Journal: Journal of Extracellular Vesicles

Article Title: Yersinia enterocolitica O:3 Outer Membrane Vesicles as a Platform for Complement Activation

doi: 10.1002/jev2.70270

Figure Lengend Snippet: (A) The impact of OMVs on germicidal activity of serum against YeO3‐c bacteria. The influence of OMVs‐associated with: LPS polysaccharide chain length (a) and pYV‐coded factors (b). Data from one of at least two experiments with the use of separately prepared culture supernatants with similar results are presented. 1 sterile cell culture supernatants (secreted by bacteria grown to OD 600 = 0.6) were used as a source of OMVs; 2 expression at 37°C only; 3 NHS inactivated 30 min at 56°C. (B) Y. enterocolitica O:3 OMV‐induced complement activation in the presence of calcium and magnesium chelators. ELISA plates were coated with 10 8 of OMVs secreted by YeS‐c bacteria grown at 4°C, 22°C and 37°C and with OMVs of YeRa‐c, YeRd1‐c and YeRe‐c variants propagated at 37°C. After incubation with/without EDTA, EGTA or EGTA/Mg 2+ (EGTA supplemented with Mg 2+ ions), products of C3 activation were detected with specific antibodies. Data from one of two experiments with similar results are presented. (C) Comparison of complement activation and MBL binding by Yersinia enterocolitica O:3 bacterial cells, LPS and OMVs. ELISA plates were coated with 50 ng/well of bacteria, LPS or OMVs. The deposition of C3 (a, possible AP, CP and LP involvement), C4 (b, possible CP and LP involvement) or C4 LP‐dependent (d) activation products, TCC formation (c, possible AP, CP and LP involvement) and MBL‐binding (e) was analyzed after preincubation with normal human serum (filled columns) or with EDTA‐treated NHS (stripped columns) or without serum (open columns, negative control). Data from one of two experiments with similar results are presented. Dots represent individual OD values for each well. (D) Recognition of Y. enterocolitica O:3 OMVs by human mannose‐binding lectin. YeS‐c_37°C bacteria (1), OMVs (2) and LPS (3) were separated in SDS‐PAGE. After transfer to nitrocellulose and incubation with NHS, the bound MBL was detected with specific mAb (a). The experiment was performed at least 5 times. To control the loading of bacteria, LPS or OMVs the presence of OPS in separated samples was confirmed with anti‐6‐deoxy‐L‐altropyranose mAbs (b).

Article Snippet: Depletion of functional C3 from serum was verified in Western blot [acc. to Younger et al. ( )], using goat antibodies against mouse C3 (MP Biomedicals, USA), HRP‐conjugated anti‐goat Ig (Dako) for detection of intact C3 α‐chain and ECL detection system.

Techniques: Activity Assay, Bacteria, Sterility, Cell Culture, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Comparison, Binding Assay, Negative Control, SDS Page, Control