Please note that this presentation aims at summarizing currently available data only without the claim of comprehensively representing established knowledge. Thus, information and conclusions presented in this course may be subject to change in the future due to the rapid evolution of the field of extracellular vesicles. The current MOOC chapter is entitled Bacterial extracellular vesicles. It has been prepared by Yong Song Gho of the Department of Life Sciences at POSTECH along with other members of POSTECH. My name is Kenneth Witwer, I'm currently the Executive Chair of Science and Meetings for ISEV, and I will be narrating this module. Now, let's begin. All living organisms including bacteria, actively shed extracellular vesicles or EVs into the extracellular environment. EVs are nano-sized, lipid bilayered particles harboring proteins, lipids, nucleic acids, metabolites, and more, and they are involved in diverse pathophysiological functions. Since they function in signaling between cells, EVs could truly be called Intercellular Communicasomes. Bacterial EVs were first identified in the 1960s by observing bacterial structures with electron microscopy. Since then, bacterial EVs have been shown to be released by various Gram-negative and Gram-positive bacteria. Bacterial EVs have been observed in all environments in which bacteria live, including laboratory cultures, natural environments, tissues and body fluids of bacterium-infected hosts. Various studies on bacterial EVs contributed to elucidating the components and functions of these vesicles. After the first discovery in the 1960s, bacterial EV-related studies have made steady progress. The field of Bacterial EVs is a recently emerging field as shown by the fast increase in the number of publications on bacterial EVs. Direct evidence of vesicle formation by Gram-positive bacteria was demonstrated through electron microscopy and proteomic analyses in 2009. Bacterial EVs harbor proteins, lipids, nucleic acids, and virulence factors, including lipopolysaccharide and lipoteichoic acid that is LPS or LTA. High-throughput analyses of bacterial components have been focused on EV-associated proteins. EVpedia, a community web portal for EV research includes bacterial EV-associated molecules identified in high-throughput analyses. Gram-negative bacterial EVs, also known as outer membrane vesicles or OMVs, have been suggested to play several pathophysiological functions in bacteria to bacteria and bacteria to host interactions by directly stimulating the target cells or delivering the vesicular cargos. The bacteria-bacteria interactions include antibiotic resistance, killing competing bacteria, and promoting bacterial survival. Whereas the bacteria host interactions include virulence factor delivery, bacterial adhesion and invasion, host cell modulation, and immune evasion. Like Gram-negative bacterial EVs, Gram-positive bacterial EVs have also been proposed to elicit several pathophysiological functions in bacteria-bacteria and bacteria-host interactions. The bacteria-bacteria interactions include antibiotic resistance and killing competing bacteria whereas the bacteria-host interactions include coagulation, virulence factor delivery, bacteria adhesion and invasion, and host cell modulation. Let's summarize this introduction to the bacterial EV module. Bacterial EVs were first observed in the 1960s and various Gram-negative and Gram-positive bacteria have been shown to release bacterial EVs. Bacterial EVs have been identified in various environments in which bacteria live, including tissues and body fluids of bacterium-infected hosts so they can be utilized in developing diagnostic tools. In addition, these EVs contain various bioactive molecules so they can be employed in developing vaccines. Bacterial EVs have been suggested to play multifaceted functions in bacteria-bacteria and bacteria-hosts interactions so they can be used in developing therapeutics. Thus, advances in the studies of bacterial EVs could shed light on the development of diagnostic tools, vaccines, and therapeutics against infectious diseases caused by bacteria. This next part of our chapter on bacterial EVs is on Proteomic analysis of Gram-negative bacterial extracellular vesicles. It has been prepared by Jaewook Lee. The field of Gram-negative bacterial EVs is a recently emerging field. During the last 10 years, mass spectrometry-based high-throughput proteomic analyses of Gram-negative bacterial EVs have identified more than 100 vesicular proteins, paving the way to identify potentially thousands of Gram-negative bacterial EV-associated proteins. Currently, mass spectrometric proteomics of Gram-negative bacterial EVs are performed in three steps. The first is the isolation of the Gram-negative bacteria EVs, second is mass spectrometry proteomics and analyses, and third is systematic approaches to the identified vesicular proteins. Mass spectrometry-based high-throughput proteomic analyses can be improved by reducing dynamic ranges of either vesicular proteins or peptides by fractionation. This fractionation results in identification of various vesicular proteins with high confidence. After fractionation, liquid chromatography and mass spectrometry can be conducted in combination, and results can be used to search proteins in various protein databases. Systematic approaches to identified vesicular proteins can reveal clues about biogenesis and functions of Gram-negative bacterial EVs. The identified Gram-negative bacterial EV proteins are listed in the EVpedia database. Through EVpedia, systematic approaches to identify vesicular proteins such as orthologous mapping, gene ontology enrichment analysis, and network analysis are available. This next table takes up several slides and list the top 50 vesicular proteins that had been most frequently identified in Gram-negative bacterial EVs. They are listed in descending order of identification counts. Identification counts of each Gram-negative bacterial EV protein are the number of identifications of that vesicular protein in mass spectrometry-based high-throughput proteomic data sets of Gram-negative bacterial EVs. Outer membrane proteins, assembly factors, transport proteins, chaperones, and metabolic enzymes are frequently identified in Gram-negative bacterial EVs. This list may provide suggestions for how to characterize Gram-negative bacterial EVs with respect to enrichment of certain proteins. Furthermore, subcellular localization and biological processes of cellular and EV proteomes of Gram-negative bacteria can be compared. Gram-negative bacterial EVs are enriched with proteins associated with the extracellular region, the outer membrane, as well as the periplasm, and a relatively depleted of inner membrane proteins. The vesicular proteins are enriched with proteins involved in outer membrane assembly, pathogenesis, protein folding, protein insertion into membranes, and siderophore transport. Thus, Gram-negative bacterial EVs are not formed randomly from the cells and their cargo are loaded selectively. Proteomic analysis of Gram-negative bacteria EVs can be improved in several aspects. Even with density gradient ultracentrifugation, flagella related proteins are frequently identified. Combinations of different isolation strategies could be a promising way to separate Gram-negative bacterial EVs from non-vesicular contaminant. In pathophysiological circumstances, Gram-negative bacteria face various exogenous stressors which can influence the biogenesis and components of Gram-negative bacterial EVs. Proteomics of these EVs derived under diverse conditions would contribute to elucidating the pathophysiological functions of Gram-negative bacteria EVs in pathophysiological circumstances. Although Gram-negative bacterial EVs have been identified in body fluids, there have been few if any high-throughput proteomic studies of body fluid derived Gram-negative bacterial EVs. Since Gram-negative bacteria significantly contribute to infectious diseases, proteomics of Gram-negative bacterial EVs derived from body fluids of bacterium infected hosts would allow Gram-negative bacterial EVs to be utilized in clinical applications. In summary, high-throughput proteomic analyses of Gram-negative bacterial EVs have provided information regarding protein composition, biogenesis, and functions of Gram-negative bacterial EVs. However, proteomic analysis of Gram-negative bacterial EVs should be improved in several aspects including better separation, studies of EVs derived under diverse conditions and more attention to body fluids of bacterium infected hosts. Advances in the proteomic analyses of Gram-negative bacterial EVs could help in the development of diagnostic tools and vaccines against infectious diseases caused by Gram-negative bacteria. We now turn our attention to Gram-positive bacteria. Historically, the lack of interest in EVs and Gram-positive bacteria relative to those of Gram-negative bacteria has been due primarily to the thick cell wall of Gram-positive bacteria. Observers simply assumed that EV biogenesis could not take place through such a barrier. However, it turns out that this assumption was not entirely justified. In 2009, Gram-positive bacteria, including Staphylococcus aureus, were shown to release EVs into the extracellular environment. The isolated EVs were spherical, bilayered, and closed membranous structures with a diameter of 20-100 nanometers. Since then, other Gram-positive bacteria have been found to produce EVs. Thus, the biogenesis of EVs occurs in different types of bacteria and is an evolutionarily conserved process. Like Gram-negative bacterial EVs, Gram-positive bacteria EVs have also been proposed to elicit several pathophysiological functions in bacteria-bacteria and bacteria-host interactions. The bacteria-bacteria interactions include antibiotic resistance and killing competing bacteria. Whereas the bacteria-host interactions include coagulation, virulence factor delivery, bacteria adhesion and invasion, and host cell modulation. In this slide, let's look at an example of bacteria to bacteria interactions that are mediated by Gram-positive bacteria EVs. S. aureus ATCC14458 is an ampicillin resistance strain due to the action of biologically active beta-lactamase namely BlaZ. EVs from this strain carry the active protein but not the BlaZ gene. They enabled ampicillin susceptible Gram-negative and Gram-positive bacteria to survive even in the presence of ampicillin. The beta-lactamase activities of the S. aureus soluble fraction and EV associated BlaZ were similar. However, only EV associated BlaZ was resistant to digestion by proteinase K. Thus, Gram-positive bacteria EVs can play functional roles in antibiotic resistance allowing the polymicrobial community to prosper against antibiotics. What about bacteria-host interactions? S. aureus produces EVs that can induce host cell death through EV associated alpha-hemolysin. In addition, S. aureus EVs induce nucleophilic pulmonary inflammation via both Th1 and Th17 cell responses upon intranasal administration. Furthermore, EVs from B. anthracis contain toxins and mice immunized with these EVs live longer than controls after being challenged with B. anthracis by producing EV-specific IgM. In addition, active immunization with S. aureus EVs effectively protects against Staphylococcal lung infections, mainly via Th1 cell-mediated immunity. Thus, Gram-positive bacterial EVs can play functional roles in bacteria host interactions. In summary, due to the perception that thick cell walls would prevent EV biogenesis, Gram-positive bacteria EVs were discovered only in 2009. Gram-positive bacteria EVs harbor proteins, lipids, nucleic acids, and virulence factors including LTA. As with Gram-negative bacterial EVs, Gram-positive bacteria. EVs have been suggested to play crucial roles in bacteria-bacteria and bacteria-host interactions. Advances in studies of Gram-positive bacteria EVs could aid development of diagnostic tools and vaccines against infectious diseases caused by Gram-positive bacteria. The next part of our module on bacterial EVs has been prepared by Youn Kim of POSTECH. This section examines bacterial extracellular vesicles as vaccines and antigen delivery systems. What is the state of the art and what are the future directions in this field? According to the World Health Organization, the second leading cause of death worldwide is infectious diseases. Pathogens are increasingly resistant to current interventions such as antibiotics. Fortunately, we have our own way of fighting infectious diseases through vaccine-induced immunity. Current vaccine delivery systems can be divided into those of biological and synthetic origin. However, only a minority of vaccine candidates are currently being used, for example, because they have not reached satisfactory clinical results. Achieving a balance between vaccine efficacy and safety is very important. Research into the outer membrane vesicles or OMVs of various bacterial strain has revealed that they induce potent protective immune responses against the pathogens from which they arise. OMVs as delivery vehicles have numerous advantages. These included their nanoscale size and also the easy expression in loading of antigens due to genetic engineering of the parent cells. Studies showing the potential ago in these as vaccine delivery vehicles are also being increasingly published. Several examples are shown here. In the next few slides, we will introduce in chronological order the clinically available OMV based vaccines for meningitis. The first clinically available OMV vaccine was called VA-MENGOC-BC, and was made available by the Finlay Institute in Cuba in 1987. Soon thereafter, MenBvac was made by the Norwegian Institute of Public Health to fight an epidemic of group B meningococcal disease. In 2004, MeNZB was developed and was widely used in children in New Zealand. The techniques developed through these vaccines consisting of OMVs complex with capsular polysaccharides would later lead to the development of Bexsero by Novartis. The Bexsero vaccine was recently approved by the European Medicines Agency and the US FDA. Despite these developments and the widespread use of OMV vaccines, the precise mechanism behind OMV vaccination remained unclear. The POSTECH group that studied the mechanism behind OMV vaccination and publish the results in 2013. In this study, the group first checked the vaccination efficacy of E. coli OMV in mice. They found effective bacterial clearance within 48 hours of challenge. Next, the POSTECH group discovered that macrophages isolated from OMV immunized mice effectively phagocytosed and killed the bacteria. Through adoptive transfer of serum and spleenocytes, CD4-positive T cells were found to be the major mediator of OMV vaccination induced responses. Among these CD4- positive T cells, interferon gamma and IL 17 cytokine induced T-cell responses were the major contributors to OMV vaccination responses. Unfortunately, the clinical use of OMVs may counter several hurdles. These include a low production rate and tremendous size diversity of OMVs. In an attempt to increase OMV production, modifications to the process have been tried. One of these is the use of detergents to induce cell disruption. A second approach is introduction of stressors to stimulate more OMV production. However, studies show that these modifications generate OMV with different protein compositions that may elicit different effects when administered in Vivo. Another problem associated with the clinical use of OMV is the potential for adverse events, in other words Biosafety. The POSTECH group, through many years of studies of bacterial EVs, have found that administration of OMVs and Vivo may cause many changes involving various inflammatory responses. Examples are increases in adhesion molecules and pro-inflammatory cytokines. Importantly, when injected into mice at very high doses, OMVs may even induce a systemic inflammatory response syndrome leading to lethality by sepsis. The final part of this module reviews the next-generation of OMV vaccines. Specifically, bacterial protoplast nanovesicles have the potential to circumvent the problems associated with previous generations of OMV vaccines. Just what is protoplast? Protoplast is a cell that has had its cell wall removed. In the case of bacteria, most of the bacterial toxins are located in the cell wall thus, removing the cell wall should remove the toxins that can complicate the use of OMVs. In fact, the bacterial inner membrane is so far known to be safe. With the cell wall removed, the POSTECH group could adapt the savable technology previously used to make nanovesicles. The combination of these two approaches yielded the PDNV or protoplast derived nanovesicle. PDNVs were found to be approximately 100 nanometers in diameter. Importantly, they were devoid of toxic outer membrane components. Furthermore, PDNVs were both more efficiently produced and safer when compared to OMVs produced from the same bacteria. Also importantly, PDNVs loaded with bacterial antigens showed effective protection against specific bacteria. This included preventing bacterial sepsis. Let's now summarize. OMVs are being used as vaccine or antigen delivery systems against various bacterial infections. However, there are problems with low productivity and safety issues. Therefore, as an alternative to OMVs, PDNVs, which lack the toxic components of the cell wall, are safer and also more efficiently produced. PDNVs when loaded with bacterial antigens could effectively prevent bacterial sepsis. We have now reached the end of our module on bacterial EVs. Here are some references for additional reading.