The approaches to precision medicine defined by the Precision Medicine Initiative and National Research Council allow doctors and researchers to predict more accurately which treatments and preventive strategies will work in patients with a particular disease. These definitions indicate that precision medicine can guide the most effective health care decisions for a given patient and thus provide the best quality of therapy while reducing unnecessary medical interventions.
With the genomic revolution of the 21st century came the ability to rapidly sequence entire genomes in a matter of days at increasingly more affordable costs. Additionally, artificial intelligence (AI) platforms have been used to optimize combination therapy in preclinical and clinical trials. Thus, the prospect of deciphering an individual’s unique genome as a roadmap for precision medicine has become a tantalizing goal for scientists and physicians around the world.
In 1991, Lynn and Fester defined the term “holobiont”, which is derived from Ancient Greek hólos ("whole") and the word biont for a unit of life. In 1994, Jefferson defined the term “hologenome” when he introduced the hologenome theory of evolution. Human genome and human microbiome projects have revealed that microbial protein-encoding genes are 360 times more abundant than human genes. According to the hologenome theory of evolution. humans might be holobionts, with 99% of their DNA attributed to microbial genomes (the microbiome) and 1% of their DNA attributed to the human genome.
Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are naturally released from almost all types of cells and cannot replicate. The vast majority of EVs are smaller than 200 nm. EVs carry cargoes of proteins, nucleic acids, lipids, metabolites, and even organelles from parent cells. Most cells that have been studied to date are thought to release EVs, including archaeal, bacterial, fungal, plant, and human cells. Microbial EVs appear to be key messengers in host cell−microbiota communication. Recent experimental evidence shows that microbial EVs are key messengers in the communication between host cells and the environmental microbiota.
The biogenesis of EVs is a very tightly regulated process governed by multiple signaling molecules and begins with receptor activation that is unique for each cell type. Eukaryotic EV biogenesis is well characterized, whereas microbial EV biogenesis has only recently been elucidated. According to the biogenesis of EVs, eukaryotic cell-derived EVs are classified as exosomes, ectosomes (or shedding vesicles), and apoptotic bodies, while prokaryotic cell-derived EVs are classified as ectosomes and apoptotic bodies. Both gram-negative and gram-positive bacteria produce ectosomes, known as outer membrane (OM) vesicles (OMVs) and membrane vesicles (MVs), respectively. The key milestones of bacterial EVs are as follows: 1) gram-negative bacteria-derived OMVs were first discovered in 1966, 2) the fact that bacterial EVs contain nucleic acids was reported in 1989, 3) gram-positive bacteria-derived EVs were first included in a publication in 2009, and 4) since 2010, much evidence has shown that both gram-negative and gram-positive bacteria release EVs that are positively or negatively involved in disease pathogenesis.
Nanometer-sized microbial EV particles contain a variety of functional components, including lipids, proteins, nucleic acids, and metabolites, within a spherical phospholipid bilayer and play a key role in cell-to-cell communication. Lipids, carbohydrates, and proteins in the microbial EV membrane are crucial for specific targeting. In particular, the physical state of the lipids in EVs is important for determining the pathways of EVs. The EV lipid bilayer is adapted to different target environment conditions, such as pH, for optimal function. The carbohydrates in the cell membrane include receptors for glycoproteins, suggesting that the tissue distribution and cellular uptake of phospholipid EVs could be controlled by carbohydrate determinants on the EV surface.
Proteomics studies have shown that there are a number of proteins in the membrane or luminal space of microbial EVs, which demonstrate complex protein organization. Generally, membrane proteins are essential components for EV functions, such as immunity, targeting, and pathogenicity, and EV proteins can play significant roles in regulatory processes, cellular responses, host−microbe interactions, and targeting. Most EV membrane proteins originate from the cytoplasmic membrane, while EV luminal proteins are cytoplasmic proteins packaged during vesiculogenesis. For example, S. aureus EVs (SaEVs) were found to have more than 200 proteins associated with EVs, and 160 of these proteins were cytoplasmic proteins. Furthermore, α-hemolysin is localized in the lumen of SaEVs. α-hemolysin in EVs induces necrotic cell death via toxin entry into the cytoplasm of keratinocytes, whereas soluble α-hemolysin induces keratinocyte death. Thus, α-hemolysin in the EV lumen enhances keratinocyte death and evasion of host immune defenses, illustrating the clinical significance of EVs in luminal protein delivery.
Recent works have shown that microbial EVs carry DNA, rRNA, tRNA, mRNA, and sRNA. The majority of RNAs in EVs are sRNAs and are noncoding. Additionally, a large proportion of sRNAs in EVs are from uncharacterized intergenic regions and may also modulate gene expression in target cells. However, there is much less evidence regarding the properties and functions of the nucleic acids in microbial EVs. The nucleic acids in EVs mediate immunomodulatory effects. Microbial nucleic acids are sensed by either endosomal or cytoplasmic receptors in host cells. Several microbial EVs trigger pattern recognition receptor signaling to promote protective immunity, a property that has been harnessed for vaccine development.
Recently, it has been reported that EVs are metabolically active. Although emerging evidence suggests that EVs can act as metabolic regulators, the involvement of EVs in metabolic activity and the existence and function of metabolites in microbial EVs have not been completely characterized.
Recent studies elucidated the role of microbial EVs in disease development and the pathogenesis of diseases, including skin, lung, and gut diseases, metabolic diseases, and cancers. Atopic dermatitis (AD) is a chronic inflammatory disease of the skin and is defined as eczematous lesions with pruritus and xerosis. AD skin lesions show distinct features, such as abnormal skin barrier function with epidermal hyperplasia and S. aureus colonization. Abnormal skin barrier function induced by the death of keratinocytes is one of the major causative factors of AD. Skin inflammation has been associated with S. aures EVs (SaEVs). α-hemolysin can induce keratinocyte cell death, consequently enhancing skin penetration of high-molecular-weight allergens. SaEV-associated α-hemolysin was found to induce keratinocyte necrosis and induce epidermal thickening and eosinophilic inflammation in the dermis. SaEVs were internalized into the cytoplasm of keratinocytes, and EVs efficiently delivered α-hemolysin to the cytoplasm. EV-associated toxins might be key molecules in disease pathogenesis.
Indoor dust containing microbial EVs induces immune dysfunction associated with neutrophilic or granulomatous inflammation in the lung. Airway exposure to microbial EVs triggers two main pathophysiological mechanisms, Th17 and Th1 responses, based on whether the parent bacterial cell is extracellular or intracellular, respectively. Specifically, extracellular bacteria-derived EVs generally cause neutrophilic inflammation through IL-17 release by polarized Th17 cells, whereas intracellular microbial EVs induce mononuclear inflammation through IFN-γ produced by polarized Th1 cells. The pathogenic microbial EV-induced neutrophilic inflammatory response leads to airway hyperreactivity and fibrosis that contribute to asthma development. The combination of increased elastase production and fibrosis induced by neutrophilic inflammation can cause emphysema, which is a key pathology of chronic obstructive pulmonary disease (COPD). Furthermore, intracellular microbial EVs lead to Th1 polarization and subsequent IFN-γ-induced mononuclear inflammation, leading to increased elastase production in the alveoli and causing pulmonary tuberculosis-associated emphysema.
Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract. Although the exact etiology of IBD is under investigation, disruption of the normal intestinal flora and normal intestinal function have been linked to the development of IBD. Alteration of the commensal gut microbial ecosystem, which interacts with gut epithelial cells and plays significant roles in the maintenance of the mucosal barrier, induces dysfunctional immunomodulation and metabolic activity in the gut. Such disruptions lead to reductions in mucus barrier functionality, including decreased tight junctions in the intestinal epithelial lining. Since the integrity of the mucosal barrier is significantly reduced, inflammatory agents such as microbial components in the intestinal lumen can induce mucosal inflammation associated with IBD.
Commensal bacteria in the gastrointestinal tract play an important role in nutrient absorption and the fermentation of dietary fibers. Additionally, the gut microbiota provides a critical function in the production of short-chain fatty acids (SCFAs) for energy sources and epigenetic regulation via histone deacetylase inhibition. Gut microbiota dysbiosis resulting in irregular SCFA production can lead to dysregulated host metabolic functionality. Moreover, microbial EVs can participate in the development of metabolic diseases. Pseudomonas panacis EVs promoted by a high-sucrose/fat diet can induce type 2 diabetes (T2D) via the induction of insulin resistance in insulin-responsive organs. Pseudomonas panacis EVs blocked the insulin signaling pathway in both skeletal muscle and adipose tissue, thereby promoting glucose intolerance in skeletal muscle, while these EVs induced typical diabetic phenotype characteristics, such as glucose intolerance after glucose administration or systemic insulin injection.
Microbial EVs are known to be associated with tumors, and cancer patients show different microbial EVs than healthy people. Previous studies have elucidated that Th17 cells play an important role in pathogen clearance during host defense reactions but induce tissue inflammation in the pathogenesis of autoimmune disease. Recent studies reported that Th17 cells played an important role in cancer pathogenesis. Airway exposure to pathogenic microbial EVs triggers the Th17 response and neutrophilic pulmonary inflammation. The neutrophilic inflammatory response induces epithelial cell dysplasia and the induction of matrix metalloproteinase expression, possibly leading to lung cancer.
Microbial EVs can penetrate the blood–brain barrier (BBB) through three possible mechanisms: 1) receptor-mediated transcytosis, 2) paracellular passage in disease states, or 3) via EVs loaded in immune cells. Once microbial EVs are inside brain tissues after penetrating the BBB, the components and cargo in EVs act as ligands of innate immune receptors, such as TLRs, and the NALP3 inflammasome, and activate the inflammatory immune response.
The pathogenesis of currently intractable diseases is related to cellular aging and increased reactive oxygen species (ROS). Intracellular ROS cause loss of proteostasis, mitochondrial dysfunction, genome instability, and telomerase exhaustion, thereby leading to aging-related diseases, such as immune, metabolic, and neurodegenerative diseases, and cancers. According to changes in the disease patterns of aging-related chronic diseases prevalence, medical needs have shifted from caring for sick patients to promoting health and from toxic or expensive drugs to safe drugs with reasonable costs. To meet medical needs, advances in microbial EV medicine enable disease prediction and tailored therapy in the near future.