Production of Outer Membrane Vesicles by the Plague Pathogen <i>Yersinia pestis</i>

<div><p>Many Gram-negative bacteria produce outer membrane vesicles (OMVs) during cell growth and division, and some bacterial pathogens deliver virulence factors to the host via the release of OMVs during infection. Here we show that <i>Yersinia pestis</i>, the causative agent of the disease plague, produces and releases native OMVs under physiological conditions. These OMVs, approximately 100 nm in diameter, contain multiple virulence-associated outer membrane proteins including the adhesin Ail, the F1 outer fimbrial antigen, and the protease Pla. We found that OMVs released by <i>Y. pestis</i> contain catalytically active Pla that is competent for plasminogen activation and α2-antiplasmin degradation. The abundance of OMV-associated proteins released by <i>Y. pestis</i> is significantly elevated at 37°C compared to 26°C and is increased in response to membrane stress and mutations in RseA, Hfq, and the major Braun lipoprotein (Lpp). In addition, we show that <i>Y. pestis</i> OMVs are able to bind to components of the extracellular matrix such as fibronectin and laminin. These data suggest that <i>Y. pestis</i> may produce OMVs during mammalian infection and we propose that dispersal of Pla via OMV release may influence the outcome of infection through interactions with Pla substrates such as plasminogen and Fas ligand.</p></div

<i>Y. pestis</i> culture supernatants contain active Pla.

<p>(A) Plg-activating ability of whole bacteria, 0.2 µm-filtered culture supernatants, or the filtrate of 100 kDa-passed culture supernatants, from wild-type or Δ<i>pla Y. pestis</i>, respectively. Materials were incubated with human glu-plg and a fluorescent substrate of plasmin for 3 h at 37°C. One experiment representative of 3 independent biological replicates is shown.</p

<i>Y. pestis</i> produces membrane blebs consistent with OMVs.

<p><i>Y. pestis</i> bacteria cultured for 6 h at 37°C were fixed and imaged via SEM. (A and B) Images reveal round membrane protrusions on the bacterial surface (arrows) that are consistent with OMVs. (C) TEM of OMVs purified from <i>Y. pestis</i> supernatants. Bar represents size in nanometers. (D) Size distribution of OMVs purified from <i>Y. pestis</i>. One hundred OMVs were measured and diameters are shown as a percent of the total. The average OMV diameter is 93.07+/−11.75 nm. Bars represent size in nanometers as indicated.</p

OMV-bound Pla is catalytically active and interacts with components of the ECM.

<p>(A) Plg-activating ability of wild-type or Δ<i>pla Y. pestis</i> bacteria or OMVs. Whole bacteria or purified OMVs were incubated with human glu-plg and a fluorescent substrate of plasmin for 3 hours at 37°C. (B) Degradation of α2-antiplasmin by wild-type or Δ<i>pla Y. pestis</i> bacteria or OMVs. Whole bacteria or purified OMVs were incubated with purified human α2-antiplasmin at 37°C and at the times indicated, the presence of uncleaved α2-antiplasmin was determined by immunoblot analysis.</p

Effects of temperature and stress response factors on OMV production.

<p>(A) OMVs were isolated from <i>Y. pestis</i> cultured at either 26°C or 37°C, and total protein abundance associated with the OMVs was measured. Protein concentrations were normalized to the OD₆₂₀ of the bacterial cultures. (B) Wild-type <i>Y. pestis</i> or strains lacking the genes for RseA, Hfq, or Lpp were cultured at 37°C as above and OMVs were isolated and total associated protein was measured and normalized to the OD₆₂₀. For cold shock experiments, bacteria were placed in an ice water bath for one h before proceeding. One experiment representative of two biological replicates is shown. The mean and SE are shown. *p<0.05, **p<0.005 (student's t–test).</p