Abundant OM proteins(OMPs; i.e., OmpA, OmpC and OmpF), membrane interstitial proteins(AcrA and alkaline phosphatase), misfolded proteins and virulence factors associated with adhesion and invasion of host organs are found in EVs (17) ( Figure 2 ). More than 3500 proteins belonging to various functional classes have been characterised using MS-based proteomic approaches after purification of EVs via ultracentrifugation. According to a global proteomic analysis, EVs lack components of the inner cell membrane and cytoplasm, implying that vesicle formation is a directed process instead of a random process (18). EVs are formed via blistering and swelling and mostly consist of interstitial proteins and OM protein envelope components. Over the past few years, high-throughput proteomic data of EVs collected from pathogens and commensal bacteria have revealed new functional proteins that can be used for biomedical applications (19). Therefore, the integration of proteomic data under various physiological conditions may facilitate the development of EV-based biomimetic materials (20).

Bacterial EVs carry lumen- and surface-associated DNA, RNA, purified plasmids, bacteriophage DNA and chromosomal DNA( Figure 2 ). A study revealed the presence of DNA in EVs by treating bacterial samples with DNase (21). Resistance to luminal DNA was maintained even after finishing the course of treatment (22). Additionally, a few different forms of Escherichia coli(E. coli), Neisseria gonorrhoeae(N. gonorrhoeae), Pseudomonas aeruginosa(P. aeruginosa)and Haemophilus influenzae(H. influenzae)have been reported (22). Genomic DNA has been found in EVs released by the commensal bacterium Enterobacter cloacae (18). Over the past few years, small noncoding RNAs(sRNAs)have been discovered in bacterial EVs (23). These sRNAs have a length of 50–400 nucleotides (24). For instance, Ghosal et al. reported that sRNAs in E. coli MG1655-derived EVs had a length of 15–40 nucleotides and performed numerous functions (25, 26). To escape degradation by RNases and perform biological functions in the related host, bacteria may carry functional sRNAs in EVs. Although functional sRNAs native to bacterial EVs have been identified in various studies via high-throughput RNA sequencing (26), their function remains unclear.

Lipids are one of the key components of the structure of bacterial EVs. Based on the enriched and excluded EV components, some lipid components may be important for EV biogenesis. In Gram-negative bacteria, lipopolysaccharides(LPSs)and phospholipids(PLs)are the most common lipid components (27) ( Figure 2 ). PLs constitute the inner layer of the OM, whereas LPSs are found only on the outer surface. The content of PLs varies among Gram-negative bacteria. Phosphatidylethanolamine(PE), phosphatidylglycerol(PG)and lysophosphate(LY)are the main PLs in E. coli EVs, whereas PG, PE and stearic acid are the major lipid components in other bacterial EVs (28). The three major classes of OM lipids in the myxobacterium Sorangium cellulosum include sphingolipids(SLs), which have long-chain amino alcohol backbone; glyceryl ether lipids(GELs)and bird-containing lipids amino acid lipid (OLs). In group A Streptococcus (GAS) -derived EVs, more than 85 glycerolipids (GLs)are present, including anionic and cationic PLs (29). In Hp-derived EVs, cardiolipin(CL)is a major lipid component. In Gram-negative bacteria, the OM contains both neutral (O-LPS)and negatively charged (A) -LPSO antigenic residues. EVs derived from Gram-negative bacteria contain only A-LPS (17). Gingivalase, a protein highly abundant in P. gingivalis, is eliminated in the A-LPS mutant strain, which highlights the importance of EV packaging. A recent study demonstrated that delivery of P. gingivalis SL via EVs can alter host inflammatory responses. Because EVs derived from Singomonas paucimobilis contain SL, a lipid class that plays an important structural and signalling role in eukaryotes, this bacterium may be used to build a system in which host–bacteria interactions are exploited.

EVs can directly communicate between bacteria and the host by delivering toxins and enhancing bacterial virulence in host cells to benefit pathogenic bacteria (30) ( Figure 2 ). As an example, EstA, a bacterial virulence factor derived from the EVs of P. aeruginosa, induces nitric oxide and pro-inflammatory cytokines during macrophage interaction with host cells (18). As a result of these virulence factors, host cells are dysregulated by proteins released by EVs, thereby affecting proteolysis, ion transport and ion binding (18). EVs derived from H. pylori exhibit Lewis antigens on their surface and can activate the immune system of the host. As a result, EVs directly bind to anti-Lewis antibodies in serum, thereby decreasing the self-defence ability of host cells (31). EVs play an important role in a wide range of biomedical applications because they can deliver various toxins and activate the defence system of the host. Therefore, EVs can be used to develop nano-platforms.

LPS is the most potent and immunostimulatory component of EVs (32). EV-based vaccines with diametrically opposed views were developed by two scientists who discovered LPS. Owing to the risk of inducing systemic reactogenicity when administered to humans(which is why LPS was historically called an endotoxin), LPS poses safety concerns. However, the presence of LPS supports the ability of EVs to stimulate the immune system effectively. Developing EV-based vaccines requires maintaining the balance between reactogenic risk and the ability to stimulate an effective immune response (33).

The lipid A subunit of LPS directly interacts with toll-like receptor (TLR) 4 (32), and changes in its composition significantly affect its binding and recognition, resulting in decreased agonistic or antagonistic effects, dimerisation of TLR4/MD-2 and downstream signalling.

The basic lipid A structure in Gram-negative bacteria is phosphorylated at positions 1 and 40 by a glucosamine disaccharide and Acylated composition at positions 2 and 3 with R-3-hydroxymyristate at positions 20 and 30 (known as lipid IV A). Several enzymes decorate this structure at various positions, especially fatty acids of varying length and saturation, or alter it by adding (or removing)phosphate groups of glucosamine (34). Bacteria use changes in the basic structure of lipid A to respond to various shocks and modulate host–pathogen interactions, thereby evading innate immune detection (34, 35). In E. coli and Shigella, the late acyltransferases HtrB (36)(also known as LpxL) and MsbB (also known as LpxM)transfer lauroyl and myristoyl fatty acids to positions 30 and 20, respectively, resulting in the highest reactivity of lipid A (phosphorylated at positions 1 and 40, acyl chain length of 12–14 carbons and asymmetric distribution) (37). PagP (38) catalyses the addition of a seventh fatty acid chain to hexaacylated lipid A via 2-O-palmitoylation, whereas LpxR (39) or PagL (40) catalyses deacylation. EptA and ArnT add phosphatidylethanolamine or arabinose to 1 and 40 phosphate groups (41), respectively, whereas LpxE degrades one phosphate group. Hydroxylation of LpxO is another widely described example of an enzymatic reaction that targets different lipid A sites (42, 43). Additionally, different enzymes can act on the same site of lipid A molecules, catalysing the substitution of some acyl chains by other amino chains(LpxP can add palm oleyl chains to the same site as HtrB) (44). Depending on the substrate specificity, enzymes have different acyl chains(acyl chains of varying lengths of 2–4 carbons are attached depending on stress conditions or the metabolites present). Therefore, the various types of lipid A provide bacteria with different abilities to activate TLR4, and hence, EVs have different abilities to activate TLR4, with different structures having different agonistic and antagonistic abilities (45).

A majority of genes involved in lipid A biosynthesis are essential for bacterial membrane integrity. Therefore, to alter the acylation state of lipid A with minimal defects in bacterial growth, genes encoding late acyltransferases should be inactivated individually or in combination. For instance, HtrB or MsbB deletion results in the production of EVs with predominantly pentaacylated lipid A, with EVs carrying wild-type lipid A for each. However, these EVs significantly decrease the levels of proinflammatory cytokines. In Salmonella enterica and Francisella tularensis (33, 46, 47), dephosphorylation of lipid A has been examined preclinically by knocking down the enzyme encoded by lpxE or by overexpressing the deacylase encoded by lpxR, which resulted in the cleavage of two fatty acid chains or PagL, which is a 3-O-degradation enzyme. For the development of meningococcal vaccines based on EVs, the endotoxin-reducing activity of lipid A is also essential. The LPS content of EVs is reduced when a detergent-based extraction method(for example, Bexsero)is used. In addition, genetically detoxified LPS can be used to generate meningococcal strains, specifically by knocking out the L1 (48) and L2 (49) genes. These genes produce pentaacylated lipid A, which significantly attenuates endotoxin activity, both preclinically and clinically.

Other PAMPs have also been found in EVs. Stably transfected human embryonic kidney cells expressing only specific TLRs consistent with their native lipoprotein content have been shown to efficiently activate TLR2 via GMMA in Shigella (46), Salmonella (50) and Neisseria meningitidis. Additionally, targeted blockade of TLRs in human PBMCs, alone or in combination, elucidates the relative contribution of TLR2, TLR4 and TLR5 to the ability of Shigella and Salmonella GMMA with modified lipid A to induce pro-inflammatory responses ( Figure 2 ). The contribution of TLR4 to pro-inflammatory responses is significantly reduced to a level where TLR2 activation mainly mediates the response, whereas the relative contribution of PRRs other than TLR2, TLR4 and TLR5 is negligible(<10%). Non-LPS PAMPs can enhance immune responses to vesicles (51) in LPS-deficient EVs in Neisseria. Flagellin and CpG DNA have been found in Salmonella GMMA and Pseudomonas aeruginosa-derived vesicles (52). However, whether they directly affect the response of the host to intact vesicles containing LPS remains unclear. Shigella GMMA preparations, possibly purified with GMMA, have low levels of cytoplasmic proteins(mainly ribosomal proteins) (53) ( Figure 3 ).

EVs can interact with other microorganisms and hosts in the environment and have specialised communication systems under variable or challenging environmental conditions, including quorum sensing(QS), biofilm formation, nutrient acquisition, antibiotic resistance and competition with or defence against other microorganisms (54). Paracoccus denitrificans- and Vibrio harveii-derived EVs use quorum sensing under activated quinolone signalling(PQS)to interact with other bacteria and host cells, such as C16-HSL and CAI-1 (54). In bacterial communities, EVs share biofilm substrates to improve their ability to absorb nutrients and survive. Plankton- and biofilm-derived EVs in P. aeruginosa differ in quantity, quality, proteolytic activity and antibiotic-binding ability and participate in some activities of biofilms (55). Additionally, EVs derived from antibiotic-resistant strains have been shown to transfer antibiotic resistance genes and proteins to susceptible strains (9). For example, Acinetobacter baumannii-derived EVs contain carbapenem-resistance-related genes that can be horizontally transferred to carbapenem-resistant organisms (56). Furthermore, E. coli produces EVs that contain colistin, which degrades antimicrobial peptides such as melittins (57). The EVs of Moraxella catarrhalis and Staphylococcus aureus carry beta-lactamase, which allows them to escape antibiotics.

Horizontal gene transfer is a primary EV-mediated mechanism. However, only a few studies have examined the gene transfer ability of EVs. There are three steps in the process as follows: DNA is released from EV-encapsulated donor bacteria, EVs are incorporated with recipient bacteria and genetic material is transferred into the cytoplasm of the recipient bacteria. Several instances and related factors have been reported;however, this review does not address them in detail. Further investigation is required to understand the mechanisms underlying the loading of EVs and their attachment to recipient bacteria. In addition, novel mechanisms underlying EV-mediated bacterial communication should be investigated in species with varying lipid compositions (58) ( Figure 4 ).

Because EVs contain many immune-reactive proteins originating from their parent bacterium, they modulate innate immune responses in the host without direct host cell–bacteria interactions (59). In terms of structure and biological activity, bacteria-derived EVs are similar to those secreted by mammalian cells. EVs are required to alter the integrity of the epithelial barrier to maintain homeostasis in mammalian cells associated with inflammatory and metabolic diseases and induce immunomodulatory activity (60). Furthermore, the gut microbiome and intestinal epithelium interact to maintain barrier integrity. For example, EVs derived from probiotic E. coli and commensal ECOR63 can enhance barrier function (61).

In addition to interacting with host epithelial cells, in which EVs derived from both Gram-negative and -positive bacteria are involved, EVs directly affect immune cells, either by activating or suppressing immune responses. The gastric mucosa of patients with H. pyroli infection has been reported to contain EVs (62). S. typhimurium-derived EVs promote flagellin-mediated caspase-1 activation and IL-1β secretion in an endocytosis-dependent manner (59). EVs derived from H. pyroli, Neisseria, Pseudomonas, Campylobacter and Vibrio induce the secretion of interleukin-8(IL-8), a chemokine produced by tissues and blood cells, in non-phagocytic epithelial cells (63). EVs derived from Gram-positive bacteria such as Lactobacillus plantarum and L. sakei can protect against pathogen infection by stimulating host defence-related genes. The TLR8 and NF-κB signalling pathways in A. actinomycetemcomitans-derived EVs can activate the pro-inflammatory cytokine TNF-α (64).

EVs derived from many pathogenic bacteria contain toxins, degrading enzymes, virulence factors and pro-inflammatory compounds. These components can be used as tools to facilitate the contact of EVs with host cells. The Apx toxin (Shiga toxin)is activated by A. pleuropneumoniae-derived EVs and induces haemolysis and cytolysis. Salmonella typhimurium, however, possesses a special secretion system called the type III secretion system (T3SS)that secretes more than 40 virulence factors (65). EV-related virulence factors are not completely understood;however, it is evident that the survival of Salmonella is reduced without these factors. Although these toxins are found on the outer surface of vesicles, they can often resist proteases (65).

Gram-negative and -positive bacteria secrete EVs with particle sizes of 20–300 nm. EVs contain biologically active proteins, toxins, virulence factors and immunogenic materials that stimulate bacteria–host interactions, through which vaccines are developed (65). The non-replicative nature of EVs makes them relatively safe, and their biological roles are unpredictable under different environmental stresses. Therefore, in vivo biomedical applications of EVs can be more effective.

The human microbiome is an inherent element in the lifelong conservation of fitness and immune system homeostasis. It plays a fundamental role in tumorigenesis, cancer course and disease feedback. Approximately 85% of cases are related to PDAC, with only 24% of patients surviving 1 year after diagnosis, and the 5-year survival rate is approximately 5–7%.

Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans have been associated with an increased risk of PDAC (66). Porphyromonas gingivalis and Actinobacillus congeners were found to increase the risk of PDAC by 59% and 50%, respectively, in a nested case–control study that involved 361 patients with PDAC and 371 control patients and spanned over a decade (67). Periodontitis plays an important role in the pathogenesis of PC, and the two abovementioned oral microbes are causative agents (66, 68). Microbes from the mouth can migrate and translocate to the pancreas and other organs through the vascular network and/or the digestive tract. When located at a distance, disruption of the immune system and inflammation can accelerate cancer progression (69).

Porphyromonas gingivalis is a major pathogen associated with oral and gastrointestinal cancers (70). It exerts its toxicity through various virulence factors;however, recent studies have revealed a unique protein called PPAD. Gabarrini et al. reported that PPAD homologues are found in other Porphyromonas species, such as P. gulae and P. loveae ( 71, 72 ). Several studies have suggested that PPAD plays a role in the tumorigenesis of PC through KRAS mutation and P53 activation. Normally, the tumour suppressor gene P53 causes cell cycle arrest, thus allowing DNA repair in damaged cells (73). If the damage cannot be repaired, the cells become apoptotic targets. Approximately 50% of human cancers have mutations in p53 (74). The KRAS gene is an oncogene that usually has GTPase activity, and mutations in this gene interfere with its activity and may lead to inappropriate cell proliferation and transformation, which are associated with tumorigenesis and a poor prognosis. KRAS mutations are associated with several pathological conditions with an aggressive phenotype and a poor prognosis. PDAC is usually associated with KRAS mutations. Studies on Porphyromonas gingivalis and PPAD have reported that rheumatoid arthritis is associated with both periodontitis and citrullination (75). PPAD can catalyse citrullination, a post-translational modification.

Chronic inflammation and changes in the diversity and function of the gut microbiota are accompanied by changes in immune responses. A major risk factor for colorectal cancer (CRC)is chronic inflammation. TLRs play an important role in inflammatory responses. They can sense microbial constituents, including nucleic acids, LPSs, peptidoglycans and bacterial EVs. EVs can carry or be decorated with these TLR activators. These microbial factors can boost tolerance or activate signalling pathways that result in chronic inflammation. In a survey, we discussed the role of the gut microbiome and EVs derived from gut bacteria in promoting the development of chronic inflammation and colitis-associated CRC. In addition, we discussed the role of TLRs ( Figure 3 ) in mediating the microbiome inflammatory axis and subsequent tumour development.

To interact with microbes and maintain a symbiotic relationship, the host requires a well-developed communication system with its symbionts and pathogens. Factors secreted by cells can be degraded by enzymes (e. g. proteases and nucleases). Prokaryotes and eukaryotes package cargoes (e. g. proteins, ribonucleotides and metabolites)in extracellular vesicles (EVs)to prevent extracellular degradation (104). A cell vesicle is a ubiquitous cellular carrier capable of transferring biomolecules and transmitting signals. Subcategories are effectively created when EVs have multiple functions, goods and compositions. The adaptive immune response is initiated by certain eukaryotic cells, such as B cells and dendritic cells, by presenting antigens through EVs. EVs are also used by other cell types, such as neurons, to improve action potentials and stimulate targets, and by adipocytes to process insulin in a paracrine manner. EVs can regulate cancer cell communication as well (105). The DNA, metabolites and proteins found in EVs secreted by normal cells can be used by tumour cells for’educating’non-cancerous cells to promote carcinogenesis through EVs. Using these vesicles, cancer cells can control the local environment and cellular processes, promoting inflammation and cancer development and evasion of the immune system.

In eukaryotes, EVs are enriched with certain classes of biomolecules, indicating the involvement of selective transport pathways. EVs can be used as novel biomarkers or therapeutic delivery systems for certain diseases and for the development of vaccines. EVs participate in various molecular pathways, including TLRs (106) ( Table 2 ). LPS(TLR4), peptidoglycan (TLR2) and nucleic acids (TLR3/7/8/9)are a few biomolecules that interact with TLRs when attached to EVs. Many bacterial EVs bind to TLRs to promote disease or maintain homeostasis;however, the underlying mechanisms remain unclear. The IEC and E. coli C25 strains are symbiotic strains that interact via EVs. EV–IEC interactions upregulate TLR2/4/5 expression and promote IL-8 production, causing mild inflammation and preventing bacterial internalisation into the epithelium. The EVs of commensal E. coli cultures can induce the expression of TLR 1/5/6/7/8/9, IL-10, IL-12A and IL-1 in intestinal epithelial cells (107). Similarly, other commensal microbes interact with immune cells to affect gut homeostasis.

PSA binds to TLR2 on DCs, thus enabling B. fragilis (NTBF)to promote intestinal immunotolerance. In the same fashion, B. fragilis(NTBF)-derived EVs are enriched with PSA, which activates TLR2-mediated increase in IL-10 production in DCs and enhances Treg production to protect against experimental colitis. Bacteroides vulgatus, an intestinal symbiont, releases EVs that activate TLR2 and TLR4 on CD11c⁺ DCs to promote anti-inflammatory responses (108). It is also possible for the health status of the host to influence how responsive the host cells are to commensal EVs. Specifically, EVs derived from Bacteroides thetaiotaomicron(Bt)stimulate the expression of IL-10 and IL-6 in DCs derived from healthy individuals, which in cell cultures from IBD patients, but EVs were ineffective, suggesting that TLR expression or components of the TLR pathway are altered. Therefore, EVs derived from commensal strains help to regulate homeostasis in the intestine through immunomodulatory antigens (109). However, interactions between pathogen-derived EVs and host cells result in the integration of microbial antigens within host cells, affecting intracellular processes and exacerbating the disease state (110).

IBD and cancer are exacerbated by EVs derived from pathogens (111). Innate immune responses are initiated by epithelial cells during immunosurveillance. P. gingivalis-derived EVs attract strong TLR2 (peptidoglycan)and TLR4 (LPS)responses and activate TLR7/8/9 (nucleic acids) (112) to a minor extent in the epithelium. H. pylori-derived EVs transfer virulence factors (i. e. CagA)that activate the TLR and NF-kB pathways in gastric and B cells, thus increasing the release of pro-inflammatory cytokines and cell proliferation in gastric neoplasm. H. pylori is a well-documented oncogenic bacterium that used EVs to initiate carcinogenesis. Other intestinal microbes may similarly use EVs to influence carcinogenesis. RNA within EVs derived from Aggregatibacter actinomycetemcomitans enhances the pro-inflammatory effects of TNF through TLR8 and NFkB (113). The delivery of DNA and RNA via EVs derived from Staphylococcus aureus can lead to potent IFN-β responses in macrophages via endosomal TLRs. EVs derived from the microbiome of rats with colitis can interact with TLR4 on IECs (114), thus triggering proinflammatory responses from macrophages, especially the release of IL-8. EVs in patients with IBD can induce pro-inflammatory responses, enhance the destruction of the intestinal epithelium and promote macrophage polarisation to M1 and M2 types (113). Peptidoglycan present in EVs can be internalised by IECs via TLR2 and transported to the basic membrane (115). After the peptidoglycan is transferred, EVs communicate with macrophages and induce the secretion of IL-6, which exacerbates inflammation in CRC. Therefore, EVs are very similar to their eukaryotic counterparts and represent an important component of intercellular interactions between microbes and host cells by interacting with TLRs (116).

Cancer immunotherapy ( Figure 5 ) has been performed using bacteria-derived vesicles. Owing to the abundance of PAMPs in bacteria, bacterial EVs are highly immunogenic and can attract and activate immune cells at tumour sites (117). However, intact bacteria pose safety concerns, and hence, bacterial ghosts whose intracellular contents have been removed are a safer alternative. Incorporation of adjuvants or antigens can enhance the efficacy and specificity of hollowed-out bacteria. Compared with their free-form counterparts, antigens incorporated into bacterial ghosts have demonstrated better retention, increased immune responses and improved tumour growth suppression after subcutaneous administration. When subcutaneously injected, EVs enable effective lymphatic drainage and enhance localisation to solid tumours through passive targeting. In addition to natural accumulation at tumour sites, EVs can be used therapeutically against multiple types of cancer because they can use local cancer cells as antigen sources in situ. For example, E. coli-derived EVs administered intravenously can eradicate CT26 and MC38 colorectal cancer, 4T1 metastatic breast cancer and B16BL6 metastatic melanoma (117).

Several lines of evidence support the potential capability of entrance of bacterial EVs into tumor microenvironment and modification of immune status. For example, Kim et al. showed that systematically administered bacterial outer membrane vesicles specifically target and accumulate in the tumor tissue, and subsequently induce the production of antitumor cytokines CXCL10 and interferon-γ. This antitumor effect is interferon-γ dependent, as interferon-γ-deficient mice could not induce such outer membrane vesicle-mediated immune response. This work showed remarkable capability of bacterial outer membrane vesicles to effectively induce long-term antitumor immune responses that can fully eradicate established tumors without notable adverse effects.

The combination of immunotherapy and EVs can synergistically enhance anti-tumour efficacy ( Figure 6 ). Because EVs can localise to tumour sites, they can be encapsulated with chemotherapeutic drugs, including doxorubicin and paclitaxel (118, 119). Compared with the use of monotherapy, this combination therapy exhibited significantly greater inhibitory effects on tumour growth. In a study, EVs were coated with polymeric micelles containing tegafur, a fluorouracil prodrug derived from attenuated Salmonella that induces tumour cell apoptosis and sensitises cancer cells to cytotoxic T lymphocytes (120). Indocyanine green is another photosensitiser used in photoimmunotherapy. During laser ablation, a large amount of cancer antigens is released for immune priming after their accumulation in tumours (121). Through thrombosis and erythrocyte extravasation, Salmonella typhimurium-derived EVs sensitise tumours to photothermal therapy even in the absence of photothermal agents (59, 100, 122). To enhance the effectiveness of EVs, radiation therapy can also be used, which is considered a purely external method for destroying tumours. The efficacy of EVs can be enhanced using external tumour-destroying methods such as radiation therapy (123). This type of therapy has been used to eradicate B78 melanoma and NXS2 neuroblastoma in previous studies. Furthermore, loading of EVs onto micromotors is an innovative combination therapy, which causes considerable physical damage and destruction to tumours. Compared with non-propulsion micromotors, EV-loaded micromotors can effectively clear tumours at the primary site and improve the control of tumour growth distally in MC38, CT26 and B16-F10 tumour models (124).