Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells.

IMPORTANCE: Enteric adenoviruses have historically been difficult to grow in cell culture, which has resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum-derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

Structure and Role of O-Linked Glycans in Viral Envelope Proteins.

N- and O-glycans are both important constituents of viral envelope glycoproteins. O-linked glycosylation can be initiated by any of 20 different human polypeptide O-acetylgalactosaminyl transferases, resulting in an important functional O-glycan heterogeneity. O-glycans are organized as solitary glycans or in clusters of multiple glycans forming mucin-like domains. They are functional both in the viral life cycle and in viral colonization of their host. Negatively charged O-glycans are crucial for the interactions between glycosaminoglycan-binding viruses and their host. A novel mechanism, based on controlled electrostatic repulsion, explains how such viruses solve the conflict between optimized viral attachment to target cells and efficient egress of progeny virus. Conserved solitary O-glycans appear important for viral uptake in target cells by contributing to viral envelope fusion. Dual roles of viral O-glycans in the host B cell immune response, either epitope blocking or epitope promoting, may be exploitable for vaccine development. Finally, specific virus-induced O-glycans may be involved in viremic spread.

Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein.

Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate the interactions of the wild-type and truncated capsid proteins with membranes with biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids, which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.

Cellular Chondroitin Sulfate and the Mucin-like Domain of Viral Glycoprotein C Promote Diffusion of Herpes Simplex Virus 1 While Heparan Sulfate Restricts Mobility.

The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus-GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.

Membrane insertion mechanism of the caveola coat protein Cavin1.

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Dissimilar Deformation of Fluid- and Gel-Phase Liposomes upon Multivalent Interaction with Cell Membrane Mimics Revealed Using Dual-Wavelength Surface Plasmon Resonance.

The mechanical properties of biological nanoparticles play a crucial role in their interaction with the cellular membrane, in particular for cellular uptake. This has significant implications for the design of pharmaceutical carrier particles. In this context, liposomes have become increasingly popular, among other reasons due to their customizability and easily varied physicochemical properties. With currently available methods, it is, however, not trivial to characterize the mechanical properties of nanoscopic liposomes especially with respect to the level of deformation induced upon their ligand-receptor-mediated interaction with laterally fluid cellular membranes. Here, we utilize the sensitivity of dual-wavelength surface plasmon resonance to probe the size and shape of bound liposomes (∼100 nm in diameter) as a means to quantify receptor-induced deformation during their interaction with a supported cell membrane mimic. By comparing biotinylated liposomes in gel and fluid phases, we demonstrate that fluid-phase liposomes are more prone to deformation than their gel-phase counterparts upon binding to the cell membrane mimic and that, as expected, the degree of deformation depends on the number of ligand-receptor pairs that are engaged in the multivalent binding.

Protein-lipid interaction at low pH induces oligomerization of the MakA cytotoxin from Vibrio cholerae.

The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.

A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion.

The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD, and MakE. MakA, MakB, and MakE were readily detected in culture supernatants of wild-type V. cholerae, whereas secretion was very much reduced from a flagellum-deficient mutant. Crystal structures of MakA, MakB, and MakE revealed a structural relationship to a superfamily of bacterial pore-forming toxins. Expression of MakA/B/E in Escherichia coli resulted in toxicity toward Caenorhabditis elegans used as a predatory model organism. None of these Mak proteins alone or in pairwise combinations were cytolytic, but an equimolar mixture of MakA, MakB, and MakE acted as a tripartite cytolytic toxin in vitro, causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining the assembly of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity toward C. elegans Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen Vibrio anguillarum We suggest that the hitherto-unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms.

Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context.

The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry.

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced cell binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

Herpes Simplex Virus Type 2 Mucin-Like Glycoprotein mgG Promotes Virus Release from the Surface of Infected Cells.

The contribution of virus components to liberation of herpes simplex virus type 2 (HSV-2) progeny virions from the surface of infected cells is poorly understood. We report that the HSV-2 mutant deficient in the expression of a mucin-like membrane-associated glycoprotein G (mgG) exhibited defect in the release of progeny virions from infected cells manifested by ~2 orders of magnitude decreased amount of infectious virus in a culture medium as compared to native HSV-2. Electron microscopy revealed that the mgG deficient virions were produced in infected cells and present at the cell surface. These virions could be forcibly liberated to a nearly native HSV-2 level by the treatment of cells with glycosaminoglycan (GAG)-mimicking oligosaccharides. Comparative assessment of the interaction of mutant and native virions with surface-immobilized chondroitin sulfate GAG chains revealed that while the mutant virions associated with GAGs ~fourfold more extensively, the lateral mobility of bound virions was much poorer than that of native virions. These data indicate that the mgG of HSV-2 balances the virus interaction with GAG chains, a feature critical to prevent trapping of the progeny virions at the surface of infected cells.

The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization.

BACKGROUND: ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. RESULTS: Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. CONCLUSIONS: We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes.

Phosphatidic acid-mediated binding and mammalian cell internalization of the Vibrio cholerae cytotoxin MakA.

Vibrio cholerae is a noninvasive intestinal pathogen extensively studied as the causative agent of the human disease cholera. Our recent work identified MakA as a potent virulence factor of V. cholerae in both Caenorhabditis elegans and zebrafish, prompting us to investigate the potential contribution of MakA to pathogenesis also in mammalian hosts. In this study, we demonstrate that the MakA protein could induce autophagy and cytotoxicity of target cells. In addition, we observed that phosphatidic acid (PA)-mediated MakA-binding to the host cell plasma membranes promoted macropinocytosis resulting in the formation of an endomembrane-rich aggregate and vacuolation in intoxicated cells that lead to induction of autophagy and dysfunction of intracellular organelles. Moreover, we functionally characterized the molecular basis of the MakA interaction with PA and identified that the N-terminal domain of MakA is required for its binding to PA and thereby for cell toxicity. Furthermore, we observed that the ΔmakA mutant outcompeted the wild-type V. cholerae strain A1552 in the adult mouse infection model. Based on the findings revealing mechanistic insights into the dynamic process of MakA-induced autophagy and cytotoxicity we discuss the potential role played by the MakA protein during late stages of cholera infection as an anti-colonization factor.

A vaccine combination of lipid nanoparticles and a cholera toxin adjuvant derivative greatly improves lung protection against influenza virus infection.

This is a proof-of-principle study demonstrating that the combination of a cholera toxin derived adjuvant, CTA1-DD, and lipid nanoparticles (LNP) can significantly improve the immunogenicity and protective capacity of an intranasal vaccine. We explored the self-adjuvanted universal influenza vaccine candidate, CTA1-3M2e-DD (FPM2e), linked to LNPs. We found that the combined vector greatly enhanced survival against a highly virulent PR8 strain of influenza virus as compared to when mice were immunized with FPM2e alone. The combined vaccine vector enhanced early endosomal processing and peptide presentation in dendritic cells and upregulated co-stimulation. The augmenting effect was CTA1-enzyme dependent. Whereas systemic anti-M2e antibody and CD4+ T-cell responses were comparable to those of the soluble protein, the local respiratory tract IgA and the specific Th1 and Th17 responses were strongly enhanced. Surprisingly, the lung tissue did not exhibit gross pathology upon recovery from infection and M2e-specific lung resident CD4+ T cells were threefold higher than in FPM2e-immunized mice. This study conveys optimism as to the protective ability of a combination vaccine based on LNPs and various forms of the CTA1-DD adjuvant platform, in general, and, more specifically, an important way forward to develop a universal vaccine against influenza.

FRET-Based Assay for the Quantification of Extracellular Vesicles and Other Vesicles of Complex Composition.

Research in the field of extracellular vesicles is rapidly expanding and finding footholds in many areas of medical science. However, the availability of methodologies to quantify the concentration of membrane material present in a sample remains limited. Herein, we present a novel approach for the quantification of vesicle material, specifically the quantification of the total lipid membrane surface area, found in a sample using Förster resonance energy transfer (FRET). In this assay, sonication is used to drive the fusion between vesicles in the sample to be quantified and liposomes containing a pair of FRET fluorophores. The change in emission spectrum upon vesicle fusion is directly related to the total membrane surface area of the sample added, and a calibration curve allows for the quantification of a variety of vesicle species, including enveloped viruses, bacterial outer membrane vesicles, and mammalian extracellular vesicles. Without extensive optimization of experimental parameters, we were able to quantify down to ∼109 vesicles/mL, using as little as 60 µL of the sample. The assay precision was comparable to that of a commercial nanoparticle tracking analysis system. While its limit of detection was slightly higher, the FRET assay is superior for the detection of small vesicles, as its performance is vesicle-size-independent. Taken together, the FRET assay is a simple, robust, and versatile method for the quantification of a variety of purified vesicle samples.

Histo-blood group antigens of glycosphingolipids predict susceptibility of human intestinal enteroids to norovirus infection.

The molecular mechanisms behind infection and propagation of human restricted pathogens such as human norovirus (HuNoV) have defied interrogation because they were previously unculturable. However, human intestinal enteroids (HIEs) have emerged to offer unique ex vivo models for targeted studies of intestinal biology, including inflammatory and infectious diseases. Carbohydrate-dependent histo-blood group antigens (HBGAs) are known to be critical for clinical infection. To explore whether HBGAs of glycosphingolipids contribute to HuNoV infection, we obtained HIE cultures established from stem cells isolated from jejunal biopsies of six individuals with different ABO, Lewis, and secretor genotypes. We analyzed their glycerolipid and sphingolipid compositions and quantified interaction kinetics and the affinity of HuNoV virus-like particles (VLPs) to lipid vesicles produced from the individual HIE-lipid extracts. All HIEs had a similar lipid and glycerolipid composition. Sphingolipids included HBGA-related type 1 chain glycosphingolipids (GSLs), with HBGA epitopes corresponding to the geno- and phenotypes of the different HIEs. As revealed by single-particle interaction studies of Sydney GII.4 VLPs with glycosphingolipid-containing HIE membranes, both binding kinetics and affinities explain the patterns of susceptibility toward GII.4 infection for individual HIEs. This is the first time norovirus VLPs have been shown to interact specifically with secretor gene-dependent GSLs embedded in lipid membranes of HIEs that propagate GII.4 HuNoV ex vivo, highlighting the potential of HIEs for advanced future studies of intestinal glycobiology and host-pathogen interactions.

Independent Size and Fluorescence Emission Determination of Individual Biological Nanoparticles Reveals that Lipophilic Dye Incorporation Does Not Scale with Particle Size.

Advancements in nanoparticle characterization techniques are critical for improving the understanding of how biological nanoparticles (BNPs) contribute to different cellular processes, such as cellular communication, viral infection, as well as various drug-delivery applications. Since BNPs are intrinsically heterogeneous, there is a need for characterization methods that are capable of providing information about multiple parameters simultaneously, preferably at the single-nanoparticle level. In this work, fluorescence microscopy was combined with surface-based two-dimensional flow nanometry, allowing for simultaneous and independent determination of size and fluorescence emission of individual BNPs. In this way, the dependence of the fluorescence emission of the commonly used self-inserting lipophilic dye 3,3'-dioctadecyl-5,5'-di(4-sulfophenyl)oxacarbocyanine (SP-DiO) could successfully be correlated with nanoparticle size for different types of BNPs, including synthetic lipid vesicles, lipid vesicles derived from cellular membrane extracts, and extracellular vesicles derived from human SH-SY5Y cell cultures; all vesicles had a radius, r, of ∼50 nm and similar size distributions. The results demonstrate that the dependence of fluorescence emission of SP-DiO on nanoparticle size varies significantly between the different types of BNPs, with the expected dependence on membrane area, r2, being observed for synthetic lipid vesicles, while a significant weaker dependence on size was observed for BNPs with more complex composition. The latter observation is attributed to a size-dependent difference in membrane composition, which may influence either the optical properties of the dye and/or the insertion efficiency, indicating that the fluorescence emission of this type of self-inserting dye may not be reliable for determining size or size distribution of BNPs with complex lipid compositions.

Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO.

Lipophilic carbocyanine dyes are widely used as fluorescent cell membrane probes in studies ranging from biophysics to cell biology. While they are extremely useful for qualitative observation of lipid structures, a major problem impairing quantitative studies is that the chemical environment of the lipid bilayer affects both the dye's insertion efficiency and photophysical properties. We present a systematic investigation of the sulphonated carbocyanine dye 3,3'-dioctadecyl-5,5'-di(4-sulfophenyl) (SP-DiO) and demonstrate how its insertion efficiency into pre-formed lipid bilayers and its photophysical properties therein determine its apparent fluorescence intensity in different lipid environments. For this purpose, we use large unilamellar vesicles (LUVs) made of lipids with distinct chain unsaturation, acyl chain length, head group charge, and with variation in membrane cholesterol content as models. Using a combination of absorbance, fluorescence emission, and fluorescence lifetime measurements we reveal that SP-DiO incorporates more efficiently into liquid disordered phases compared to gel phases. Moreover, incorporation into the latter phase is most efficient when the mismatch between the length of the lipid and dye hydrocarbon chains is small. Furthermore, SP-DiO incorporation is less efficient in LUVs composed of negatively charged lipids. Lastly, when cholesterol was included in the LUV membranes, we observed significant spectral shifts, consistent with dye aggregation. Taken together, our study highlights the complex interplay between membrane composition and labeling efficiency with lipophilic dyes and advocates for careful assessment of fluorescence data when attempting a quantitative analysis of fluorescence data with such molecules.

Gel Phase 1,2-Distearoyl-sn-glycero-3-phosphocholine-Based Liposomes Are Superior to Fluid Phase Liposomes at Augmenting Both Antigen Presentation on Major Histocompatibility Complex Class II and Costimulatory Molecule Display by Dendritic Cells in Vitro.

Lipid-based nanoparticles have in recent years attracted increasing attention as pharmaceutical carriers. In particular, reports of them having inherent adjuvant properties combined with their ability to protect antigen from degradation make them suitable as vaccine vectors. However, the physicochemical profile of an ideal nanoparticle for vaccine delivery is still poorly defined. Here, we used an in vitro dendritic cell assay to assess the immunogenicity of a variety of liposome formulations as vaccine carriers and adjuvants. Using flow cytometry, we investigated liposome-assisted antigen presentation as well as the expression of relevant costimulatory molecules on the cell surface. Cytokine secretion was further evaluated with an enzyme-linked immunosorbent assay (ELISA). We show that liposomes can successfully enhance antigen presentation and maturation of dendritic cells, as compared to vaccine fusion protein (CTA1-3Eα-DD) administered alone. In particular, the lipid phase state of the membrane was found to greatly influence the vaccine antigen processing by dendritic cells. As compared to their fluid phase counterparts, gel phase liposomes were more efficient at improving antigen presentation. They were also superior at upregulating the costimulatory molecules CD80 and CD86 as well as increasing the release of the cytokines IL-6 and IL-1ß. Taken together, we demonstrate that gel phase liposomes, while nonimmunogenic on their own, significantly enhance the antigen-presenting ability of dendritic cells and appear to be a promising way forward to improve vaccine immunogenicity.

Bacteria-Based Production of Thiol-Clickable, Genetically Encoded Lipid Nanovesicles.

Despite growing research efforts on the preparation of (bio)functional liposomes, synthetic capsules cannot reach the densities of protein loading and the control over peptide display that is achieved by natural vesicles. Herein, a microbial platform for high-yield production of lipidic nanovesicles with clickable thiol moieties in their outer corona is reported. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented, and customizable corona of transmembrane polypeptides. Furthermore, this approach enables encapsulation of soluble proteins into the nanovesicles. Due to the mild preparation and loading conditions (absence of organic solvents, pH gradients, or detergents) and their straightforward surface functionalization, which takes advantage of the diversity of commercially available maleimide derivatives, bacteria-based proteoliposomes are an attractive eco-friendly alternative that can outperform currently used liposomes.