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. Author manuscript; available in PMC: 2016 Nov 16.
Published in final edited form as: Nat Chem Biol. 2016 May 16;12(7):516–522. doi: 10.1038/nchembio.2083

Figure 4. Membrane-anchoring favors secretion of NDM-1 into outer membrane vesicles (OMVs).

Figure 4

(a) Negative stain TEM of OMVs from E. coli cells expressing NDM- 1. (b) MBL levels in whole cells expressing NDM-1, NDM-1 C26A or N-VIM and OMVs purified from their cell culture supernatants. Gel lanes were loaded with equal amounts of total protein. (c) Western blot of intact (–) and lysed OMVs (+, treated with 0.1 % Triton X-100) upon treatment with 100 μg/mL proteinase K. (d) Imipenem hydrolysis by OMVs purified from E. coli cells expressing NDM-1, NDM-1 C26A and N-VIM. Data correspond to three independent experiments and are shown as mean ± s.e.m. (e) Effect of calprotectin on the imipenemase activity of OMVs (0.8 μg) containing NDM-1 or NDM-1 C26A, compared to recombinant NDM-1 (6 nM). Data correspond to mean values from two independent experiments. (f) Effect of calprotectin on NDM-1 levels in OMVs. NDM-1 OMVs were incubated for 20 minutes at 37ºC in 10 mM HEPES, 200 mM NaCl at pH 7.4 with or without addition of calprotectin 500 μg/mL. Top, relative imipenem hydrolysis rates of OMVs; bottom, Western blot showing NDM-1 levels in OMVs. Data correspond to mean values from two independent experiments. Original Western Blots for panels (b), (c) and (f) are displayed in Supplementary Figure 11.