Abstract
In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.
Highlights
We have found that in P. gingivalis OMV, only virulence factors were enriched, suggesting the existence of a specific cargo selection process
The specific protein packing process into OMV was aberrant in two LPS mutant strains
We propose that P. gingivalis has developed a mechanism that utilizes LPS to enable the preferential packing of a select subset of proteins into the OMV
Summary
Grown either on blood agar plates containing 5% defibrinated horse blood or brain-heart infusion broth supplemented with hemein (5 g mlϪ1) and meniadone (1 g mlϪ1) in an anaerobic atmosphere of 90% N2, 5% H2, and 5% CO2. LPS Analysis (Immunoblotting/Silver Staining)—LPS was prepared as described previously by Marolda et al (18), and the details are given in the supplemental data. OMV Purification—Cells corresponding to 10 OD600 units of overnight cultures of P. gingivalis wild type and mutant strains were removed from the suspension by centrifugation at 6,000 ϫ g. Half of the supernatant was subjected to ultracentrifugation to recover the OM (OM t0 fraction), as described above. This analysis was performed from at least three independent sample preparations to ensure reproducibility. Imaging of Bacterial Cells with Atomic Force Microscopy (AFM)—The cells of P. gingivalis W50 were strongly bound to the surface of glass slides coated with 3-aminopropyltrimethoxysilane (Genorama, Asper Biotech, Tartu, Estonia) as described in the supplemental data. Every set of AFM experiments was conducted with new tips
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