Churning Out OMVs

 

Gram-negative bacteria (such as E. coli) produce outer membrane vesicles (OMVs) when put under environmental stress. This type of bacteria is characterized by the presence of a peptidoglycan layer within its membrane bilayer. (Don’t worry, we’ll talk more about this soon.)

For our endeavors, we would ideally work with an overload of OMVs so that we’ll have a greater probability of loading them with Cas9 proteins. The methods to our hyper-blebbing madness are described in this lab note.

We are planning on using two methods to overproduce outer membrane vesicles:

1) The introduction of the pseudomonas quinolone signal molecule (we call this molecular blessing “PQS”).

2) A hypervesicular* strain of E. coli (mutant ninja bacteria ftw).

Gram-negative bacteria have two membranes: an outer lipid membrane and the inner cytoplasmic membrane. Between these membranes is the peptidoglycan (PG) layer. The area sandwiched between the PG layer and the inner/outer membranes is called the periplasm. We plan to load Cas9 into the periplasmic space between the PG layer and outer membrane. For now, let’s focus on the membranes and PG layer.

Notice the protrusions on the outer lipid membrane that look like a grassy lawn in the springtime. These molecules are lipopolysaccharides (LPS). The LPS molecule has a negatively charged head and a nonpolar tail. The anionic heads give the membrane a collective negative charge. We can take advantage of this property of E. coli by introducing the PQS molecule.

 

Figure 2: PQS Molecule (a.k.a. “pseudomonas quinolone signal” if you’re feeling fancy)

When dumped on E. coli, the nonpolar tail of PQS will “wedge” itself between the LPS molecules, leading to an increase in anionic repulsion in the outer membrane (1). A bulge forms in the membrane and is eventually pinched off to make an outer membrane vesicle.

 Figure 3: OMV Production with Introduction of PQS (from Schertzer JW, Whiteley M) 

LPS provokes an immune response from a host organism, so the use of PQS for accelerated OMV production is just a tool for our lab use. For OMV production in vivo (crazytalk for “in a living animal"), we would rely on a hypervesicular strain of E. coli.

Hypervesicu-what?

It’s a genetically modified form of E. coli that will churn out OMVs. How did E. coli become mutant ninja bacteria that fling OMVs into the environment? Well, we’re not responsible for it, but scientists from other labs have found that deletion/modification of a certain membrane cross-linking protein (one of the proteins in the Tol-Pal system pictured in Figure 1) will make the cell flexible enough and encourage greater OMV production (3). (The more rigid the membrane, the lower the amount of OMV production.)

With this method, we would let the mutant E. coli do the work and we’d harvest the fruits of its glorious encapsulated periplasmic goodness.

*A "hypervesicular" strain of bacteria is one that will produce a greater quantity of outer membrane vesicles at a higher rate than what is seen in wild-type (non-mutant) strains.

1. Schwechheimer C, Kuehn MJ. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nature Rev Microbiol. 2015;13(10):605–19.

2. Schertzer JW, Whiteley M. A Bilayer-Couple Model of Bacterial Outer Membrane Vesicle Biogenesis. mBio. 2012;3(2):e00297-11.

3. Bernadac A, Gavioli M, Lazzaroni JC, Raina S, Lloubes R. Escherichia coli tol-pal Mutants Form Outer Membrane Vesicles. J Bacteriol. 1998;180(18):4872-4878.