Microbes spontaneously release membrane vesicles (MVs), which play roles in nutrient acquisition and microbial interactions. Iron is indispensable for microbes, but is a difficult nutrient to acquire. However, whether MVs are also responsible for efficient iron uptake and therefore involved in microbial interaction remains to be elucidated. Here, we used a Gram-positive strain, Dietzia sp. DQ12-45-1b, to analyze the function of its MVs in heme-iron recycling and sharing between species. We determined the structure and constituent of MVs and showed that DQ12-45-1b releases MVs originating from the mycomembrane. When comparing proteomes of MVs between iron-limiting and iron-rich conditions, we found that under iron-limiting conditions, heme-binding proteins are enriched. Next, we proved that MVs participate in extracellular heme capture and transport, especially in heme recycling from environmental hemoproteins. Finally, we found that the heme carried in MVs is utilized by multiple species, and we further verified that membrane fusion efficiency and species evolutionary distance determine heme delivery. Together, our findings strongly suggest that MVs act as a newly identified pathway for heme recycling, and represent a public good shared between phylogenetically closely related species.

Background: Plasma extracellular vesicles (EVs), especially exosome-like vesicles (ELVs), are being increasingly explored as a source of potential noninvasive disease biomarkers. The discovery of blood-based biomarkers associated with ELVs reguires methods that isolate high yields of these EVs without significant contamination with highly abundant plasma proteins and lipoproteins. The rising interest in blood-based EV-associated biomarkers has led to the rapid development of novel EV isolation methods. However, the field suffers from a lack of standardization and often, new technigues are used without critical evaluation. Size exclusion chromatography (SEC) has become the method of choice for rapid isolation of relatively pure EVs from plasma, yet it has technical limitations for certain downstream applications. The recently released exoEasy kit (Qiagen) is a new membrane affinity spin column method for the isolation of highly pure EVs from biofluids with the potential to overcome most of the limitations of SEC.Methods: By using multiple complementary technigues we assessed the performance of the exoEasy kit in isolating ELVs from 2 ml of human plasma and compared it with the SEC qEV column (Izon Science).Results: Our data show that exoEasy kit isolates a heterogenous mixture of particles with a larger median diameter, broader size range and a higher yield than the SEC gEV column. The exclusive presence of small RNAs in the particles and the total RNA yield were comparable to the SEC gEV column. Despite being less prone to low density lipoprotein contamination than the SEC gEV column, the overall purity of exoEasy kit EV preparations was suboptimal.The low particle-protein ratio, significant amount of albumin, very low levels of exosome-associated proteins and propensity to triglyceride-rich lipoprotein contamination suggest isolation of mainly non-ELVs and co-isolation of plasma proteins and certain lipoproteins by the exoEasy kit.Conclusions: We demonstrate that performance of exoEasy kit for the isolation of ELVs for biomarker discovery is inferior to the SEC gEV column. This comprehensive evaluation of a novel EV isolation method contributes to the acceleration of the discovery of EV-associated biomarkers and the development of EV-based diagnostics.

The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, 1× phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at ∼1.3 V AC. A375 melanoma exosome recovery was approximately 70-80% after each buffer substitution, and recovery was independent of whether the sample was substituted into 1× PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using 1× PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from ∼-1.6 to -0.1 μm cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from ∼-5.5 to -0.1 μm cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.

Although many properties for small extracellular vesicles (sEVs, formerly termed "exosomes") isolated at ∼100 000 are known, a wide range of values are reported for their electrophoretic mobility (EM) measurements. This paper reports for the first time the effect of dilution on the EM of U87 glioblastoma cell-derived and plasma-derived sEVs and medium size EVs (mEVs, commonly termed "oncosomes") preisolated by differential centrifugation. Furthermore, the effect of resalting on the EM of sEVs and mEVs was evaluated. The EM of U87 sEVs and U87 mEVs showed an increase as the salt concentration decreased to 0.005% of the initial salt concentration. However, for the plasma sEVs and plasma mEVs, the electrophoretic mobility increased as the salt concentration decreased to 0.01% of the initial salt concentration and then increased to its initial value when the salt concentration decreased to 0.005% of the initial salt concentration. For both U87 and plasma sEVs and mEVs, the EM remained almost constant when the concentration of the particles changed and the salt concentration was kept the same as its initial value. This indicates that the EM of EVs is only a function of the salt concentration of the buffer and is independent of the concentration of the particles. The sEVs and mEVs were separated with cyclical ElFFF for the first time. The results indicate that ElFFF was able to fractionate the EVs, and a crescent-shaped trend was found for the retention time when the applied AC voltage was altered (increased).

Immunomagnetic EpCAM based methods are used to enrich circulating tumor cells (CTCs) in metastatic breast cancer (mBC) patients. EpCAM negative CTCs may be missed. We addressed the question of the reliability of an EpCAM dependent assay to enrich CTCs.To elucidate this issue, our study has been designed to assess two different CTC enrichment technologies (i) in EpCAM positive (+) and EpCAM negative cell lines and (ii) in mBC patients in dependency on their respective EpCAM expression. These two technologies encompass one anti-EpCAM immunomagnetic enrichment technology, MACS HEA MicroBeads(®) (MACS), and one EpCAM independent density centrifugation method, OncoQuick(®) plus (OQ+). Furthermore, the coherence between EpCAM expression in the primary tumor tissue of mBC patients and the CTC detection rates in the corresponding patients is analyzed.(i) MACS recovered significantly more EpCAM (+) than EpCAM (-) tumor cells (p < 0.001) in spiked blood samples. With OQ+ no significantly different recovery rates between EpCAM (+) and EpCAM (-) tumor cells (p = 0.796) were detected. (ii) In mBC patients MACS yielded a significantly higher (p = 0.024) detection rate of EpCAM (+) CTCs. No statistically significant difference (p = 0.070) was found concerning the EpCAM status-based detection rate of CTCs by OQ+. (iii) CTC detection rates are independent of the primary tumors' EpCAM expression.EpCAM (-) CTCs can not be detected by immunomagnetic EpCAM dependent enrichment methods. EpCAM independent enrichment technologies seem to be superior to detect the entire CTC population. Evaluation of CTCs as prognostic marker should compromise EpCAM (+) and (-) subpopulations.

Epithelial cell adhesion molecule (Ep-CAM; CD326) is used as a target by many immunotherapeutic approaches, but little data are available about Ep-CAM expression in major human malignancies with respect to level, frequency, tumour stage, grade, histologic tumour type and impact on survival. We analysed by immunohistochemical staining tissue microarrays with 4046 primary human carcinoma samples from colon, stomach, prostate and lung cancers for both frequency and intensity of Ep-CAM expression under highly standardised conditions. A total of 3360 samples were analysable. High-level Ep-CAM expression was observed in 97.7% (n=1186) of colon, 90.7% of gastric (n=473), and 87.2% of prostate cancers (n=414), and in 63.9% of lung cancers (n=1287). No detectable Ep-CAM staining was found with only 0.4% of colon, 2.5% of gastric, 1.9% of prostate cancers, and 13.5% of lung cancers. The only significant correlation of Ep-CAM expression with tumour grading was observed in colon cancer where high-level Ep-CAM expression on grade 3 tumours was down to 92.1% (P<0.0001). Adenosquamous and squamous carcinomas of the lung had a lower percentage of high-level Ep-CAM expression compared to adenocarcinomas with 35.4 and 53.6%, respectively, and with 45.5 and 17.3% of tumours being Ep-CAM negative. With the exception of moderately differentiated colon carcinoma, where patients not expressing Ep-CAM on their tumours showed an inferior survival (P=0.0014), correlation of Ep-CAM expression with survival did not reach statistical significance for any of the other cancer indications and subgroups. In conclusion, the data strongly support the notion that Ep-CAM is a prime target for immunotherapies in major human malignancies. This is because the most common human cancers show (i) a low frequency of Ep-CAM-negative tumours, (ii) a high frequency of Ep-CAM expression on cells of a given tumour, and (iii) for most cancers, an insignificant influence of tumour staging, grading and histology on Ep-CAM expression.

The use of exosomes in clinical settings is progressively becoming a reality, as clinical trials testing exosomes for diagnostic and therapeutic applications are generating remarkable interest from the scientific community and investors. Exosomes are small extracellular vesicles secreted by all cell types playing intercellular communication roles in health and disease by transferring cellular cargoes such as functional proteins, metabolites and nucleic acids to recipient cells. An in-depth understanding of exosome biology is therefore essential to ensure clinical development of exosome based investigational therapeutic products. Here we summarise the most up-to-date knowkedge about the complex biological journey of exosomes from biogenesis and secretion, transport and uptake to their intracellular signalling. We delineate the major pathways and molecular players that influence each step of exosome physiology, highlighting the routes of interest, which will be of benefit to exosome manipulation and engineering. We highlight the main controversies in the field of exosome research: their adequate definition, characterisation and biogenesis at plasma membrane. We also delineate the most common identified pitfalls affecting exosome research and development. Unravelling exosome physiology is key to their ultimate progression towards clinical applications. Video Abstract.

Conditions that impair protein folding in the Gram-negative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism.

Organophosphate compounds are ubiquitously employed as agricultural pesticides and maintained as chemical warfare agents by several nations. These compounds are highly toxic, show environmental persistence and accumulation, and contribute to numerous cases of poisoning and death each year. While their use as weapons of mass destruction is rare, these never fully disappear into obscurity as they continue to be tools of fear and control by governments and terrorist organizations. Beyond weaponization, their wide-scale dissemination as agricultural products has led to environmental accumulation and intoxication of soil and water across the globe. Therefore, there is a dire need for rapid and safe agents for environmental bioremediation, personal decontamination, and as therapeutic detoxicants. Organophosphate hydrolyzing enzymes are emerging as appealing targets to satisfy decontamination needs owing to their ability to hydrolyze both pesticides and nerve agents using biologically-derived materials safe for both the environment and the individual. As the release of genetically modified organisms is not widely accepted practice, researchers are exploring alternative strategies of organophosphate bioremediation that focus on cell-free enzyme systems. In this review, we first discuss several of the more prevalent organophosphorus hydrolyzing enzymes along with research and engineering efforts that have led to an enhancement in their activity, substrate tolerance, and stability. In the later half we focus on advances achieved through research focusing on enhancing the catalytic activity and stability of phosphotriesterase, a model organophosphate hydrolase, using various approaches such as nanoparticle display, DNA scaffolding, and outer membrane vesicle encapsulation.Copyright © 2019 Thakur, Medintz and Walper.

Some pathogens and pests deliver small RNAs (sRNAs) into host cells to suppress host immunity. Conversely, hosts also transfer sRNAs into pathogens and pests to inhibit their virulence. Although sRNA trafficking has been observed in a wide variety of interactions, how sRNAs are transferred, especially from hosts to pathogens and pests, is still unknown. Here, we show that host cells secrete exosome-like extracellular vesicles to deliver sRNAs into fungal pathogen These sRNA-containing vesicles accumulate at the infection sites and are taken up by the fungal cells. Transferred host sRNAs induce silencing of fungal genes critical for pathogenicity. Thus, has adapted exosome-mediated cross-kingdom RNA interference as part of its immune responses during the evolutionary arms race with the pathogen.Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Quorum sensing is a process of bacterial cell-to-cell chemical communication that relies on the production, detection and response to extracellular signalling molecules called autoinducers. Quorum sensing allows groups of bacteria to synchronously alter behaviour in response to changes in the population density and species composition of the vicinal community. Quorum-sensing-mediated communication is now understood to be the norm in the bacterial world. Elegant research has defined quorum-sensing components and their interactions, for the most part, under ideal and highly controlled conditions. Indeed, these seminal studies laid the foundations for the field. In this Review, we highlight new findings concerning how bacteria deploy quorum sensing in realistic scenarios that mimic nature. We focus on how quorums are detected and how quorum sensing controls group behaviours in complex and dynamically changing environments such as multi-species bacterial communities, in the presence of flow, in 3D non-uniform biofilms and in hosts during infection.

The classical quorum-sensing (QS) model is based on the assumption that diffusible signaling molecules accumulate in the culture medium until they reach a critical concentration upon which expression of target genes is triggered. Here we demonstrate that the hydrophobic signal N-hexadecanoyl-L-homoserine lactone, which is produced by Paracoccus sp., is released from cells by the aid of membrane vesicles (MVs). Packed into MVs, the signal is not only solubilized in an aqueous environment but is also delivered with varying propensities to different bacteria. We propose a novel MV-based mechanism for binary trafficking of hydrophobic signal molecules, which may be particularly relevant for bacteria that live in open aqueous environments.

Microbes have evolved over millennia to become adapted and specialized to the environments that they occupy. These environments may include water or soil, extreme environments such as hydrothermal vents, and can even include a host organism. To become adapted to these locations, microbes have evolved specific tools to mediate interactions with the environment. One such tool that prokaryotes have evolved includes the production of membrane vesicles (MVs). MVs are 10-300 nm spherical blebs derived from the outermost membrane and have known functions in protein secretion, immune activation and suppression, stress response, attachment, internalization and virulence. In this review, we consider the highly conserved role of membrane vesicles derived from Gram-negative, Gram-positive and archaeal species as a mechanism to facilitate intermicrobial and microbe-host interaction. We examine both the offensive and defensive capabilities of MVs in regard to the interaction of MVs with both host and microbial cells in their environment.Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Conventionally used antibiotics are present in low concentrations at the infection site and require multiple administrations to sustain a continuous bactericidal effect, which not only increases their systemic toxicity but also results in bacterial drug resistance. In this study, we first identified an interesting drug resistance mechanism mediated by bacterial outer membrane vesicles (OMVs) and then designed novel antibiotic-loaded OMVs using this mechanism. We show that these antibiotic-loaded OMVs can effectively enter and kill pathogenic bacteria in vitro. In a mouse model of intestinal bacterial infection, one low-dose oral administration of antibiotic-loaded OMVs showed that the drug was retained in the intestine for 36 h, and no systemic spread was detected 12 h after drug administration. The antibiotic-loaded OMVs significantly reduced the bacterial load in the small intestine and feces of infected mice. Safety experiments confirmed that the antibiotic-loaded OMVs had excellent biocompatibility. This study extends the application range of OMVs and provides new ideas for the development of antibacterial drugs.Copyright © 2019 Elsevier B.V. All rights reserved.

细胞外囊泡（extracellular vesicles,EVs）是细胞旁分泌释放到细胞外的膜性小囊泡,几乎所有类型的细胞均可以产生并释放细胞外囊泡,外泌体（exosomes,Exo）、微囊泡（microvesicles,MVs）等均在其范畴,作为细胞间信号传导的重要方式,其功能已成为当前生命科学研究中的热点。研究表明,细胞外囊泡在多种寄生虫中均能分泌,且广泛参与虫体感染宿主及致病的生理过程,有望成为相关疾病诊断的生物标志物、治疗靶点或工具,具有广阔的应用前景。本文重点对主要的致病寄生虫分泌的细胞外囊泡及其生理功能进行综述。

The twenty-first century has witnessed major developments in the field of extracellular vesicle (EV) research, including significant steps towards defining standard criteria for the separation and detection of EVs. The recent recognition that EVs have the potential to function as biomarkers or as therapeutic tools has attracted even greater attention to their study. With this progress in mind, an updated comprehensive overview of the roles of EVs in the immune system is timely. This Review summarizes the roles of EVs in basic processes of innate and adaptive immunity, including inflammation, antigen presentation, and the development and activation of B cells and T cells. It also highlights key progress related to deciphering the roles of EVs in antimicrobial defence and in allergic, autoimmune and antitumour immune responses. It ends with a focus on the relevance of EVs to immunotherapy and vaccination, drawing attention to ongoing or recently completed clinical trials that aim to harness the therapeutic potential of EVs.© 2022. Springer Nature Limited.

Extracellular vesicles (EVs) are important for cell-to-cell communication in animals. EVs also play important roles in plant-microbe interactions, but the underlying mechanisms remain elusive. Here, proteomic analyses of EVs from the soybean (Glycine max) root rot pathogen Phytophthora sojae identify the tetraspanin family proteins PsTET1 and PsTET3, which are recognized by Nicotiana benthamiana to trigger plant immune responses. Both proteins are required for the full virulence of P. sojae. The large extracellular loop (EC2) of PsTET3 is the key region recognized by N. benthamiana and soybean cells in a plant receptor-like kinase NbSERK3a/b dependent manner. TET proteins from oomycete and fungal plant pathogens are recognized by N. benthamiana thus inducing immune responses, whereas plant-derived TET proteins are not due to the sequence divergence of sixteen amino acids at the C-terminal of EC2. This feature allows plants to distinguish self and non-self EVs to trigger active defense responses against pathogenic eukaryotes.© 2023. Springer Nature Limited.