Although production of outer membrane vesicles by Gram-negative bacteria has been described by microscopic observations for decades, only recently have genetic and biochemical studies begun to reveal its physiological utility. Far from an artifact of experimental design or a by-product of imbalanced cell division, vesicle release is a specific mechanism for secretion of envelope components. Production of vesicles offers the cell an effective method for the elimination and remodelling of envelope components, particularly during times of stress. The data presented in this work demonstrate that vesiculation is an independent, flexible process for stress management.
The quantity of material released as vesicles is positively and negatively regulated in direct correlation with the degree of stress product build-up in the envelope. For example, a slight increase in DegP expression increases the housekeeping capacity of the envelope, lowering the quantity of misfolded products and consequently reducing the need for the cell to release this toxic material in vesicles. Conversely, the DegP protein itself becomes an unwelcome envelope component when overexpressed at a high level in the periplasm, and the cell increases vesiculation to expunge the additional material. The same response is observed upon overexpression of increasing quantities of MBP. Thus, the increased vesiculation response is both general and dose-dependent.
The DegS truncate produced by the MK6D31 and MK557 degS
::Tn5 mutants is presumably unstable or impaired in binding to misfolded OMPs, rendering it sensing deficient. Impaired stress sensing and an inability to upregulate extracytoplasmic stress response factors in these mutants would result in the accumulation of misfolded envelope proteins (Mogensen and Otzen, 2005
). Similarly, degP
mutants are unable to effectively reduce the normally produced levels of misfolded envelope proteins. DegP becomes essential at high temperatures and is the primary protease responsible for degradation of misfolded proteins in the periplasm (Clausen et al., 2002
). An lpp–
mutation that causes outer membrane instability and leakage of periplasmic proteins suppresses degP–
high-temperature lethality, in theory by allowing stress products to leak out of the cell (Strauch et al., 1989
). The heightened vesiculation phenotype of the degP
mutant is stress-dependent, increasing with higher temperatures that amplify protein misfolding. We propose that degP
mutants use increased vesiculation as a survival mechanism to rid the cell of toxic misfolded proteins under sublethal stress conditions. This appears to be a common mechanism, as vesiculation of uropathogenic E. coli
increases upon deletion of the gene encoding SurA, a periplasmic OMP chaperone (A. McBroom, D. Hunstad and M. Kuehn, unpubl. data). OMP misfolding is elevated in surA
mutants (Missiakas et al., 1996
; Rouviere and Gross, 1996
The vesiculation process is not controlled by any of the previously described envelope stress responses. Instead, impairment of either σE
or Cpx results in increased vesicle production. σE
and Cpx appear to be the primary signal transduction pathways responsible for monitoring and responding to envelope protein misfolding. When these systems are impaired, the cell is unable to properly manage protein misfolding and aggregation in the envelope compartment. Potentially damaging material accumulates, and vesicle production is upregulated to compensate for the inability of the cell to fully address folding imbalances. Vesiculation in response to the bulk accumulation of material in the envelope is not mediated by σE
, because the increase in vesiculation upon the overexpression of DegP or MBP occurs without an increase in σE
activity ( and Mecsas et al., 1993
). Although vesiculation is independent of known envelope stress pathways, recent work in our laboratory indicates that σE
pathway stimulation is sufficient to increase vesiculation (A. J. McBroom and M. J. Kuehn, unpubl. data). At first glance, this would seem to be a paradoxical finding. However, we propose that in such situations, vesiculation increases as a result of increased envelope products: either an increase of σE
-regulated proteins accumulating in the envelope, an increase in proteolytic products in the envelope due to σE
-induced protease activity, and/or the accumulation of the σE
pathway stimulus in the envelope.
We studied whether the incorporation of lumenal cargo into vesicles is selective or occurs by bulk-flow. Preferential vesicle packaging of the cyt-YYF stress protein analogue relative to MBP () suggests that misfolded protein cargo may be selectively enriched in vesicles. However, selective packaging does not appear to occur for all vesiculation-stimulating cargo: an increase in the quantity of the stressor product in the periplasm also leads to a correspondingly high level of incorporation into vesicles, suggestive of bulk-flow movement of protein into these structures (). Either selective or bulk-flow methods of cargo loading coupled with increased vesicle production would enable removal of toxic material from the cell envelope.
Our data testing the relationship of vesiculation to protein misfolding and overexpression, thermal stress and modulation of the folding capacity of the envelope can be distilled to a unifying model in which vesicle production serves to maintain a periplasmic content ‘equilibrium’. In this respect, it is not surprising that maximal vesicle production occurs during exponential growth (Bauman and Kuehn, 2006
, reviewed in McBroom and Kuehn, 2005
), a period in which cells likely experience the largest disturbance of this equilibrium due to high levels of protein expression, folding and misfolding. A build-up of damaged proteins may lead to distension of the periplasm and subsequent bulging of material into vesicles, or the accumulated misfolded material may upregulate vesicle production through a more indirect means. Cultures of wild-type and vesicle-overproducing strains examined by scanning and transmission electron microscopy revealed intact membranes and vesicles with similar diameters (20–200 nm) (), suggesting a common physical mechanism for vesicle-mediated stress relief.
Release of outer membrane vesicles offers the cell an effective mechanism for removal of material as a macromolecular complex, allowing the cell to discard unwanted material or alter the composition of the envelope under conditions where remodelling would be advantageous. Bacteria, particularly pathogens, encounter a variety of envelope stresses. Vesicle production offers a mechanism for the cell to protect itself from such insults. For example, antimicrobials can bind to vesicle decoys surrounding the cell, or they can bind to the outer membrane and become detached upon vesicle release. Our chemical, antibiotic and lethal protein challenges of vesiculation mutants demonstrate that vesiculation correlates positively with survival.
Transport of material via membrane-bound vesicles is a common phenomenon for eukaryotic cells, and it is clear that this capability extends to prokaryotes as well. An interesting parallel can be drawn between vesiculation by Gram-negative bacteria and the release of vesicles (called ectosomes or microparticles) formed from the plasma membrane of eukaryotic cells. Microparticle release has been observed for a wide range of eukaryotic cell types in response to activating stimuli (Martinez et al., 2005
). Eukaryotic cells also release exosomes, which are endosomal in origin and whose release appears to increase upon heat stress (Clayton et al., 2005
). Activities attributed to eukaryotic vesicles include involvement in angiogenesis, development and immune response, as well as specific elimination of undesirable components from the cell (Thery et al., 2002
; Pilzer et al., 2005
Our work demonstrating regulated, specific release of envelope material via outer membrane vesicles extends this paradigm to Gram-negative bacteria and fulfil the proposed key requirements of a genuine prokaryotic secretion process (Economou et al., 2006
). Further work is required to identify how envelope stress transmits into the mechanics of outer membrane bulging and fission, as well as the mechanism of cargo selection.