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Friday, 11 November 2016 19:57

Lipid A Modifications: The Whole is Greater than the Sum of its Modifications

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OMV640Gram-negative bacteria form OMV from their outer membrane, leading to an enrichment of periplasmic contents in the OMV lumen. Source

All cellular microbes thus far investigated release vesicles to their extracellular environment. These vesicles are given a variety of names: outer membrane vesicles, exosomes, extracellular vesicles, microvesicles, and shed vesicles are used in various fields and for various organisms. Although the name, number, size, and cargo can fluctuate, these structures seem to be made ubiquitously. These formations remain incompletely understood and are in many ways mysterious, but a recent flurry of research focused on gram-negative vesicles has shed a bit of light on their function and formation.

What are Outer Membrane Vesicles?

Outer membrane vesicles, or OMVs, are produced by gram-negative bacteria, as the "outer membrane”"part of the name would suggest. These membrane-encased structures, ranging from 20-200 nm in diameter, carry many different types of cargo, but are especially enriched for periplasmic contents—the bacterial components found in-between the two membranes of the gram-negative bacterium (see figure, right). Importantly, OMVs are actively made by growing cells, a point addressed by meticulously designed protocols with multiple controls used to avoid artifacts from particle or lipid aggregates.

What do Outer Membrane Vesicles do?

Containing a myriad of bacterial proteins, lipids, and even nucleic acids, OMVs are an energetically expensive way to release cellular contents. How might microbes benefit by using these instead of a less costly bacterial secretion system?

One hypothesis suggests that OMVs act as molecular decoys. By releasing a structure that contains similar pathogen-associated molecular patterns as its parent cell, OMVs may ‘distract’ the immune system and the white blood cells that find and eliminate bacterial invaders. These decoys can also absorb certain antimicrobial defenses—from cationic antimicrobial peptides to antibodies to certain antibacterial compounds, OMVs may act as a titrating force to lower the effective concentration near the bacterial cell.

LPS2Lipid A is the anchoring lipid portion of lipopolysaccharide, found in the outer membrane of Gram-negative bacteria. Source

OMVs may also serve to protect the cargo they carry. Individual molecules released into the extracellular environment are vulnerable to degradation and the surrounding environmental conditions. Cargo inside the OMV lipid membrane, however, can be carried further in harsh conditions. OMVs can even act as a delivery system: Some OMVs target host cells via lipid rafts, where they can dock, fuse, and release their cargo.

The properties of OMVs that enable them to act as decoys also make them ideal for biotech applications. Because they contain important antigens in the natural context of the outer membrane, Neisseria meningitidis OMVs were used to generate a meningitis vaccine. OMV vaccines are safe and effective, and the N. meningitidis vaccine helped quell a New Zealand N. meningitidis outbreak.

Pathogenic bacteria may use OMVs to deliver bacterial toxins, but OMVs from engineered strains might be used as drug delivery systems. The protective nature of OMVs has led some researchers to engineer OMVs and extend the enzymatic life of proteins used in bioremediation practices. But despite potential applications in medicine and nature, exactly how these structures are made remains a mystery.

Lipid Modification and OMVs

To understand how OMVs form, we have to take a closer look at the structure from which they are made: the bacterial outer membrane. The gram-negative outer membrane contains lipopolysaccharide, which is anchored to the membrane via lipid A (see figure, right). Two recent reports suggest lipid A modification is important during OMV formation but also suggest contrasting orders of operations: in one, the lipid modification is necessary for OMV formation, and in the other, OMV formation is necessary to eliminate disadvantageous lipids.

In July 2016, Wael Elhenawy, working with a team of scientists lead by Mario Feldman, reported that LPS remodeling triggers Salmonella OMV formation. This idea is built on lipid geometry: acylated lipid A has a cylindrical shape, while deacylated lipid A has a conical shape. The conical shape is important to facilitate membrane curvature, and thus deacetylation is needed to form OMVs, which have tight, curved membranes. The authors tested their hypothesis by correlating lipid A deacylase PagL activity with vesiculation. A pagL mutant produced fewer OMVs, but when pagL was over expressed, they were abundant. The lipid profiles of OMVs further supported their hypothesis, since deacylated lipid A forms were found in higher proportions in OMV membranes compared to bacterial outer membranes.

Lipd ALipid A modifications likely work in tandem to modify its geometrical shape and contribute to outer membrane turnover. Source

Soon after, in October 2016, Katherine Bonnington and Meta Kuehn reported that Salmonella OMV formation maintain outer membrane integrity by eliminating unfavorable lipopolysaccharide glycoforms. In their studies, the researchers looked at how Salmonella respond to various stresses that induce changes to its LPS. Bacteria exposed to acidic pH, toxic metals, antimicrobial peptides, and decreased divalent cation access modify their lipid A with phosphoethanolamine and L-4-aminoarabinose additions. The authors hypothesized that OMV formation is an expensive but fast way to eliminate non-modified LPS and make way for the modified forms. They found bacteria exposed to these conditions increased OMV production and diameter (to more efficiently eliminate old membrane), with the modified lipid A preferentially retained in the bacterial outer membrane.

So do lipid A modifications cause OMV formation or does OMV formation promote outer membrane lipid A modification? That’s a trick question, since the situation isn’t quite so black-and-white as the question implies. The modifications described by Bonnington also affect lipid architecture (highlighted in this commentary) and can also influence membrane curvature. In reality, a single modification is less likely to be the lone factor. Rather, different modification types must work in concert to affect lipid characteristics like shape and charge (see figure, right). Lipid A can undergo many additional, often species-specific modifications to alter its properties; as researchers tease out the effects of these modifications one-by-one, scientists can refine the models of how this dynamic molecule influences these fascinating bacterial structures.


Last modified on Friday, 11 November 2016 20:55
Julie Wolf

ASM Communications Social Media Specialist Julie Wolf spent her research career focused on medical mycology and infectious disease. Broadly interested in microbiology and scientific communication, she has taught at Long Island University and the community biolab Genspace and has written for the Scientista Foundation and Scholastic’s Science World magazine. Follow her on Twitter for more ASM and Microbiology highlights at @JulieMarieWolf.

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