Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Summary

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Abstract

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures.

The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Introduction

Extracellular vesicles (EVs) are nanometer-sized, liposome-like structures comprised of lipids, proteins, glycans, and nucleic acids, secreted by both prokaryotic and eukaryotic cells¹. Since the early studies visualizing the release of EVs from gram-negative bacteria², the number of biological functions attributed to bacterial EVs (20-300 nm in diameter) has constantly been growing in the past decades. Their functions include transferring antibiotic resistance³, biofilm formation⁴, quorum sensing⁵, and toxin delivery⁶. There ....

Protocol

NOTE: Ensure that all work involving bacteria and recombinant DNA follows best practices for biosafety containment appropriate for the biosafety hazard level of each strain. Work should be done in accordance with local, national, and international biosafety regulations.

1. Bacterial strains and culturing conditions

NOTE: Bacterial strains used in this study were Escherichia coli MP1¹³, Akkermansia mucinophila, Bacteroides thetaiotaomicron, Bifidobacterium breve, and Bifidobacterium dentium.

1. For E. coli,....

Results

To assess which SEC chromatography fractions were enriched for EVs, the SEC column was loaded with 2 mL of E. coli MP1-conditioned culture medium that had been concentrated 1,000-fold by TFF, and sequential fractions were collected. Using MRPS, it was found that Fractions 1-6 contained the most EVs (Figure 2A). Subsequent fractions contained very few EVs, comprising instead of EV-free proteins (Figure 2B). EVs were primarily <100 nm in diameter (

Discussion

In the protocol above, a method is described that is scalable and reliably isolates EVs from various gram-negative/positive and aerobic/anaerobic bacteria. It has several potential stopping points throughout the procedure, although it is better to avoid taking longer than 48 h to isolate EVs from conditioned bacterial culture media.

First, it consists of culturing bacteria to generate conditioned bacterial culture medium. It was found that increasing the culture time to at least 48 h and using.......

Materials

Name	Company	Catalog Number	Comments
0.5 mL flat cap, thin-walled PCR tubes	Thermo Scientific	3430	it is important to use thin-walled PCR tubes to obtain accurate readings with Qubit
16% Paraformaldehyde (formaldehyde) aqueous solution	Electron microscopy sciences	15700
250 mL Fiberlite polypropylene centrifuge bottles	ThermoFisher	010-1495
500 mL Fiberlite polypropylene centrifuge bottles	ThermoFisher	010-1493
65 mm Polypropylene Round-Bottom/Conical Bottle Adapter	Beckman Coulter	392077	Allows Vivacell to fit in rotor
Akkermansia mucinophila	ATCC	BAA-835
Amicon-15 (100 kDa MWCO)	MilliporeSigma	UFC910024
Avanti J-20 XPI centrifuge	Beckman Coulter		No longer sold by Beckman. Avanti J-26XP is closest contemporary model.
Bacteroides thetaiotaomicron VPI 5482	ATCC	29148
Bifidobacterium breve	NCIMB	B8807
Bifidobacterium dentium	ATCC	27678
Brain Heart infusion (BHI) broth	Himedia	M2101	After autoclaving, Both BHI broth and agar were introduced into the anaerobic chamber, supplemented with Menadione (1 µg/L), hematin (1.2 µg/L), and L-Cysteine Hydrochloride (0.05%). They were then incubated for at least 24 h under anaerobic conditions before inoculation with the anaerobic bacterial strains.
C-300 microfluidics cartridge	Spectradyne
Chloramphenicol	MP Biomedicals	ICN19032105
Escherichia coli HST08 (Steller competent cells)	Takara	636763
Escherichia coli MP1	Dr. Mark Goulian (gift)		commensal bacteria derived from mouse gut
Fiberlite 500 mL to 250 mL adapter	ThermoFisher	010-0151-05	used with Fiberlite rotor to enable 250 mL bottles to be used for smaller size of starting bacterial culture
Fiberlite fixed-angle centrifuge rotor	ThermoFisher	F12-6x500-LEX	fits 6 x 500 mL bottles
Formvar Carbon Film 400 Mesh, Copper	Electron microscopy sciences	FCF-400-CU
Glutaraldehyde (EM-grade, 10% aqeous solution)	Electron microscopy sciences	16100
Hematin	ChemCruz	207729B	Stock solution was made in 0.2 M L-histidine solution as  1.2 mg/mL
Infinite M Nano+ Microplate reader	Tecan		This equibment was used to measure the mCherry fluorescence
In-Fusion  HD Cloning Plus	Takara	638909	For cloning of the PCR fragements into the PCR-lineraized vectors
JS-5.3 AllSpin Swinging-Bucket Rotor	Beckman Coulter	368690
Lauria Bertani (LB) broth, Miller	Difco	244620
L-Cysteine Hydrochloride	J.T. Baker	2071-05	It should be weighed and added directly to the autoclaved BHI media inside the anaerobic chamber
Masterflex Fitting, Polypropylene, Straight, Female Luer to Hose Barb Adapter, 1/8" ID; 25/PK	cole-parmer - special	HV-30800-08	connection adapters for filtration tubing circuit
Masterflex Fitting, Polypropylene, Straight, Male Luer to Hose Barb Adapter, 1/8" ID; 25/PK	cole-parmer - special	HV-30800-24	connection adapters for filtration tubing circuit
Masterflex L/S Analog Variable-Speed Console Drive, 20 to 600 rpm	Masterflex	HV-07555-00
Masterflex L/S Easy-Load Head for Precision Tubing, 4-Roller, PARA Housing, SS Rotor	Masterflex	EW-07514-10
Masterflex L/S Precision Pump Tubing, PharmaPure, L/S 16; 25 ft	Cole Palmer	EW-06435-16	low-binding/low-leaching tubing
Menadione (Vitamin K3)	MP	102259	Stock solution was made in ethanol as 1 mg/mL
MIDIKROS 41.5CM 100K MPES 0.5MM FLL X FLL 1/PK	Repligen	D04-E100-05-N	TFF device we have used to filter up to 2 L of E. coli culture supernatant
Nano-Glo Luciferase Assay System	Promega	N1110	This assay kit was used to measure the luminescence of the nluc reporter protein
NanoLuc (Nluc) Luciferase Antibody, clone 965808	R&D Systems	MAB10026
nCS1 microfluidics resistive pulse sensing instrument	Spectradyne
nCS1 Viewer	Spectradyne		Analysis software for particle size distribution
OneTaq 2x Master Mix with Standard Buffer	NEB	M0482	DNA polymerase master mix used to perform the routine PCR reactions for colony checking
Protein LoBind, 2.0 mL, PCR clean tubes	Eppendorf	30108450
Q5 High-Fidelity 2x Master Mix	NEB	M0492	DNA polymerase master mix used to perform the PCR reactions needed for cloning
qEV original, 35 nm	Izon		maximal loading volume of 0.5 mL
qEV rack	Izon		for use with the qEV-original SEC columns
qEV-2, 35 nm	Izon		maximal loading volume of 2 mL
Qubit fluorometer	ThermoFisher		Item no longer available. Closest available product is Qubit 4.0 Fluorometer (cat. No. Q33238)
Qubit protein assay kit	ThermoFisher	Q33211	Store kit at room temperature. Standards are stored at 4 °C.
Sorvall Lynx 4000 centrifuge	ThermoFisher	75006580
SpectraMax i3x Microplate reader	Molecular Devices		This equipment was used to measure the nanoluciferase bioluminescence
Stericup Quick-release-GP Sterile Vacuum Filtration system (150, 250, or 500 mL)	MilliporeSigma	S2GPU01RE S2GPU02RE S2GPU05RE	One or multiple filters can be used to accommodate working volumes. In our experience, you can filter twice the volume listed on the product size.
Uranyl acetate	Electron microscopy sciences	22400
Vinyl anaerobic chamber	Coy Lab
Vivacell 100, 100,000 MWCO PES	Sartorius	VC1042
Whatman Anotop 10 Plus syringe filters (0.02 micron)	MilliporeSigma	WHA68093002	to filter MRPS diluent