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Gas vesicle nanoparticles (GVNPs) are hollow, buoyant prokaryotic organelles used for cell flotation. GVNPs are encoded by a large gas vesicle protein ( gvp ) gene cluster in the haloarchaeon, Halobacterium sp. NRC-1, including one gene, gvpC , specifying a protein bound to the surface of the nanoparticles. Genetically engineered GVNPs in the Halobacterium sp. have been produced by fusion of foreign sequences to gvpC . To improve the versatility of the GVNP platform, we developed a method for displaying exogenously produced GvpC fusion proteins on the haloarchaeal nanoparticles. The streptococcal IgG-binding protein domain was fused at or near the C-terminus of GvpC, expressed and purified from E. coli , and shown to bind to wild-type GVNPs. The two fusion proteins, GvpC3GB and GvpC4GB, without or with a highly acidic GvpC C-terminal region, were found to be able to bind nanoparticles equally well. The GVNP-bound GvpC-IgG-binding fusion protein was also capable of binding to an enzyme-linked IgG-HRP complex which retained enzyme activity, demonstrating the hybrid system capability for display and delivery of protein complexes. This is the first report demonstrating functional binding of exogenously produced GvpC fusion proteins to wild-type haloarchaeal GVNPs which significantly expands the capability of the platform to produce bioengineered nanoparticles for biomedical applications. Key points • Haloarchaeal gas vesicle nanoparticles (GVNPs) constitute a versatile display system. • GvpC-streptococcal IgG-binding fusion proteins expressed in E. coli bind to GVNPs. • IgG-binding proteins displayed on floating GVNPs bind and display IgG-HRP complex. Graphical abstract

Background Various bacteria and archaea, including halophilic archaeon Halobacterium sp. NRC-1 produce gas vesicle nanoparticles (GVNPs), a unique class of stable, air-filled intracellular proteinaceous nanostructures. GVNPs are an attractive tool for biotechnological applications due to their readily production, purification, and unique physical properties. GVNPs are spindle- or cylinder-shaped, typically with a length of 100 nm to 1.5 μm and a width of 30–250 nm. Multiple monomeric subunits of GvpA and GvpC proteins form the GVNP shell, and several additional proteins are required as minor structural or assembly proteins. The haloarchaeal genetic system has been successfully used to produce and bioengineer GVNPs by fusing several foreign proteins with GvpC and has shown various applications, such as biocatalysis, diagnostics, bioimaging, drug delivery, and vaccine development. Results We demonstrated that native GvpC can be removed in a low salt buffer during the GVNP purification, leaving the GvpA-based GVNP's shell intact and stable under physiological conditions. Here, we report a genetic engineering and chemical modification approach for functionalizing the major GVNP protein, GvpA. This novel approach is based on combinatorial cysteine mutagenesis within GvpA and genetic expansion of the N-terminal and C-terminal regions. Consequently, we generated GvpA single, double, and triple cysteine variant libraries and investigated the impact of mutations on the structure and physical shape of the GVNPs formed. We used a thiol–maleimide chemistry strategy to introduce the biotechnological relevant activity by maleimide-activated streptavidin–biotin and maleimide-activated SpyTag003-SpyCatcher003 mediated functionalization of GVNPs. Conclusion The merger of these genetic and chemical functionalization approaches significantly extends these novel protein nanomaterials' bioengineering and functionalization potential to assemble catalytically active proteins, biomaterials, and vaccines onto one nanoparticle in a modular fashion. Graphical Abstract

To evaluate the effect of conditioned media (CM) and Extracellular Vesicles (EVs) derived from bovine oviduct epithelial cell (BOEC) lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E), together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150-200 nm) by Nanosight® and electron microscopy. Zygotes were cultured in the presence of 3x10(5) EVs/mL, 1.5x10(5) EVs/mL or 7.5x10(4) EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2) and blastocyst development (Day 7-9) was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the oviduct and the embryo in the early stages of development.

Extra-intestinal Pathogenic Escherichia coli (ExPEC) is defined as an extra-intestinal foodborne pathogen, and several dominant sequence types (STs) ExPEC isolates are highly virulent, with zoonotic potential. Bacteria extracellular vesicles (EVs) carry specific subsets of molecular cargo, which affect various biological processes in bacteria and host. The mechanisms of EVs formation in ExPEC remains to be elucidated. Here, the purified EVs of ExPEC strains of different STs were isolated with ultracentrifugation processes. A comparative analysis of the strain proteomes showed that cytoplasmic proteins accounted for a relatively high proportion of the proteins among ExPEC EVs. The proportion of cytoplasm-carrying vesicles in ExPEC EVs was calculated with a simple green fluorescent protein (GFP) expression method. The RecA/LexA-dependent SOS response is a critical mediator of generation of cytoplasm-carrying EVs. The SOS response activates the expression of prophage-associated endolysins, Epel1, Epel2.1, and Epel2.2, which triggered cell lysis, increasing the production of ExPEC cytoplasm-carrying EVs. The repressor LexA controlled directly the expression of these endolysins by binding to the SOS boxes in the endolysin promoter regions. Reducing bacterial viability stimulated the production of ExPEC EVs, especially cytoplasm-carrying EVs. The imbalance in cell division caused by exposure to H.sub.2 O.sub.2, the deletion of ftsK genes, or t.sup.6 A synthesis defects activated the RecA/LexA-dependent SOS response, inducing the expression of endolysins, and thus increasing the proportion of cytoplasm-carrying EVs in the total ExPEC EVs. Antibiotics, which decreased bacterial viability, also increase the production of ExPEC cytoplasm-carrying EVs through the SOS response. Changes in the proportion of cytoplasm-carrying EVs affected the total DNA content of ExPEC EVs. When macrophages are exposed to a higher proportion of cytoplasm-carrying vesicles, ExPEC EVs were more cytotoxic to macrophages, accompanied with more-severe mitochondrial disruption and a higher level of induced intrinsic apoptosis. In summary, we offered comprehensive insight into the proteome analysis of ExPEC EVs. This study demonstrated the novel formation mechanisms of E. coli cytoplasm-carrying EVs.

Key Points Rab GTPases control all aspects of intracellular vesicle trafficking by acting as regulatable switches that recruit effector molecules when in their active GTP-bound form. There are approximately 60 different Rab GTPases in humans that are associated with distinct intracellular membranes. Rab GTPases are turned on and off by specific guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively. Rab effectors come in many forms and range from vesicle tethers to motors, kinases, phosphatases and various adaptor proteins. Different Rab GTPases are restricted to distinct subdomains of the same organelle membrane and can crosstalk through effectors that are coupled to GEFs or GAPs. Rab GTPases are essential for physiological processes such as immunity, hormone secretion and neurotransmission, and their genetic inactivation is associated with diseases. Rab GTPases control intracellular vesicle traffic by acting as regulatable switches that recruit effector molecules when in their GTP-bound form. The functional coupling between multiple Rab GTPases ensures the spatiotemporally coordinated regulation of vesicle traffic. Membrane trafficking between organelles by vesiculotubular carriers is fundamental to the existence of eukaryotic cells. Central in ensuring that cargoes are delivered to their correct destinations are the Rab GTPases, a large family of small GTPases that control membrane identity and vesicle budding, uncoating, motility and fusion through the recruitment of effector proteins, such as sorting adaptors, tethering factors, kinases, phosphatases and motors. Crosstalk between multiple Rab GTPases through shared effectors, or through effectors that recruit selective Rab activators, ensures the spatiotemporal regulation of vesicle traffic. Functional impairments of Rab pathways are associated with diseases, such as immunodeficiencies, cancer and neurological disorders.

Cells release extracellular vesicles (EVs) in their environment and cellular lipids play an important role in their formation, secretion and uptake. Besides, there is also evidence that EV transferred lipids impact on recipient's cell signaling. Cellular senescence is characterized by a state of permanent proliferation arrest and represents a barrier towards the development of neoplastic lesions. A peculiar feature of senescence is the release of many soluble factors, the so-called Senescence-Associated Secretory Phenotype, which play a key role in triggering paracrine senescence signals. Recently, evidences have suggested that this phenotype includes not only soluble factors, but also EVs. To identify lipid signatures associated with H-Ras-induced senescence in EVs, we expressed active H-Ras (H-RasV12) in human fibroblasts and investigated how it affects EV release and lipid composition. An enrichment of hydroxylated sphingomyelin, lyso- and ether-linked phospholipids and specific H-Ras-induced senescence signatures, e.g. sphingomyelin, lysophosphatidic acid and sulfatides, were found in EVs compared to cells. Furthermore, H-RasV12 expression in fibroblasts was associated with higher levels of tetraspanins involved in vesicle formation.

The tumor microenvironment (TME) is an essential intrinsic portion of hepatocellular carcinoma (HCC) for the regulation of its origination, development, invasion, and metastasis. As emerging components of the tumor-host interaction, exosomes are increasingly recognized as professional carriers of information in TME and as pivotal molecular entities involved in tumorigenic microenvironment setup. However, much remains unknown about the role of the exosome communication system within TME in the development and progression of HCC. In this review, we focus on the roles and probable mechanisms of TME in HCC and show the exosome-based immune regulation in TME to promote HCC. Multiple processes are involved in HCC, including tumor survival, growth, angiogenesis, invasion, and metastasis. We also discuss the specific roles of exosomes in HCC processes by molding hospitable TME for HCC, such as providing energy, transmitting protumor signals, and evading inhibitory signals. In addition, exosomes induce angiogenesis by changing the biological characteristics of endothelial cells and directly regulating proangiogenic and propermeability factors. Furthermore, exosomes may lead to HCC metastatic invasion by epithelial-mesenchymal transformation, extracellular matrix degradation, and vascular leakage. Finally, we summarize the therapeutic usage of exosomes in the HCC microenvironment and attempt to provide a theoretical reference for modern antitumor agents designed to target these mechanisms.

Key Points Many bacteria and archaea synthesize intracellular gas-filled proteinaceous structures known as gas vesicles to act as flotation devices in aqueous environments. Gas vesicles provide buoyancy to cells and might help cells survive under stress conditions. Large amounts of gas vesicles reduce the size of the cytoplasm and thereby increase the cell surface-to-volume ratio. Gas vesicles add hollow spaces to the cells, and these spaces are filled, by diffusion, with gases that are dissolved in the surrounding medium; no storage of gas occurs. Gas vesicles of cyanobacteria are permeable to oxygen, nitrogen, hydrogen, carbon dioxide, carbon monoxide, methane and even perfluorocyclobutane. The archaeal gas vesicle wall is formed solely of protein, and the small hydrophobic protein gas vesicle protein A (GvpA) is the main constituent. Another structural protein is GvpC, which stabilizes the wall by attaching to the outside. In the halophilic archaeon Halobacterium salinarum , 12 additional Gvp proteins are required for gas vesicle formation, two of which, GvpD and GvpE, are involved in the regulation of gvp gene expression. GvpE increases transcription by 60–100-fold, whereas GvpD acts as a sink for GvpE. High salt concentrations, high cell densities or growth at low temperatures increase the formation of archaeal gas vesicles, whereas low salt concentrations, anoxic conditions or high light intensities decrease the production of these vesicles. Bacterial gvp gene clusters share many of the essential gvp genes with haloarchaea, but also contain genes with no haloarchaeal homologues. The formation of bacterial gas vesicles is influenced by cell density and light intensities. The formation of the gas vesicle wall is not yet fully understood. Initial information on the structure of GvpA came from solid-NMR studies and suggests a coil–α-helix–β-strand–β-strand–α-helix–coil peptide backbone. The GvpA monomer contains an antiparallel β-sheet (formed by the two β-strands), which might be extended by antiparallel associations with the β-sheets in adjacent monomers. Many bacteria and archaea synthesize intracellular gas-filled proteinaceous structures known as gas vesicles to act as flotation devices in aqueous environments. Here, Felicitas Pfeifer describes the basic properties of these interesting structures, the proteins that form them, the gene clusters that encode them and the regulation of their production. A range of bacteria and archaea produce intracellular gas-filled proteinaceous structures that function as flotation devices in order to maintain a suitable depth in the aqueous environment. The wall of these gas vesicles is freely permeable to gas molecules and is composed of a small hydrophobic protein, GvpA, which forms a single-layer wall. In addition, several minor structural, accessory or regulatory proteins are required for gas vesicle formation. In different organisms, 8–14 genes encoding gas vesicle proteins have been identified, and their expression has been shown to be regulated by environmental factors. In this Review, I describe the basic properties of gas vesicles, the genes that encode them and how their production is regulated. I also discuss the function of these vesicles and the initial attempts to exploit them for biotechnological purposes.

PDL1 blockade therapy holds great promise in cancer immunotherapy. Ultrasound imaging of PDL1 expression in the tumor is of great importance in predicting the therapeutic efficacy. As a proof‐of‐concept study, a novel ultrasound contrast agent has been innovated here to image and block PDL1 in the tumor tissue. Briefly, extracellular vesicles (EVs) are engineered to display truncated PD1 (tPD1) on the surface to bind PDL1 with high affinity by fusion to EV‐abundant transmembrane protein PTGFRN. The engineered EVs are then encapsulated with Ca(HCO3)2 via electroporation and designated as Gp‐EVtPD1, which would recognize PDL1 highly expressed cells and produce gas in the endosomes and lysosomes. On the one hand, the echogenic signal intensity correlates well with the PDL1 expression and immune response inhibition in the tumor. On the other hand, during the trajectory of Gp‐EVtPD1 in the recipient cells, tPD1 on the EV binds PDL1 and triggers the PDL1 endocytosis and degradation in endosomes/lysosomes in a sequential manner, and thus boosts the anti‐tumor immunity of cytotoxic T cells. In summary, Gp‐EVtPD1 serves as a novel ultrasound contrast agent and blocker of PDL1, which might be of great advantage in imaging PDL1 expression and conquering immune checkpoint blocker resistance. Extracellular vesicles (EVs) are engineered with fusion protein truncated PD1‐PTGFRN (tPD1‐PTGFRN) and then loaded Ca(HCO3)2 via electroporation, designated as Gp‐EVtPD1. Gp‐EVtPD1 would recognize PDL1 highly expressed cells and produce CO2 gas bubbles to realize ultrasound imaging of PDL1 and trigger PDL1 degradation.

Background Extracellular vesicles (EVs) play important roles in intercellular communication by delivering RNA, lipid, and proteins to neighboring or distant cells. Identification and classification of EVs secreted from diverse cell types are essential for understanding their signaling properties. Methods In this study, EVs from the culture media were isolated by ultracentrifugation and analyzed by electron microscopy (EM) and nanoparticle tracking analyses. Conditioned media (CM) from HEK293 cells culture grown either in serum-free (SF) or 10% fetal bovine serum (FBS) containing media were centrifuged at 100,000×g to separate the SN Δ supernatant and the P100 pellet in which exosomes are enriched. Then, the SN Δ fraction was centrifuged at 200,000×g to yield the P200 pellet fraction containing novel EVs smaller than exosomes. The exosomal markers in the EV subgroups were examined by western blotting and immune-EM, and the functional analyses of EVs were conducted on HEK293 and THP-1 cell culture. Results We identified a new group of EVs in the P200 fraction that was smaller than exosomes in size. Typical exosome markers such as Hsp70, TSG101, and CD63 were found in both P100 exosomes and the P200 vesicles, but CD81 was highly enriched in exosomes but not in the P200 vesicles. Furthermore, chemicals that inhibit the major exosome production pathway did not decrease the level of P200 vesicles. Therefore, these small EVs indeed belong to a distinguished group of EVs. Exosomes and the P200 vesicles were found in CM of human cell lines as well as FBS. Addition of the exosomes and the P200 vesicles to human cell cultures enhanced exosome production and cell proliferation, respectively. Conclusions Our study identifies a novel population of EVs present in the P200 fraction. This EV population is distinguished from exosomes in size, protein contents, and biogenesis pathway. Furthermore, exosomes promote their own production whereas the P200 vesicles support cell proliferation. In sum, we report a new group of EVs that are distinct physically, biologically and functionally from exosomes.