Envelope control of outer membrane vesicle production in Gram-negative bacteria

Abstract

All Gram-negative bacteria studied to date have been shown to produce outer membrane vesicles (OMVs), which are budded, released spheres of outer membrane with periplasmic content. OMVs have been implicated in the delivery of virulence factors in pathogenesis. However, OMVs also benefit nonpathogenic species by delivering degradative enzymes to defend an ecological niche against competing bacterial species, and they can serve as an envelope stress response. Despite these important roles, very little is known about the mechanism of production of OMVs. Here we review the advantage of vesiculation, particularly in a nonpathogenic context, as well as the hurdles that have to be overcome in Gram-negative envelope architecture before a vesicle can form and bud. Lastly, we address the question of whether OMV production is a stochastic or regulated process.

Figure 1. Atomic force microscopy images of pure OMVs and OMV budding by E. coli

A. Purified OMVs from an E. coli nlpI transposon mutant [95]. OMVs were purified from an overnight culture as described [95]. A 50 µl aliquot of the vesicle suspension was applied to gelatin-coated mica for 20 minutes before thoroughly rinsing with deionized water. The sample was dried under a stream of dry nitrogen and imaged in air using contact mode atomic force microscopy. Scanning speed ranged from 6–10 microns per second. B. Time-course of an E. coli nlpI transposon mutant [95] producing an OMV. The bacteria were grown to log phase (shaking, Luria Broth, 37°C). Cells were immobilized on gelatin-coated mica as described previously [96]. Continuous MacMode™ atomic force microscopy was performed in buffer. The images shown were collected at room temperature at the indicated times using speeds ranging from 1–7 microns per second.

Figure 2. OMV production model

Overview of Gram-negative envelope architecture in the context of OMV production.

Figure 3. Involvement of cargo and PG-OM crosslinks in OMV production

A. OMVs as cellular garbage cans. Proteinaceous waste is shed via OMVs and the accumulation of misfolded protein may aid in vesicle formation. B. OMV cargo enrichment. Cargo is secreted via OMVs and its localized accumulation may aid in OMV formation by occurring in regions lacking crosslinks and/or preventing crosslink formation. C. Destabilized envelope due to the lack of Lpp. The lack of this major OM component results in a discontinuous, leaky OM, and the absence of its ability to crosslink the OM with the PG results in a looser OM.

Figure 4. Free Lpp contributes to membrane integrity

OMVs were purified from the indicated bacterial mutants grown overnight in LB at 37°C. OMVs were quantified by densitometry of major Omps as per [95] and compared to OMV production by the wild-type (WT). n=4, *, p=0.01, **, p=0.001.