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Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10 Page 43

log fold increase at T2 for the fatigued cohort.
Implications: There is an association between EV markers and severity of fatigue in men treated with EBRT
for prostate cancer. EV-associated and soluble analytes should be used for early detection of persistent CRF
that can continue for months after treatment completion.

30. Mechanistic analysis of protein transport to extracellular membrane vesicles of a
hypervesiculating bacterial strain, Shewanella vesiculosa HM13

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2
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Authors: Tatsuo Kurihara , Kouhei Kamasaka , Chen Chen , Fumiaki Yokoyama , Tomoya Imai , Takuya
1
Ogawa , Jun Kawamoto 1
E-mail: kurihara@scl.kyoto-u.ac.jp
Affiliations:
1 Institute for Chemical Research, Kyoto University, Gokasho, Uji, Japan.
2 Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, Japan.
Abstracts: Shewanella vesiculosa HM13 is a hypervesiculating Gram-negative bacterium isolated from the
intestine of a horse mackerel. The strain produces extracellular membrane vesicles (EMVs) that contain a
single major cargo protein, P49, of unknown function. P49 is expected to be useful as a carrier to deliver
foreign proteins to EMVs. To develop an EMV-based heterologous protein production system by using S.
vesiculosa HM13 as the host, we analyzed the mechanism of transport of P49 to EMVs. Whole genome
sequencing of S. vesiculosa HM13 revealed that the P49 gene is located downstream of a gene cluster
coding for homologs of components of a type II protein secretion system (T2SS). Disruption of the genes
coding for these proteins caused disappearance of P49 from EMVs. Thus, it is very likely that the T2SS-like
machinery functions as a protein translocon to transport P49 to EMVs. In the vicinity of the P49 gene in
the genome, we also found the genes coding for proteins that are presumably involved in the synthesis of
surface glycolipids/polysaccharides. When these genes were disrupted, P49 was secreted to the extracellular
space without being loaded to EMVs. The results raised the possibility that P49 is loaded onto EMVs
through interaction with surface glycolipids/polysaccharides of EMVs. We further studied the transport
mechanism in vitro by incubating purified P49 with P49-free EMVs prepared from various gene-disrupted
mutants. We found that P49 was loaded onto EMVs prepared from the mutants that lack the T2SS-like
machinery, whereas the loading efficiency was markedly decreased for EMVs prepared from the mutants
that lack the putative surface glycolipid/polysaccharide biosynthesis genes in the P49 gene cluster. These
results suggest that P49 is translocated across the outer membrane through the T2SS-like machinery and
loaded onto EMVs through interaction with surface glycolipids/polysaccharides of EMVs.

31. Mycobacterial dynamin like proteins are necessary for extracellular vesicle release in M.
tuberculosis

Authors: Shamba Gupta, Ainhoa Palacios, Atul Khataokar, Madhuri Bhagavathula, Matthew B. Neiditch,
Padmini Salgame, Rafael Prados-Rosales, G. Marcela Rodríguez
E-mail: sg1318@njms.rutgers.edu
Affiliations: Public Health Research Institute Center and New Jersey Medical School - Rutgers, The State
University of New Jersey, Newark, NJ, USA.
Abstracts: Mycobacterium tuberculosis (Mtb) secretes pathogenicity factors and immunologically active
molecules via extracellular vesicles. However, little is known regarding the mechanisms and molecules
involved in mycobacterial vesicle biogenesis. This study investigates molecular determinants of vesicle