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Article ; Online: Capturing of extracellular vesicles derived from single cells of

Yokoyama, Fumiaki / Kling, André / Dittrich, Petra S

2024 Volume 24, Issue 7, Page(s) 2049–2057

Abstract: Bacteria secrete extracellular vesicles (EVs), also referred to as bacterial membrane vesicles, which carry, among other compounds, lipids, nucleic acids and virulence factors. Recent studies highlight the role of EVs in the emergence of antibiotic ... ...

Abstract	Bacteria secrete extracellular vesicles (EVs), also referred to as bacterial membrane vesicles, which carry, among other compounds, lipids, nucleic acids and virulence factors. Recent studies highlight the role of EVs in the emergence of antibiotic resistance,
MeSH term(s)	Humans ; Escherichia coli ; Cells, Cultured ; Oligonucleotide Array Sequence Analysis ; Extracellular Vesicles/metabolism ; Anti-Bacterial Agents/pharmacology
Chemical Substances	Anti-Bacterial Agents
Language	English
Publishing date	2024-03-26
Publishing country	England
Document type	Journal Article
ZDB-ID	2056646-3
ISSN	1473-0189 ; 1473-0197
ISSN (online)	1473-0189
ISSN	1473-0197
DOI	10.1039/d3lc00707c
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Article ; Online: Structural factors governing binding of curvature-sensing peptides to bacterial extracellular vesicles covered with hydrophilic polysaccharide chains.

Kawano, Kenichi / Kamasaka, Kouhei / Yokoyama, Fumiaki / Kawamoto, Jun / Ogawa, Takuya / Kurihara, Tatsuo / Matsuzaki, Katsumi

Biophysical chemistry

2023 Volume 299, Page(s) 107039

Abstract: Extracellular vesicles (EVs) have attracted an attention as important targets in the fields of biology and medical science because they contain physiologically active molecules. Curvature-sensing peptides are currently used as novel tools for marker- ... ...

Abstract	Extracellular vesicles (EVs) have attracted an attention as important targets in the fields of biology and medical science because they contain physiologically active molecules. Curvature-sensing peptides are currently used as novel tools for marker-independent EV detection techniques. A structure-activity correlation study demonstrated that the α-helicity of the peptides is prominently involved in peptide binding to vesicles. However, whether a flexible structure changing from a random coil to an α-helix upon binding to vesicles or a restricted α-helical structure is an important factor in the detection of biogenic vesicles is still unclear. To address this issue, we compared the binding affinities of stapled and unstapled peptides for bacterial EVs with different surface polysaccharide chains. We found that unstapled peptides showed similar binding affinities for bacterial EVs regardless of surface polysaccharide chains, whereas stapled peptides showed substantially decreased binding affinities for bacterial EVs covered with capsular polysaccharides. This is probably because curvature-sensing peptides must pass through the layer of hydrophilic polysaccharide chains prior to binding to the hydrophobic membrane surface. While stapled peptides with restricted structures cannot easily pass through the layer of polysaccharide chains, unstapled peptides with flexible structures can easily approach the membrane surface. Therefore, we concluded that the structural flexibility of curvature-sensing peptides is a key factor for governing the highly sensitive detection of bacterial EVs.
MeSH term(s)	Peptides/chemistry ; Extracellular Vesicles/metabolism ; Polysaccharides ; Protein Conformation, alpha-Helical
Chemical Substances	Peptides ; Polysaccharides
Language	English
Publishing date	2023-05-12
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	185052-0
ISSN	1873-4200 ; 0301-4622
ISSN (online)	1873-4200
ISSN	0301-4622
DOI	10.1016/j.bpc.2023.107039
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Article ; Online: Extracellular-Vesicle Catch-and-Release Isolation System Using a Net-Charge Invertible Curvature-Sensing Peptide.

Kawano, Kenichi / Kuzuma, Yuki / Yoshio, Koichi / Hosokawa, Kenta / Oosugi, Yuuto / Fujiwara, Takahiro / Yokoyama, Fumiaki / Matsuzaki, Katsumi

Analytical chemistry

2024 Volume 96, Issue 9, Page(s) 3754–3762

Abstract: Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. These components are utilized for cell-cell communication between donor and recipient cells. EVs have ... ...

Abstract	Extracellular vesicles (EVs) carry various informative components, including signaling proteins, transcriptional regulators, lipids, and nucleic acids. These components are utilized for cell-cell communication between donor and recipient cells. EVs have shown great promise as pharmaceutical-targeting vesicles and have attracted the attention of researchers in the fields of biological and medical science because of their importance as diagnostic and prognostic markers. However, the isolation and purification of EVs from cell-cultured media remain challenging. Ultracentrifugation is the most widely used method, but it requires specialized and expensive equipment. In the present study, we proposed a novel methodology to isolate EVs using a simple and convenient method,
MeSH term(s)	Humans ; Extracellular Vesicles/metabolism ; Ultracentrifugation ; Exosomes ; Cell Line ; Peptides/metabolism
Chemical Substances	Peptides
Language	English
Publishing date	2024-02-25
Publishing country	United States
Document type	Journal Article
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.3c03756
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Article: Parallel study of transient dosing of antibiotics in a microfluidic device.

Rackus, Darius G / Jusková, Petra / Yokoyama, Fumiaki / Dittrich, Petra S

Biomicrofluidics

2022 Volume 16, Issue 4, Page(s) 44105

Abstract: Microfluidic tools are well suited for studying bacteria as they enable the analysis of small colonies or single cells. However, current techniques for studying bacterial response to antibiotics are largely limited to static dosing. Here, we describe a ... ...

Abstract	Microfluidic tools are well suited for studying bacteria as they enable the analysis of small colonies or single cells. However, current techniques for studying bacterial response to antibiotics are largely limited to static dosing. Here, we describe a microfluidic device and a method for entrapping and cultivating bacteria in hydrogel plugs. Ring-shaped isolation valves are used to define the shape of the plugs and also to control exposure of the plugs to the surrounding medium. We demonstrate bacterial cultivation, determination of the minimum inhibitory concentration of an antibiotic, and transient dosing of an antibiotic at sub-1-h doses. The transient dosing experiments reveal that at dose durations on the order of minutes, ampicillin's bactericidal effect has both a time and concentration dependency.
Language	English
Publishing date	2022-08-01
Publishing country	United States
Document type	Journal Article
ISSN	1932-1058
ISSN	1932-1058
DOI	10.1063/5.0091704
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Article: Identification of a Putative Sensor Protein Involved in Regulation of Vesicle Production by a Hypervesiculating Bacterium,

Yokoyama, Fumiaki / Imai, Tomoya / Aoki, Wataru / Ueda, Mitsuyoshi / Kawamoto, Jun / Kurihara, Tatsuo

Frontiers in microbiology

2021 Volume 12, Page(s) 629023

Abstract: Bacteria secrete and utilize nanoparticles, called extracellular membrane vesicles (EMVs), for survival in their growing environments. Therefore, the amount and components of EMVs should be tuned in response to the environment. However, how bacteria ... ...

Abstract	Bacteria secrete and utilize nanoparticles, called extracellular membrane vesicles (EMVs), for survival in their growing environments. Therefore, the amount and components of EMVs should be tuned in response to the environment. However, how bacteria regulate vesiculation in response to the extracellular environment remains largely unknown. In this study, we identified a putative sensor protein, HM1275, involved in the induction of vesicle production at high lysine concentration in a hypervesiculating Gram-negative bacterium,
Language	English
Publishing date	2021-02-18
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2587354-4
ISSN	1664-302X
ISSN	1664-302X
DOI	10.3389/fmicb.2021.629023
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Article ; Online: Design of the N-Terminus Substituted Curvature-Sensing Peptides That Exhibit Highly Sensitive Detection Ability of Bacterial Extracellular Vesicles.

Kawano, Kenichi / Yokoyama, Fumiaki / Kamasaka, Kouhei / Kawamoto, Jun / Ogawa, Takuya / Kurihara, Tatsuo / Futaki, Shiroh

Chemical & pharmaceutical bulletin

2021 Volume 69, Issue 11, Page(s) 1075–1082

Abstract: Extracellular vesicles (EVs) have emerged as important targets in biological and medical studies because they are involved in diverse human diseases and bacterial pathogenesis. Although antibodies targeting the surface biomarkers are widely used to ... ...

Abstract	Extracellular vesicles (EVs) have emerged as important targets in biological and medical studies because they are involved in diverse human diseases and bacterial pathogenesis. Although antibodies targeting the surface biomarkers are widely used to detect EVs, peptide-based curvature sensors are currently attracting an attention as a novel tool for marker-free EV detection techniques. We have previously created a curvature-sensing peptide, FAAV and applied it to develop a simple and rapid method for detection of bacterial EVs in cultured media. The method utilized the fluorescence/Förster resonance energy transfer (FRET) phenomenon to achieve the high sensitivity to changes in the EV amount. In the present study, to develop a practical and easy-to-use approach that can detect bacterial EVs by peptides alone, we designed novel curvature-sensing peptides, N-terminus-substituted FAAV (nFAAV) peptides. The nFAAV peptides exerted higher α-helix-stabilizing effects than FAAV upon binding to vesicles while maintaining a random coil structure in aqueous solution. One of the nFAAV peptides showed a superior binding affinity for bacterial EVs and detected changes in the EV amount with 5-fold higher sensitivity than FAAV even in the presence of the EV-secretory bacterial cells. We named nFAAV5, which exhibited the high ability to detect bacterial EVs, as an EV-sensing peptide. Our finding is that the coil-α-helix structural transition of the nFAAV peptides serve as a key structural factor for highly sensitive detection of bacterial EVs.
MeSH term(s)	4-Chloro-7-nitrobenzofurazan ; Amino Acid Sequence ; Basidiomycota/chemistry ; Biosensing Techniques ; Extracellular Vesicles/chemistry ; Extracellular Vesicles/ultrastructure ; Fluorescence Resonance Energy Transfer ; Kinetics ; Liposomes/chemistry ; Peptides/chemistry ; Protein Conformation
Chemical Substances	Liposomes ; Peptides ; 4-Chloro-7-nitrobenzofurazan (EQF2794IRE)
Language	English
Publishing date	2021-10-29
Publishing country	Japan
Document type	Journal Article
ZDB-ID	213307-6
ISSN	1347-5223 ; 0009-2363
ISSN (online)	1347-5223
ISSN	0009-2363
DOI	10.1248/cpb.c21-00516
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Article: Genetic characterization and functional implications of the gene cluster for selective protein transport to extracellular membrane vesicles of Shewanella vesiculosa HM13

Kamasaka, Kouhei / Kawamoto, Jun / Chen, Chen / Yokoyama, Fumiaki / Imai, Tomoya / Ogawa, Takuya / Kurihara, Tatsuo

Biochemical and biophysical research communications. 2020 May 28, v. 526, no. 2

2020

Abstract: A hyper-vesiculating Gram-negative bacterium, Shewanella vesiculosa HM13, secretes a protein of unknown function (P49) as a major cargo of the extracellular membrane vesicles (EMVs). Here, we analyzed the transport mechanism of P49 to EMVs. The P49 gene ... ...

Abstract	A hyper-vesiculating Gram-negative bacterium, Shewanella vesiculosa HM13, secretes a protein of unknown function (P49) as a major cargo of the extracellular membrane vesicles (EMVs). Here, we analyzed the transport mechanism of P49 to EMVs. The P49 gene is found in a gene cluster containing the genes encoding homologs of surface glycolipid biosynthesis proteins (Wza, WecA, LptA, and Wzx), components of type II secretion system (T2SS), glycerophosphodiester phosphodiesterase (GdpD), and nitroreductase (NfnB). We disrupted the genes in this cluster and analyzed the productivity and morphology of EMVs and the localization of P49. EMV production and morphology were only moderately affected by gene disruption, demonstrating that these gene products are not essential for EMV synthesis. In contrast, the localization of P49 was significantly affected by gene disruption. The lack of homologs of the T2SS components resulted in deficiency in secretion of P49. When gdpD, wzx, lptA, and nfnB were disrupted, P49 was released to the extracellular space without being loaded to the EMVs. These results suggest that P49 is translocated across the outer membrane through the T2SS-like machinery and subsequently loaded onto EMVs through interaction with surface glycolipids of EMVs.
Keywords	Gram-negative bacteria ; Shewanella ; biosynthesis ; extracellular space ; gene targeting ; glycolipids ; multigene family ; nitroreductases ; protein transport ; research ; secretion
Language	English
Dates of publication	2020-0528
Size	p. 525-531.
Publishing place	Elsevier Inc.
Document type	Article
Note	NAL-AP-2-clean
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2020.03.125
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Article: Development of a Simple and Rapid Method for In Situ Vesicle Detection in Cultured Media

Kawano, Kenichi / Yokoyama, Fumiaki / Kawamoto, Jun / Ogawa, Takuya / Kurihara, Tatsuo / Futaki, Shiroh

Journal of molecular biology. 2020 Nov. 06, v. 432, no. 22

2020

Abstract: Extracellular membrane vesicles (EMVs) are biogenic secretory lipidic vesicles that play significant roles in intercellular communication related to human diseases and bacterial pathogenesis. They are being investigated for their possible use in ... ...

Abstract	Extracellular membrane vesicles (EMVs) are biogenic secretory lipidic vesicles that play significant roles in intercellular communication related to human diseases and bacterial pathogenesis. They are being investigated for their possible use in diagnosis, vaccines, and biotechnology. However, the existing methods suffer from a number of issues. High-speed centrifugation, a widely used method to collect EMVs, may cause structural artifacts. Immunostaining methods require several steps and thus the separation and detection of EMVs from the secretory cells is time-consuming. Furthermore, detection of EMVs using these methods requires specific and costly antibodies. To tackle these problems, development of a simple and rapid detection method for the EMVs in the cultured medium without separation from the secretory cells is a pressing task. In this study, we focused on the Gram-negative bacterium Shewanella vesiculosa HM13, which produces a large amount of EMVs including a cargo protein with high purity, as a model. Curvature-sensing peptides were used for EMV-detection tools. FAAV, a peptide derived from sorting nexin protein 1, selectively binds to the EMVs even in the presence of the secretory cells in the complex cultured medium. FAAV can fully detect the EMVs within a few minutes, and the resistance of FAAV to proteases enables it to withstand prolonged use in the cultured medium. Fluorescence/Förster resonance energy transfer was used to develop a method to detect changes in the amount of the EMVs with high sensitivity. Overall, our results indicate the potential applicability of FAAV for in situ EMV detection in cultured media.
Keywords	Gram-negative bacteria ; Shewanella ; antibodies ; biotechnology ; cell communication ; centrifugation ; energy transfer ; fluorescence ; human diseases ; models ; pathogenesis ; peptides ; proteinases ; rapid methods ; vaccines
Language	English
Dates of publication	2020-1106
Size	p. 5876-5888.
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	80229-3
ISSN	1089-8638 ; 0022-2836
ISSN (online)	1089-8638
ISSN	0022-2836
DOI	10.1016/j.jmb.2020.09.009
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Article ; Online: A Novel Lysophosphatidic Acid Acyltransferase of

Toyotake, Yosuke / Nishiyama, Masayoshi / Yokoyama, Fumiaki / Ogawa, Takuya / Kawamoto, Jun / Kurihara, Tatsuo

Biomolecules

2020 Volume 10, Issue 5

Abstract: Lysophosphatidic acid acyltransferase (LPAAT) introduces fatty acyl groups into ... ...

Abstract	Lysophosphatidic acid acyltransferase (LPAAT) introduces fatty acyl groups into the
MeSH term(s)	Acyltransferases/metabolism ; Escherichia coli/enzymology ; Escherichia coli/growth & development ; Escherichia coli/ultrastructure ; Escherichia coli Proteins/metabolism ; Flagella/metabolism ; Flagella/ultrastructure ; Membrane Lipids/metabolism ; Mutation/genetics ; Phospholipids/metabolism ; Substrate Specificity ; Temperature
Chemical Substances	Escherichia coli Proteins ; Membrane Lipids ; Phospholipids ; Acyltransferases (EC 2.3.-) ; 2-acylglycerophosphate acyltransferase (EC 2.3.1.52)
Language	English
Publishing date	2020-05-11
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2701262-1
ISSN	2218-273X ; 2218-273X
ISSN (online)	2218-273X
ISSN	2218-273X
DOI	10.3390/biom10050745
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Article ; Online: Detailed Structural Characterization of the Lipooligosaccharide from the Extracellular Membrane Vesicles of

Di Guida, Rossella / Casillo, Angela / Yokoyama, Fumiaki / Kawamoto, Jun / Kurihara, Tatsuo / Corsaro, Maria Michela

Marine drugs

2020 Volume 18, Issue 5

Abstract: Bacterial extracellular membrane vesicles (EMVs) are membrane-bound particles released during cell growth by a variety of microorganisms, among which are cold-adapted bacteria. ...

Abstract	Bacterial extracellular membrane vesicles (EMVs) are membrane-bound particles released during cell growth by a variety of microorganisms, among which are cold-adapted bacteria.
MeSH term(s)	Animals ; Extracellular Vesicles/chemistry ; Extracellular Vesicles/metabolism ; Lipopolysaccharides/chemistry ; Lipopolysaccharides/metabolism ; Mass Spectrometry ; Perciformes ; Shewanella/metabolism ; Structure-Activity Relationship
Chemical Substances	Lipopolysaccharides ; lipid-linked oligosaccharides
Language	English
Publishing date	2020-04-27
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2175190-0
ISSN	1660-3397 ; 1660-3397
ISSN (online)	1660-3397
ISSN	1660-3397
DOI	10.3390/md18050231
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