OMVs contain outer membrane-associated virulence factors.

<p>(A) Whole cell lysates (WCL) or density centrifugation gradient fractions from OMVs isolated from <i>Y. pestis</i> were separated by SDS-PAGE and gels were silver stained. (♦) denotes an enriched band and (•) denotes reduced bands. (B) WCL or gradient fractions (4–6) from OMVs isolated from <i>Y. pestis</i> and <i>Y. pestis</i> Δ<i>pla</i> were examined for the presence of the virulence factors Pla, Ail, and Caf1 by immunoblot. Immunoblots for RpoA and Hfq, two cytoplasmic proteins, are shown to demonstrate the absence of contaminating proteins from the OMV preparation. (C) OMVs were immuno-gold labeled with an anti-Caf1 antibody conjugated to gold beads and examined by TEM. Black arrows indicate representative gold particles. Bar represents 50 nm. (D) Subcellular distribution of proteins present in <i>Y. pestis</i> OMVs as a percentage of total proteins identified by mass spectrometry listed in (<a href="" target="_blank">Table S3</a>).</p>