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Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10 Page 25

probe type III secretion system-independent immune responses. Uncovering the mechanisms through
which pathogen-and beneficial-derived vesicles mediate plant immune responses and exploring their role
as natural nanoparticle vehicles will reveal new targets for agricultural disease management and highlight
novel roles for vesicles in overall ecosystem function.

5. Staphylococcus aureus secretes immunomodulatory RNA via extracellular membrane
vesicles

Authors: Blanca V. Rodriguez, Meta J. Kuehn
E-mail: bvr6@duke.edu
Affiliations: Department of Biochemistry, Duke University, Durham, NC, USA.
Abstracts: Bacterial-derived RNA can function as ligands for intracellular receptor activation and induce
downstream signaling to modulate the host response to bacterial infection. The mechanisms underlying
the secretion of immunomodulatory RNA by human pathogens, such as Staphylococcus aureus, and their
delivery to intracellular host cell receptors are not well understood. Recently, extracellular membrane
vesicle (MV) production has been proposed as a general secretion mechanism that could facilitate the
delivery of functional bacterial nucleic acids into host cells. S. aureus produce membrane-bound, spherical,
nano-sized, MVs packaged with a select array of bherefore, in the context of neurotrophic vioactive
macromolecules and they have been shown to play important roles in bacterial virulence and in immune
modulation through the transmission of biologic signals to host cells. The present study sought to examine
the nature of the association between RNA and MVs produced by S. aureus. We also sought to analyze the
immunostimulatory potential of MV- associated RNA and to evaluate receptor-mediated recognition of
MV-associated RNA and DNA molecules by innate immune cells. Here we report that S. aureus produces
MV-associated RNA molecules that are protected from nuclease degradation. MV-associated nucleic acids
were transferred to cultured murine macrophages and induced significant Interferon-b mRNA expression
largely through endosomal Toll-like receptor (TLR) signaling. Upon exposure to nuclease-treated MVs,
TLR3-/- and TLR7-/- macrophages produced very little IFN-b mRNA. TLR3 recognizes dsRNA, which
points to the possibility that S. aureus MVs are packaged with immunostimulatory dsRNA molecules.
TLR7 has previously been found to recognize S. aureus tRNA, as well as ssRNA molecules. Altogether,
these data indicate that endosomal nucleic acid receptors are activated in cultured mouse macrophages
upon MV exposure, likely due to immunostimulatory properties of MV-associated nucleic acids. Our
findings show for the first time an MV-mediated pathway by which S. aureus-derived immunomodulatory
RNA molecules are delivered to host cells. How MV-associated nucleic acids are trafficked intracellularly
and recognized by endosomal TLRs will be examined in future experiments.

6. Separation of EVs from virions in coronavirus infections

1
2
1
1
4
1,3
Authors: James Erickson , Maria Cowen , Yuriy Kim , Anoop Pal , Heather Branscome , Archana Gupta ,
Fatah Kashanchi 1
E-mail: jericks@gmu.edu
Affiliations:
1 Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA,
USA;
2 Izon Science (IZON), Medford, MA, USA.
3 American Type Culture Collection (ATCC), Manassas, VA, USA.
4 Systems Biosciences (SBI), Palo Alto, CA, USA.