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Graner. Extracell Vesicles Circ Nucleic Acids 2020;1:3-19 I http://dx.doi.org/10.20517/evcna.2020.08 Page 9

regulates vesiculation; MV impacts’ on host cells frequently involve immune modulation associated with
pathogenesis. Searching for common genes involved in conditional upregulation of MV production, they
associated the IniBAC operon with increased vesiculation. This encodes dynamin-like proteins (DLPs)
necessary for cargo loading and vesicle release, and thus may be targets for therapeutics.

Tatsuo Kurihara (Kyoto University, Japan) also discussed cargo loading into extracellular membrane
vesicles (EMVs) from Shewanella vesiculosa strain HM13. The most abundant protein in this bacterium’s
vesicles is called P49, encoded in a gene cluster that also produces components that are likely to be
membrane associated transport systems. Gene disruption analyses of various cluster members led to cellular
accumulation or non-vesicular release of P49, and gene deletion of P49 of course deleted the protein entirely.
Some of the genes encode cell surface polysaccharides that are also on EMV surfaces,which appears to be
required for P49 loading into EMVs. P49 likely binds to those EMV surface polysaccharides. Collectively,
this system could be exploited to load foreign proteins into a putative delivery system.

Jean C Lee (Brigham and Women’s Hospital, Harvard Medical School, US) followed next with a talk
concerning the impact of Staphylococcus aureus (SA) EVs on host macrophage inflammatory responses.
SA causes a diverse array of infections due to their varieties of virulence factors, which may serve as cargos
in EVs such as pore-forming toxins and small peptide toxins. Macrophages uptake SA EVs via dynamin-
dependent mechanisms. SA EVs trigger inflammasome activation via toll-like receptor (TLR) triggering
(essentially as a priming step that causes IL6 release), and toxin-mediated inflammasome-driven cleavage
of pro-IL1B and pro-IL18 to mature secreted forms. These were dependent on NLRP3 and CASP1
inflammasome components. These outputs are due to the toxin cargo in the SA EVs, which do not affect the
TLR-driven IL6 release. The TLR signaling/IL6 release is driven by SA EV surface lipoproteins. Curiously,
CASP1 activity was also dependent on lipoproteins, suggesting thereby that lipoproteins modulate the toxin
content of EVs, which was verified by mutagenesis. The lipoproteins also modulate EV biogenesis and EV
biophysical characteristics. The EVs thus function to protect the cargo and mediate virulence potentially
through unmitigated inflammasome activation.

In a very interesting presentation on potential bio-utility of bacterial outer membrane vesicles (OMVs),
Allison Z Werner’s (National Renewable Energy Laboratory, US) particular project involves generation of
green products from biology; in this case, it is the conversion of lignin (e.g., in corn stalks) to usable and
sustainable solid products. Lignin is the second most common biopolymer on earth (after cellulose) that
remains behind from industrial processes and might be used to develop rigid products rather than form
large quantities of burned waste. The material’s heterogeneity makes its deconstruction and re-utilization
problematic. Pseudomonas putida, KT2440, is a bacterium capable of lignin catabolism as a part of the
“biological funneling”, which is necessary to obtain simpler molecules for further development. Lignin
promotes P. putida extracellular secretion in the form of OMVs. The research group characterized and
performed exoproteomics on these vesicles over time under different lignin conditions. These included
aromatic catabolic enzymes in the beta-ketoadipate pathway. There are many directions for future work in
this new area.

Simon R Carding (The Quadram Institute and University of East Anglia, UK) presented information on the
roles of Bacteroides thetaiotaomicron(Bt)-derived OMV, in microbiota-host crosstalk in the gastrointestinal
tract. Bt is prominent in microbiomes residing at the mucosal interface in the gut. Bt OMVs are abundant
and remarkably stable and naturally produced in the GI tract, where they are taken up by gut epithelium by
dynamin-mediated endocytosis and macropinocytosis. Intracellular trafficking puts OMVs at the ER, Golgi,
endosomal compartments and the nuclear membrane, but can apparently transcytose the epithelial barrier
by passing between cells. Bt OMVs biodistribute beyond the GI tract into the liver with lower quantities in
kidneys and lung, much like other EVs and nanoparticles. Unlike pathogenic bacterial OMVs, Bt OMVs tend