A Robust Protocol to Isolate Outer Membrane Vesicles from Nontypeable Haemophilus influenzae

Abstract

Outer membrane vesicles (OMVs) are lipid structures containing various biomolecules in their native environment and are spontaneously shed by gram-negative bacteria. OMVs perform several biological functions critical to both bacterial physiology and pathogenicity. Scientific research on OMV function and biogenesis requires a standardized and robust method of isolating these vesicles from bacterial cultures that reliably provide high-purity OMVs. Herein, we describe an optimized protocol to isolate OMVs from overnight cultures of three different strains of nontypeable Haemophilus influenzae (NTHi) for use in different downstream applications. Involving mainly differential centrifugation of the culture supernatant, the procedure described is relatively simple, efficient, and generates high-quality OMV preparations from each strain tested with sufficient yields, while preserving the native outer membrane composition.

Keywords: isolation protocol; nontypeable Haemophilus influenzae; outer membrane vesicles.

Figure 1

(A) OMV yield as measured by protein concentration obtained from three different NTHi strains using the protocol described above. Bars represent Mean ± SEM, n = 3. (B) Tris-Glycine SDS-PAGE analysis of the protein profile of OMVs isolated from the indicated NTHi strain. Heated samples were boiled at 100 °C for 10 min, while unheated samples remained at room temperature. A total of ~10 μg protein was loaded onto each well. Red arrowheads indicate the position of the heat-modifiable OmpP5.

Figure 2

Transmission electron micrographs (TEM) images of OMVs isolated from three different NTHi strains as indicated. Images are taken at 67,000× magnification.