Figure 1.
Y. pestis culture supernatants contain active Pla.
(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 Δpla Y. pestis, 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.
Figure 2.
Y. pestis produces membrane blebs consistent with OMVs.
Y. pestis 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 Y. pestis supernatants. Bar represents size in nanometers. (D) Size distribution of OMVs purified from Y. pestis. 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.
Figure 3.
OMVs contain outer membrane-associated virulence factors.
(A) Whole cell lysates (WCL) or density centrifugation gradient fractions from OMVs isolated from Y. pestis 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 Y. pestis and Y. pestis Δpla 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 Y. pestis OMVs as a percentage of total proteins identified by mass spectrometry listed in (Table S3).
Figure 4.
Effects of temperature and stress response factors on OMV production.
(A) OMVs were isolated from Y. pestis 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 Y. pestis 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).
Figure 5.
OMV-bound Pla is catalytically active and interacts with components of the ECM.
(A) Plg-activating ability of wild-type or Δpla Y. pestis 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 Δpla Y. pestis 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.
Figure 6.
Binding of Y. pestis OMVs to components of the extracellular matrix.
Wild-type or Δpla OMVs were examined by ELISA for the ability to bind the ECM components Matrigel, fibronectin, laminin, and collagen. BSA was used as a negative control for binding and differences in fluorescence are presented as fold change compared to BSA (set at 1). The combined mean and SE of 3 independent experiments are shown. *p<0.05, **p<0.005 (two-way ANOVA).