Extracellular vesicle production in Gram‐positive bacteria

Summary This is a highlight on the article ‘Extracellular vesicle formation in Lactococcus lactis is stimulated by prophage‐encoded holin‐lysin system’ by Yue Liu, Eddy Smid and Tjakko Abee.

production has been investigated in a number of species, where mutation of certain genes including broad gene regulators such as sigB and two-component systems have been shown to significantly impact both EV production and characteristics (Briaud and Carroll, 2020). In Staphylococcus aureus, vesicle production was shown to depend on the production of amphipathic, alpha-helical peptides called phenol-soluble modulins that disrupt the membrane and promote EV formation (Wang et al., 2018). Release of EVs from the cell has been demonstrated in some species to depend on lipid composition, where dissimilarities between phospholipids in EVs and the parent bacteria suggest that production and release may occur at specific locations on the membrane. In many Gram-positives, mass spectrometry proteomic analyses of EVs have shown the presence of penicillinbinding proteins and autolysins suggesting their cell-wall modification role is connected to vesicle release (Briaud and Carroll, 2020). Therefore, the current scheme for Gram-positive EV production involves a complicated interplay of these mechanisms in addition to cell-wall degrading enzymes allowing their release (Liu et al., 2018).
Despite the apparent hurdles to their production, a wide array of Gram-positives have been demonstrated to produce EVs, including S. aureus, Listeria monocytogenes, Streptococcus pneumoniae, Clostridium perfringens and Bacillus anthracis as well as probiotic species Lactobacillus acidophilus and Lactobacillus plantarum among others (Dean et al., 2019;Bose et al., 2020). These EVs have begun to be well characterized over the past two decades, including early studies that reported proteomics profiling of the membrane-bound and lumen-carried proteins in various bacteria that have highlighted some potential mechanisms for Gram-positive EV production, including the presence of peptidoglycan hydrolyase in EVs from S. aureus and phage-associated endolysin from S. pneumoniae (Lee et al., 2009;Resch et al., 2016). In another interesting study, Toyofuku et al. (2017) found that Bacillus subtilis EV release was enabled by a prophage-encoded endolysin.
The EV contents have begun to be viewed in the context of interaction with other cells and other functions in the Gram-positive's environment. In addition to proteins,   Gram-positive EVs have been shown to carry diverse cargo, including DNA that can be horizontally transferred between different species (Cao and Lin, 2021). In studies on their role in virulence, B. anthracis vesicles have shown to deliver their cargowhich include toxin components that are involved in cytotoxicityto macrophages by phagocytosis or direct fusion (Wolf and Rivera, 2012). In opposition to the virulent effects of EVs originating from pathogens, the vesicles produced by probiotic bacteria have been implicated in protection against infection (Caruana and Walper, 2020), where, in one example, EVs of Lactobacillus species were shown to inhibit HIV-1 infection of several human tissues ex vivo (Nahui Palomino et al., 2019). In another example, Lactobacillus paracasei vesicles significantly decreased the intestinal inflammatory response to lipopolysaccharide (Choi et al., 2020). This study is particularly interesting when considering the yet uncharacterized Gram-positive EV architecture, as these EVs potentially maintain extracellular polysaccharides that have been shown to have health-beneficial activities (Spangler et al., 2021). These anti-inflammatory, antiinfection and other positive effects of probiotic EVs are therefore of particular interest in the context of biotechnology and the emerging field of engineering probiotic bacteria (Spangler et al., 2021), where probiotics and the vesicles they produce can be engineered to further include therapeutic biomolecules or display characteristics favourable to vaccinations.
In the highlighted paper by Liu et al., they report investigations into the questions of vesiculogenesis in Lactococcus lactis EVs. In looking at the EVs produced by the lysogenic L. lactis strain FM-YL11 they saw that prophage-inducing conditions increased the production of EVs by greater than 10-fold. They confirmed the role of the holin-lysin system by creation of a prophageencoded holin-lysin knock-out mutant and prophagecured mutant which showed stable low-level of vesicle production. Following further experimentation, including proteomic analysis of the EVs and amazing transmission electron microscopic images showing the presence of phage heads within the L. lactis vesicles, the group clearly demonstrated the function of the prophageencoded holin-lysin system. Critically, the described system can be applied in the production of vesicles from other Gram-positives, including other probiotic species (Liu and Smid, 2021). In the context of probiotic engineering, the findings by Liu et al. are particularly exciting as they enable both hypervesiculation and controlled production of the EVs, supplying a valuable engineering tool for a field where tools are currently lacking.
Future work stemming from the work of Liu et al. and other groups will likely further elucidate the questions surrounding Gram-positive vesiculogenesis, the contents of EVs and their functions. The mechanism reported by Liu et al. could be widely applied among Gram-positives, and probiotic bacteria in particular, and therefore deserves further investigation. These results may serve as a starting point for synthetic biologists to work on a means of control for EV production from probiotic species such as L. lactis and species of Lactobacillus. An engineered controllable, inducible system could allow for increased production of EVs for therapeutic or other biotechnology purposes, depending on their contents. While mysteries concerning the underlying mechanisms of Gram-positive EVs remain unexplained, especially in the area of EV biogenesis, explanations of this component of the process, such as this report by Liu et al., will likely further encourage interesting new work that has clear applications in medicine and biotechnology.

Conflict of interest
None declared.