A Bilayer-Couple Model of Bacterial Outer Membrane Vesicle Biogenesis

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Gram-negative bacteria naturally produce outer membrane vesicles (OMVs) that arise through bulging and pinching off of the outer membrane. OMVs have several biological functions for bacteria, most notably as trafficking vehicles for toxins, antimicrobials, and signaling molecules. While their biological roles are now appreciated, the mechanism of OMV formation has not been fully elucidated. We recently demonstrated that the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (PQS) is required for OMV biogenesis in<jats:named-content content-type="genus-species">P. aeruginosa</jats:named-content>. We hypothesized that PQS stimulates OMV formation through direct interaction with the outer leaflet of the outer membrane. To test this hypothesis, we employed a red blood cell (RBC) model that has been used extensively to study small-molecule–membrane interactions. Our results revealed that addition of PQS to RBCs induced membrane curvature, resulting in the formation of membrane spicules (spikes), consistent with small molecules that are inserted stably into the outer leaflet of the membrane. Radiotracer experiments demonstrated that sufficient PQS was inserted into the membrane to account for this curvature and that curvature induction was specific to PQS structure. These data suggest that a low rate of interleaflet flip-flop forces PQS to accumulate in and expand the outer leaflet relative to the inner leaflet, thus inducing membrane curvature. In support of PQS-mediated outer leaflet expansion, the PQS effect was antagonized by chlorpromazine, a molecule known to be preferentially inserted into the inner leaflet. Based on these data, we propose a bilayer-couple model to describe<jats:named-content content-type="genus-species">P. aeruginosa</jats:named-content>OMV biogenesis and suggest that this is a general mechanism for bacterial OMV formation.</jats:p><jats:p><jats:bold>IMPORTANCE</jats:bold>Despite the ubiquity and importance of outer membrane vesicle (OMV) production in Gram-negative bacteria, the molecular details of OMV biogenesis are not fully understood. Early experiments showed that 2-heptyl-3-hydroxy-4-quinolone (PQS) induces OMV formation through physical interaction with the membrane but did not elucidate the mechanism. The present study demonstrates that PQS specifically and reversibly promotes blebbing of model membranes dependent upon the same properties that are required for OMV formation in<jats:named-content content-type="genus-species">P. aeruginosa</jats:named-content>. These results are consistent with a mechanism where expansion of the outer leaflet relative to the inner leaflet induces localized membrane curvature. This “bilayer-couple” model can account for OMV formation under all conditions and is easily generalized to other Gram-negative bacteria. The model therefore raises the possibility of a universal paradigm for vesicle production in prokaryotes with features strikingly different from what is known in eukaryotes.</jats:p>

Role of Pseudomonas aeruginosa Peptidoglycan-Associated Outer Membrane Proteins in Vesicle Formation

“…Several models, which are not necessarily mutually exclusive, describe the molecular mechanisms of OMV formation (8, 13, 26-29, 31, 33), but few studies have clarified which models apply to different species and/or growth conditions. These data have allowed us to refine the P. aeruginosa bilayer-couple model (31) for OMV biogenesis through demonstration that OprI reduces PQS-mediated OMV formation.…”

“…To test this hypothesis, OMV formation of the P. aeruginosa PA14 oprF, oprI, and oprL mutants was assessed as previously described using a spectrophotometric lipid assay (31). It is important to note that in strain PA14, oprI is reported to have a premature stop codon (TAA) at position ϩ139 relative to the ATG start codon (64); however, when we sequenced oprI from P. aeruginosa PA14, it was found that the codon encompassing position ϩ139 instead encodes glutamic acid (139T ¡ G), indicating that the open reading frame is intact.…”

“…Despite their biological importance, the molecular mechanism of OMV formation has not been fully elucidated, though multiple factors have been reported to affect the process (13,(22)(23)(24)(25)(26)(27)(28) and numerous models encompassing these factors have been proposed (9,19,(28)(29)(30)(31). A primary hurdle to elucidating the mechanism of OMV formation has been the inability to identify factors that contribute to OMV production.…”

“…Surprisingly, signaling by PQS was not required for OMV formation (10); instead, OMV formation proceeds through direct interaction of PQS with the LPS component of the outer membrane (29). Based on these results, we recently proposed a detailed P. aeruginosa OMV biogenesis model, dubbed the bilayer-couple model, in which PQS induces membrane curvature by stably inserting and expanding the outer leaflet of the outer membrane relative to the inner leaflet (31), resulting in localized membrane curvature and ultimately vesiculation.…”

Pseudomonas Aeruginosa AmpR Transcriptional Regulatory Network

“…The lasI (PA1432) and rhlI (PA3476) genes encode the N-3-oxododecanoylhomoserine lactone (3-oxo-C12-AHL) synthetase (359,626) and Nbutyrylhomoserine lactone (C4-AHL) synthetase, respectively (360,362,627,628 (224). PQS also acts independently of MvfR to induce expression of the Fur regulon through its ability to bind iron (378,384) and membrane vessicle formation by inducing membrane curvature (576,632). PmpR (PA0964), a YebC member, negatively regulates MvfR ( Figure 6.2) (383).…”