Outer membrane vesicles derived from Escherichia coli induce systemic inflammatory response syndrome

Abstract

Sepsis, characterized by a systemic inflammatory state that is usually related to Gram-negative bacterial infection, is a leading cause of death worldwide. Although the annual incidence of sepsis is still rising, the exact cause of Gram-negative bacteria-associated sepsis is not clear. Outer membrane vesicles (OMVs), constitutively secreted from Gram-negative bacteria, are nano-sized spherical bilayered proteolipids. Using a mouse model, we showed that intraperitoneal injection of OMVs derived from intestinal Escherichia coli induced lethality. Furthermore, OMVs induced host responses which resemble a clinically relevant condition like sepsis that was characterized by piloerection, eye exudates, hypothermia, tachypnea, leukopenia, disseminated intravascular coagulation, dysfunction of the lungs, hypotension, and systemic induction of tumor necrosis factor-alpha and interleukin-6. Our study revealed a previously unidentified causative microbial signal in the pathogenesis of sepsis, suggesting OMVs as a new therapeutic target to prevent and/or treat severe sepsis caused by Gram-negative bacterial infection.

Figure 1. Characterization of intestinal E. coli-derived OMVs.

A. Electron micrographs of thin sections of E. coli, showing the formation of OMVs (arrows) on the cell surface. Scale bars, 100 nm (left) and 25 nm (right). B. Transmission electron microscopy image of purified OMVs. Scale bar, 100 nm. C. Size distribution of OMVs according to diameter as determined by dynamic light scattering.

Figure 2. Induction of lethality in mice by intestinal E. coli-derived OMVs.

Mice were administered intraperitoneally once with 200 µl PBS that contained 0, 15, 25, or 50 µg OMVs derived from E. coli. Survival was monitored every 12 h for 5 days (n = 20; ***P<0.001, compared to the PBS group).

Figure 3. Induction of SIRS signs in mice by intestinal E. coli-derived OMVs.

A. Study protocol for investigation of SIRS. Sublethal dose of OMVs (5 µg) derived from E. coli was injected intraperitoneally three times at 12-h intervals, and the mice were checked for SIRS index. All experiments in (B–E) were performed following this scheme using 5 µg OMVs derived from E. coli. B. Piloerection (left) and eye exudates (right). C and D. Body temperature (C) and respiratory rate (D) examined after OMVs injection (n = 5; **P<0.01 and ***P<0.001, compared to the PBS group). E. Leukocyte number in blood collected from mice after OMV injection (n = 5; ***P<0.001, compared to the 0 h group).

Figure 4. Induction of lung dysfunction, disseminated intravascular coagulation and hypotension in mice by intestinal E. coli-derived OMVs.

A and B. Bronchoalveolar lavage fluid and lung organs were prepared from mice injected with OMVs derived from E. coli following Fig. 3A. The total number of leukocytes in bronchoalveolar lavage fluid (A) and wet-to-dry ratio of the lungs (B) (n = 5; *P<0.05, **P<0.01, and ***P<0.001, compared to the 0 h group). C and D. Measurement of the number of platelets in peripheral blood (C) and D-dimer levels in plasma (D) after OMV injection, as indicated in Fig. 3A (n = 5; **P<0.01 and ***P<0.001, compared to the 0 h group). E. Systolic blood pressure examined after OMV injection (n = 5; *P<0.05, compared to the PBS group).

Figure 5. Onset of systemic inflammation by intestinal E. coli-derived OMVs.

Measurement of cytokines levels in serum (A) and bronchoalveolar lavage fluid (B) by ELISA after OMV injection, as indicated in Fig. 3A (n = 5; *P<0.05, **P<0.01, and ***P<0.001, compared to the 0 h group).

Figure 6. Both vesicular LPS and other vesicular components are key factors in OMV-induced lethality.

A. Coomassie-brilliant-blue-staining of whole-cell lysates (WC), periplasmic proteins (PP), outer membrane proteins (OMP), and OMVs, each 10 µg. Molecular weight standards are indicated on the left (kDa). Note that OMVs harbor 75 ng of LPS per 100 ng of OMV proteins. B. Survival curve of wild-type mice from a single intraperitoneal injection of various samples as indicated (n = 10; *P<0.05 and ***P<0.001, compared with PBS group). C. Survival curve of wild-type and CD14^−/− mice after single injection of 25 µg OMVs (n = 10; *P<0.05 and ***P<0.001, compared to the PBS group).