Outer Membrane Vesicle Production byEscherichia coliIs Independent of Membrane Instability

Abstract

It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over-or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the E cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.Release of outer membrane (OM) vesicles has been observed for all gram-negative bacteria studied to date (reviewed in references 5, 15, and 16). Native vesicles are rounded structures with lumenal periplasmic components bounded by an outer layer of outer membrane proteins (Omps) and lipids (16). For Escherichia coli, our laboratory has reported that strain DH5␣ releases 0.23% of Omps F and C and 0.14% of OmpA into vesicles (12); other investigators have found vesicles to account for 0.2 to 0.5% of bacterial culture material (9,20). Electron microscopy studies reveal bulging of the OM and subsequent fission of vesicles containing electron-dense material (16). These biochemical and microscopic observations suggest that OM vesicles are formed from protrusions that are pinched off from the OM in a manner that leads to the inclusion of periplasmic material.The wide variety of strains and diversity of environments for which vesiculation has been observed suggest an important role for vesicle production in gram-negative bacterial growth and survival (5,15,16). Vesicle production varies with growth phase and nutrient availability, and vesicle-associated enzymes may aid in nutrient scavenging. Vesicle-mediated transfer of toxic components to other bacteria can eliminate competing species. In addition, interactions between eukaryotic cells and vesicles from pathogenic bacteria suggest a role for vesicles in pathogenesis (13).We have conducted a screen to generate and identify mutants in E. coli that exhibit altered vesiculation levels. Both vesicle-overproducing and -underproducing mutants are of interest, ...

Identification of a Novel Francisella tularensis Factor Required for Intramacrophage Survival and Subversion of Innate Immune Response

“…The support for our hypothesis comes from a recent report demonstrating that Francisella produces outer membrane vesicles that may serve as a "vesicle-mediated secretion" system to deliver bacterial proteins in the extracellular milieu (68). Outer membrane vesicles are spherical fragments of bacterial outer membrane that are produced continuously without the concomitant bacterial lysis by several Gram-negative bacteria to mediate inflammatory response and virulence in vivo (49,69). Interestingly, proteome characterization of outer membrane vesicles from F. novicida revealed the presence of FTN_0346, an ortholog of FTT0831c/FTL_0325 proteins of F. tularensis LVS and SchuS4, respectively (68).…”

Membrane Vesicles Are Immunogenic Facsimiles of Salmonella typhimurium That Potently Activate Dendritic Cells, Prime B and T Cell Responses, and Stimulate Protective Immunity In Vivo

“…Optimally, to definitively determine the influence of MVs on DC activation during live infection in vivo would require experimental infection with a MV-null Salmonella strain. However, due to the intractable nature of this type of experiment, we are unable to address this issue at this time (3,4,7). In another respect, many DC studies have focused on their interaction with purified microbial products that may not necessarily be relevant form(s) of PAMPs encountered in vivo; thus, it is likely that DCs detect complexes of PAMPs or TLR ligands (28 -33, 35, 36).…”

“…MV production is ubiquitous for all Gram-negative bacteria investigated to date (2,3). The essential nature of MV production is supported by the lack of reports for Gram negatives that fail to make MVs (MV-null) and that an Escherichia coli transposon library screened for MV production failed to produce MV-null mutants (3,4). These observations and the fact that MV production may be linked to bacterial replication (5,6) support at least two nonmutually exclusive models in which MV production is either intrinsic to bacterial growth/viability or a multigene process unaffected by single-gene mutations (3,7).…”

Small RNAs endow a transcriptional activator with essential repressor functions for single-tier control of a global stress regulon

“…However, porins are only 30% of the sRNA targets, indicating that the scope of the sRNA response extends considerably beyond porin control. The sRNAs regulate several genes previously found to be involved in increased production of outer membrane vesicles (porins, ycsF, pal, ybgF), which enhance bacterial survival during exposure to stress or toxic unfolded proteins, by providing a mechanism for release of the unwanted periplasmic component (36)(37)(38). Interestingly, the σ E controlled VrrA sRNA of Vibrio cholera, which is evolutionary unrelated to MicA/RybB, also regulates major two porins and controls OMV production (39,40).…”