva and were more superior in distinguishing periodontitis than gingivitis, in comparison to healthy patients. Of note, global 5mC hypermethylation in salivary sEVs can distinguish periodontitis patients from both healthy controls and gingivitis patients with high sensitivity and specificity (AUC = 1). The research findings suggest that assessing global sEV methylation may be a useful biomarker for periodontitis.     

Shigella Outer Membrane Vesicles as Promising Targets for Vaccination

The clinical symptoms of shigellosis, a gastrointestinal infection caused by Shigella spp. range from watery diarrhea to fulminant dysentery. Endemic infections, particularly among children in developing countries, represent the majority of clinical cases. The situation is aggravated due to the high mortality rate of shigellosis, the rapid dissemination of multi-resistant Shigella strains and the induction of only serotype-specific immunity. Thus, infection prevention due to vaccination, encompassing as many of the circulating serotypes as possible, has become a topic of interest. However, vaccines have turned out to be ineffective so far. Outer membrane vesicles (OMVs) are promising novel targets for vaccination. OMVs are constitutively secreted by Gram-negative bacteria including Shigella during growth. They are composed of soluble luminal portions and an insoluble membrane and can contain toxins, bioactive periplasmic and cytoplasmic (lipo-) proteins, (phospho-) lipids, nucleic acids and/or lipopolysaccharides. Thus, OMVs play an important role in bacterial cell–cell communication, growth, survival and pathogenesis. Furthermore, they modulate the secretion and transport of biomolecules, the stress response, antibiotic resistance and immune responses of the host. Thus, OMVs serve as novel secretion machinery. Here, we discuss the current literature and highlight the properties of OMVs as potent vaccine candidates because of their immunomodulatory, antigenic and adjuvant properties.     

Gene Transfer Potential of Outer Membrane Vesicles of Gram-Negative Bacteria

The increasing spread of multidrug-resistant pathogenic bacteria is one of the major threats to public health worldwide. Bacteria can acquire antibiotic resistance and virulence genes through horizontal gene transfer (HGT). A novel horizontal gene transfer mechanism mediated by outer membrane vesicles (OMVs) has been recently identified. OMVs are rounded nanostructures released during their growth by Gram-negative bacteria. Biologically active toxins and virulence factors are often entrapped within these vesicles that behave as molecular carriers. Recently, OMVs have been reported to contain DNA molecules, but little is known about the vesicle packaging, release, and transfer mechanisms. The present review highlights the role of OMVs in HGT processes in Gram-negative bacteria.     

Microbiota-Derived Extracellular Vesicles Detected in Human Blood from Healthy Donors

The microbiota constitutes an important part of the holobiont in which extracellular vesicles (EVs) are key players in health, especially regarding inter- and intra-kingdom communications. Analysis of EVs from the red blood cell concentrates of healthy donors revealed variable amounts of OmpA and LPS in 12 of the 14 analyzed samples, providing indirect experimental evidence of the presence of microbiota EVs in human circulating blood in the absence of barrier disruption. To investigate the role of these microbiota EVs, we tracked the fusion of fluorescent Escherichia coli EVs with blood mononuclear cells and showed that, in the circulating blood, these EVs interacted almost exclusively with monocytes. This study demonstrates that bacterial EVs constitute critical elements of the host–microbiota cellular communication. The analysis of bacterial EVs should thus be systematically included in any characterization of human EVs.     

Bacterial Membrane Vesicles in Pneumonia: From Mediators of Virulence to Innovative Vaccine Candidates

Pneumonia due to respiratory infection with most prominently bacteria, but also viruses, fungi, or parasites is the leading cause of death worldwide among all infectious disease in both adults and infants. The introduction of modern antibiotic treatment regimens and vaccine strategies has helped to lower the burden of bacterial pneumonia, yet due to the unavailability or refusal of vaccines and antimicrobials in parts of the global population, the rise of multidrug resistant pathogens, and high fatality rates even in patients treated with appropriate antibiotics pneumonia remains a global threat. As such, a better understanding of pathogen virulence on the one, and the development of innovative vaccine strategies on the other hand are once again in dire need in the perennial fight of men against microbes. Recent data show that the secretome of bacteria consists not only of soluble mediators of virulence but also to a significant proportion of extracellular vesicles—lipid bilayer-delimited particles that form integral mediators of intercellular communication. Extracellular vesicles are released from cells of all kinds of organisms, including both Gram-negative and Gram-positive bacteria in which case they are commonly termed outer membrane vesicles (OMVs) and membrane vesicles (MVs), respectively. (O)MVs can trigger inflammatory responses to specific pathogens including S. pneumonia, P. aeruginosa, and L. pneumophila and as such, mediate bacterial virulence in pneumonia by challenging the host respiratory epithelium and cellular and humoral immunity. In parallel, however, (O)MVs have recently emerged as auspicious vaccine candidates due to their natural antigenicity and favorable biochemical properties. First studies highlight the efficacy of such vaccines in animal models exposed to (O)MVs from B. pertussis, S. pneumoniae, A. baumannii, and K. pneumoniae. An advanced and balanced recognition of both the detrimental effects of (O)MVs and their immunogenic potential could pave the way to novel treatment strategies in pneumonia and effective preventive approaches.     

Extracellular Vesicles as Emerging Players in Intercellular Communication: Relevance in Mast Cell-Mediated Pathophysiology

Mast cells are major effector cells in eliciting allergic responses. They also play a significant role in establishing innate and adaptive immune responses, as well as in modulating tumor growth. Mast cells can be activated upon engagement of the high-affinity receptor FcεRI with specific IgE to multivalent antigens or in response to several FcεRI-independent mechanisms. Upon stimulation, mast cells secrete various preformed and newly synthesized mediators. Emerging evidence indicates their ability to be a rich source of secreted extracellular vesicles (EVs), including exosomes and microvesicles, which convey biological functions. Mast cell-derived EVs can interact with and affect other cells located nearby or at distant sites and modulate inflammation, allergic response, and tumor progression. Mast cells are also affected by EVs derived from other cells in the immune system or in the tumor microenvironment, which may activate mast cells to release different mediators. In this review, we summarize the latest data regarding the ability of mast cells to release or respond to EVs and their role in allergic responses, inflammation, and tumor progression. Understanding the release, composition, and uptake of EVs by cells located near to or at sites distant from mast cells in a variety of clinical conditions, such as allergic inflammation, mastocytosis, and lung cancer will contribute to developing novel therapeutic approaches.     

Small Extracellular Vesicles from Hypoxic Triple-Negative Breast Cancer Cells Induce Oxygen-Dependent Cell Invasion

Hypoxia, a condition of low oxygenation frequently found in triple-negative breast tumors (TNBC), promotes extracellular vesicle (EV) secretion and favors cell invasion, a complex process in which cell morphology is altered, dynamic focal adhesion spots are created, and ECM is remodeled. Here, we investigated the invasive properties triggered by TNBC-derived hypoxic small EV (SEVh) in vitro in cells cultured under hypoxic (1% O₂) and normoxic (20% O₂) conditions, using phenotypical and proteomic approaches. SEVh characterization demonstrated increased protein abundance and diversity over normoxic SEV (SEVn), with enrichment in pro-invasive pathways. In normoxic cells, SEVh promotes invasive behavior through pro-migratory morphology, invadopodia development, ECM degradation, and matrix metalloprotease (MMP) secretion. The proteome profiling of 20% O₂-cultured cells exposed to SEVh determined enrichment in metabolic processes and cell cycles, modulating cell health to escape apoptotic pathways. In hypoxia, SEVh was responsible for proteolytic and catabolic pathway inducement, interfering with integrin availability and gelatinase expression. Overall, our results demonstrate the importance of hypoxic signaling via SEV in tumors for the early establishment of metastasis.     

Mouse Gastric Epithelial Cells Resist CagA Delivery by the Helicobacter pylori Type IV Secretion System

The initial step in bacterial infection is adherence of the bacterium to the target cell surface. Helicobacter pylori exploits the interaction of bacterial adhesin protein HopQ with human epithelial CEACAMs (CEACAM1, 5, and 6) to stably adhere to gastric epithelial cells, which is necessary for delivery of the H. pylori CagA oncoprotein into the epithelial cells via a type IV secretion system. In contrast to human CEACAMs, however, HopQ does not interact with Ceacam1 (mouse CEACAM1) in vitro or in CHO cells ectopically expressing Ceacam1. Since the mouse genome lacks Ceacam5 and Ceacam6, no significant HopQ–Ceacam interaction may occur in mouse gastric epithelial cells. Here, we found that the mouse stomach has a much lower expression level of Ceacam1 than the expression level of CEACAM1 in the human stomach. Consistently, mouse gastric epithelial cells resist CagA delivery by cagA-positive H. pylori, and the delivery is restored by ectopic expression of human CEACAM1 or CEACAM5 in mouse gastric epithelial cells. Thus, despite the fact that mice are routinely used for H. pylori infection studies, a low expression level of Ceacam1 in the mouse stomach together with the loss or greatly reduced interaction of HopQ with Ceacams make the mouse an inappropriate model for studying the role of H. pylori-delivered CagA in gastric pathogenesis, including the development of gastric cancer.     

Extracellular Vesicles from 50,000 Generation Clones of the Escherichia coli Long-Term Evolution Experiment

Extracellular vesicles (EVs) are critical elements of cell–cell communication. Here, we characterized the outer membrane vesicles (OMVs) released by specific clones of Escherichia coli isolated from the Long-Term Evolution Experiment after 50,000 generations (50K) of adaptation to glucose minimal medium. Compared with their ancestor, the evolved clones produce small OMVs but also larger ones which display variable amounts of both OmpA and LPS. Tracking ancestral, fluorescently labelled OMVs revealed that they fuse with both ancestral- and 50K-evolved cells, albeit in different proportions. We quantified that less than 2% of the cells from one 50K-evolved clone acquired the fluorescence delivered by OMVs from the ancestral strain but that one cell concomitantly fuses with several OMVs. Globally, our results showed that OMV production in E. coli is a phenotype that varies along bacterial evolution and question the contribution of OMVs-mediated interactions in bacterial adaptation.     

Focus on Extracellular Vesicles: Physiological Role and Signalling Properties of Extracellular Membrane Vesicles

Extracellular vesicles (EVs) are a heterogeneous population of secreted membrane vesicles, with distinct biogenesis routes, biophysical properties and different functions both in physiological conditions and in disease. The release of EVs is a widespread biological process, which is conserved across species. In recent years, numerous studies have demonstrated that several bioactive molecules are trafficked with(in) EVs, such as microRNAs, mRNAs, proteins and lipids. The understanding of their final impact on the biology of specific target cells remains matter of intense debate in the field. Also, EVs have attracted great interest as potential novel cell-free therapeutics. Here we describe the proposed physiological and pathological functions of EVs, with a particular focus on their molecular content. Also, we discuss the advances in the knowledge of the mechanisms regulating the secretion of EV-associated molecules and the specific pathways activated upon interaction with the target cell, highlighting the role of EVs in the context of the immune system and as mediators of the intercellular signalling in the brain.     

Epigenetics and Helicobacter pylori

Epigenetics regulates gene expression, cell type development during differentiation, and the cell response to environmental stimuli. To survive, bacteria need to evade the host immune response. Bacteria, including Helicobacter pylori (Hp), reach this target epigenetically, altering the chromatin of the host cells, in addition to several more approaches, such as DNA mutation and recombination. This review shows that Hp prevalently silences the genes of the human gastric mucosa by DNA methylation. Epigenetics includes different mechanisms. However, DNA methylation persists after DNA replication and therefore is frequently associated with the inheritance of repressed genes. Chromatin modification can be transmitted to daughter cells leading to heritable changes in gene expression. Aberrant epigenetic alteration of the gastric mucosa DNA remains the principal cause of gastric cancer. Numerous methylated genes have been found in cancer as well as in precancerous lesions of Hp-infected patients. These methylated genes inactivate tumor-suppressor genes. It is time for us to complain about our genetic and epigenetic makeups for our diseases.     

Extracellular Vesicles Isolated from Malignant Mesothelioma Cancer-Associated Fibroblasts Induce Pro-Oncogenic Changes in Healthy Mesothelial Cells

Malignant mesothelioma is an aggressive tumour of the pleura (MPM) or peritoneum with a clinical presentation at an advanced stage of the disease. Current therapies only marginally improve survival and there is an urgent need to identify new treatments. Carcinoma-associated fibroblasts (CAFs) represent the main component of a vast stroma within MPM and play an important role in the tumour microenvironment. The influence of CAFs on cancer progression, aggressiveness and metastasis is well understood; however, the role of CAF-derived extracellular vesicles (CAF-EVs) in the promotion of tumour development and invasiveness is underexplored. We purified CAF-EVs from MPM-associated cells and healthy dermal human fibroblasts and examined their effect on cell proliferation and motility. The data show that exposure of healthy mesothelial cells to EVs derived from CAFs, but not from normal dermal human fibroblasts (NDHF) resulted in activating pro-oncogenic signalling pathways and increased proliferation and motility. Consistent with its role in suppressing Yes-Associated Protein (YAP) activation (which in MPM is a result of Hippo pathway inactivation), treatment with Simvastatin ameliorated the pro-oncogenic effects instigated by CAF-EVs by mechanisms involving both a reduction in EV number and changes in EV cargo. Collectively, these data determine the significance of CAF-derived EVs in mesothelioma development and progression and suggest new targets in cancer therapy.     

Extracellular Vesicles as Players in the Anti-Inflammatory Inter-Cellular Crosstalk Induced by Exercise Training

Extracellular Vesicles (EVs) are circulating particles surrounded by a plasma membrane carrying a cargo consisting of proteins, lipids, RNAs, and DNA fragments, stemming from the cells from which they originated. EV factors (i.e., miRNAs) play relevant roles in intercellular crosstalk, both locally and systemically. As EVs increasingly gained attention as potential carriers for targeted genes, the study of EV effects on the host immune response became more relevant. It has been demonstrated that EVs regulate the host immune response, executing both pro- and anti-inflammatory functions. It is also known that physical exercise triggers anti-inflammatory effects. This review underlines the role of circulating EVs as players in the anti-inflammatory events associated with the regulation of the host’s immune response to physical exercise.     

Characterisation of Extracellular Vesicles from Equine Mesenchymal Stem Cells

Extracellular vesicles (EVs) are nanosized lipid bilayer-encapsulated particles secreted by virtually all cell types. EVs play an essential role in cellular crosstalk in health and disease. The cellular origin of EVs determines their composition and potential therapeutic effect. Mesenchymal stem/stromal cell (MSC)-derived EVs have shown a comparable therapeutic potential to their donor cells, making them a promising tool for regenerative medicine. The therapeutic application of EVs circumvents some safety concerns associated with the transplantation of viable, replicating cells and facilitates the quality-controlled production as a ready-to-go, off-the-shelf biological therapy. Recently, the International Society for Extracellular Vesicles (ISEV) suggested a set of minimal biochemical, biophysical and functional standards to define extracellular vesicles and their functions to improve standardisation in EV research. However, nonstandardised EV isolation methods and the limited availability of cross-reacting markers for most animal species restrict the application of these standards in the veterinary field and, therefore, the species comparability and standardisation of animal experiments. In this study, EVs were isolated from equine bone-marrow-derived MSCs using two different isolation methods, stepwise ultracentrifugation and size exclusion chromatography, and minimal experimental requirements for equine EVs were established and validated. Equine EVs were characterised using a nanotracking analysis, fluorescence-triggered flow cytometry, Western blot and transelectron microscopy. Based on the ISEV standards, minimal criteria for defining equine EVs are suggested as a baseline to allow the comparison of EV preparations obtained by different laboratories.     

Mesenchymal Stem Cells and Extracellular Vesicles in Osteosarcoma Pathogenesis and Therapy

Osteosarcoma (OS) is an aggressive bone tumor that mainly affects children and adolescents. OS has a strong tendency to relapse and metastasize, resulting in poor prognosis and survival. The high heterogeneity and genetic complexity of OS make it challenging to identify new therapeutic targets. Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into adipocytes, osteoblasts, or chondroblasts. OS is thought to originate at some stage in the differentiation process of MSC to pre-osteoblast or from osteoblast precursors. MSCs contribute to OS progression by interacting with tumor cells via paracrine signaling and affect tumor cell proliferation, invasion, angiogenesis, immune response, and metastasis. Extracellular vesicles (EVs), secreted by OS cells and MSCs in the tumor microenvironment, are crucial mediators of intercellular communication, driving OS progression by transferring miRNAs/RNA and proteins to other cells. MSC-derived EVs have both pro-tumor and anti-tumor effects on OS progression. MSC-EVs can be also engineered to deliver anti-tumor cargo to the tumor site, which offers potential applications in MSC-EV-based OS treatment. In this review, we highlight the role of MSCs in OS, with a focus on EV-mediated communication between OS cells and MSCs and their role in OS pathogenesis and therapy.     

Selective Inhibition of Helicobacter pylori Carbonic Anhydrases by Carvacrol and Thymol Could Impair Biofilm Production and the Release of Outer Membrane Vesicles

Helicobacter pylori, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti-H. pylori activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of H. pylori CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative H. pylori CA inhibitors or to be used in association with current drugs for the management of H. pylori infection and limiting the spread of antibiotic resistance.     

Extracellular Vesicles Inhibit the Response of Pancreatic Ductal Adenocarcinoma Cells to Gemcitabine and TRAIL Treatment

Pancreatic ductal adenocarcinoma remains an aggressive cancer with a low 5-year survival rate. Although gemcitabine has been a standard treatment for advanced pancreatic cancer, patients often develop resistance to this therapeutic. We have previously shown that treating pancreatic cancer cells in vitro with a combination of gemcitabine and the cytokine TRAIL significantly reduced both cell viability and survival. The data presented here demonstrate that this response to treatment is inhibited when cells are incubated with a conditioned medium derived from untreated cells. We show that this inhibition is specifically mediated by extracellular vesicles present in the conditioned medium, as seen by a significant decrease in apoptosis. Additionally, we further demonstrate that this effect can be reversed in the presence of GW4869, an inhibitor of exosome biogenesis and release. These results show that pancreatic cancer cell-derived extracellular vesicles can confer resistance to treatment with gemcitabine and TRAIL. The implications of these findings suggest that removal of EVs during treatment can improve the response of cells to gemcitabine and TRAIL treatment in vitro.     

The Role of Cancer Stem Cells and Their Extracellular Vesicles in the Modulation of the Antitumor Immunity

Cancer stem cells (CSCs) are a population of tumor cells that share similar properties to normal stem cells. CSCs are able to promote tumor progression and recurrence due to their resistance to chemotherapy and ability to stimulate angiogenesis and differentiate into non-CSCs. Cancer stem cells can also create a significant immunosuppressive environment around themselves by suppressing the activity of effector immune cells and recruiting cells that support tumor escape from immune response. The immunosuppressive effect of CSCs can be mediated by receptors located on their surface, as well as by secreted molecules, which transfer immunosuppressive signals to the cells of tumor microenvironment. In this article, the ability of CSCs to regulate the antitumor immune response and a contribution of CSC-derived EVs into the avoidance of the immune response are discussed.     

PCSK9 Confers Inflammatory Properties to Extracellular Vesicles Released by Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) are key participants in both early- and late-stage atherosclerosis and influence neighbouring cells possibly by means of bioactive molecules, some of which are packed into extracellular vesicles (EVs). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is expressed and secreted by VSMCs. This study aimed to unravel the role of PCSK9 on VSMCs-derived EVs in terms of content and functionality. EVs were isolated from human VSMCs overexpressing human PCSK9 (VSMC^PCSK9-EVs) and tested on endothelial cells, monocytes, macrophages and in a model of zebrafish embryos. Compared to EVs released from wild-type VSMCs, VSMC^PCSK9-EVs caused a rise in the expression of adhesion molecules in endothelial cells and of pro-inflammatory cytokines in monocytes. These acquired an increased migratory capacity, a reduced oxidative phosphorylation and secreted proteins involved in immune response and immune effector processes. Concerning macrophages, VSMC^PCSK9-EVs enhanced inflammatory milieu and uptake of oxidized low-density lipoproteins, whereas the migratory capacity was reduced. When injected into zebrafish embryos, VSMC^PCSK9-EVs favoured the recruitment of macrophages toward the site of injection. The results of the present study provide evidence that PCSK9 plays an inflammatory role by means of EVs, at least by those derived from smooth muscle cells of vascular origin.