In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

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Biological properties of extracellular vesicles and their physiological functions

Microbial etiological agents of destructive periodontal diseases

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia: The 'red complex', a prototype polybacterial pathogenic consortium in periodontitis

Outer membrane (OM) vesicles are ubiquitously produced by Gram-negative bacteria during all stages of bacterial growth. OM vesicles are naturally secreted by both pathogenic and nonpathogenic bacteria. Strong experimental evidence exists to categorize OM vesicle production as a type of Gram-negative bacterial virulence factor. A growing body of data demonstrates an association of active virulence factors and toxins with vesicles, suggesting that they play a role in pathogenesis. One of the most popular and best-studied pathogenic functions for membrane vesicles is to serve as natural vehicles for the intercellular transport of virulence factors and other materials directly into host cells. The production of OM vesicles has been identified as an independent bacterial stress response pathway that is activated when bacteria encounter environmental stress, such as what might be experienced during the colonization of host tissues. Their detection in infected human tissues reinforces this theory. Various other virulence factors are also associated with OM vesicles, including adhesins and degradative enzymes. As a result, OM vesicles are heavily laden with pathogen-associated molecular patterns (PAMPs), virulence factors, and other OM components that can impact the course of infection by having toxigenic effects or by the activation of the innate immune response. However, infected hosts can also benefit from OM vesicle production by stimulating their ability to mount an effective defense. Vesicles display antigens and can elicit potent inflammatory and immune responses. In sum, OM vesicles are likely to play a significant role in the virulence of Gram-negative bacterial pathogens.

Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles

Virulence factors of Porphyromonas gingivalis.

A chymotrypsinlike protease with an Mr of 95,000 was extracted from Treponema denticola ATCC 35405 and was partially purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteolytic activity was detected in an electrophoretogram containing polyacrylamide that was conjugated to bovine serum albumin. A single band of activity was detected when the T. denticola extract was solubilized and electrophoresed in the presence of sodium dodecyl sulfate. No activity was found in extracts of Treponema vincentii. The enzyme hydrolyzed transferrin, fibrinogen, alpha 1-antitrypsin, immunoglobulin A, immunoglobulin G, gelatin, bovine serum albumin, and a synthetic peptide containing phenylalanine. It did not degrade collagen or synthetic substrates containing arginine or proline. For the hydrolysis of azocoll, the pH optimum of the enzyme was 7.5. Heating at temperatures above 50 degrees C destroyed the activity. Reducing agents and the chelators EDTA and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid increased the enzyme activity, while phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethyl chloromethyl ketone, sulfhydryl reagents, and human serum reduced activity. The ability of the enzyme to hydrolyze a number of humoral proteins suggests that it may be involved in spirochete invasiveness and tissue destruction.

Isolation of a chymotrypsinlike enzyme from Treponema denticola.

A number of immunological methods were used to localize a cell-associated Treponema denticola chymotrypsinlike protease. Indirect immunofluorescence staining, immunogold labeling, and an enzyme-linked immunosorbent assay all indicated that the protease was attached to the outside of the cell envelope. The invasive capability of T. denticola was evaluated by following the degradation of a reconstituted basement membrane material (Matrigel) and the release of spirochetes from the gel. Under conditions where the chymotrypsinlike activity was increased, more spirochetes migrated from the gel. Protease inhibitors strongly reduced the number of cells that moved out of the gel. The purified chymotrypsinlike protease degraded the basement membrane components type IV collagen, laminin, and fibronectin. The study suggests that the T. denticola chymotrypsinlike protease may play an important role in the invasion and destruction of basement membrane.

https://iai.asm.org/content/iai/58/2/347.full.pdf

Cellular location of a Treponema denticola chymotrypsinlike protease and importance of the protease in migration through the basement membrane.

A protease was isolated from an outer membrane vesicle preparation of Porphyromonas gingivalis ATCC 33277 and assessed for its ability to inactivate the bactericidal activity of normal human serum. The enzyme, which was activated by reducing agents, was found to be a trypsinlike protease with a molecular mass of approximately 80 kDa. Prior to being tested in the bactericidal assay, pooled human serum was preincubated with the partially purified enzyme. Under conditions in which the trypsinlike protease was activated, a strong reduction of the serum bactericidal activity against Capnocytophaga ochracea was noted. On the other hand, no reduction of the bactericidal action of serum was observed when the serum-protease mixture was preincubated in the presence of an inhibitor of the enzyme. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protease was shown to degrade immunoglobulins G and M as well as complement factor C3. This study confirms the previous hypothesis that the proteases of P. gingivalis can interfere with the protective action of human serum.

https://iai.asm.org/content/iai/60/5/1854.full.pdf

Inactivation of human serum bactericidal activity by a trypsinlike protease isolated from Porphyromonas gingivalis.

The present study was undertaken to evaluate the effect of Porphyromonas gingivalis outer membrane vesicles on the bactericidal activity of human serum. Human serum was pretreated with extracellular vesicles and then incubated with a cell suspension of Capnocytophaga ochracea. After 2 h at 37 degrees C, the percent viability of C. ochracea was determined by cultivation on blood agar plates. At a final concentration of 0.3 mg/ml, outer membrane vesicles completely inhibited the serum bactericidal activity against C. ochracea. Boiling the vesicles prevented this inhibition. However, partial inhibition of the serum lethal action was obtained when a higher concentration (1.5 mg/ml) of boiled vesicles was used, which indicates the involvement of both heat-labile and heat-stable components associated with vesicles. Combining vesicles at a suboptimal concentration (0.1 mg/ml) with a reducing agent brought back inhibition of the bactericidal activity, whereas combining vesicles at an optimal concentration (0.3 mg/ml) with a thiol-blocking reagent caused a restoration of the bactericidal activity. When a purified preparation of P. gingivalis lipopolysaccharides was used instead of vesicles, inhibition of the bactericidal activity was also observed. These results indicate that the lipopolysaccharides and the proteolytic enzyme(s) associated with P. gingivalis outer membrane vesicles are likely to represent the heat-stable and the heat-labile components, respectively. It is possible that outer membrane vesicles released by P. gingivalis protect other bacterial species from complement action, thus favoring the pathogenic process of periodontal disease.

https://iai.asm.org/content/iai/59/9/3004.full.pdf

D. Grenier

Papers

Isolation of a chymotrypsinlike enzyme from Treponema denticola.

Cellular location of a Treponema denticola chymotrypsinlike protease and importance of the protease in migration through the basement membrane.

Inactivation of human serum bactericidal activity by a trypsinlike protease isolated from Porphyromonas gingivalis.

Protective effect of Porphyromonas gingivalis outer membrane vesicles against bactericidal activity of human serum.