Figure 1.
Sub-lethal AMP treatment and OMVs formation of V. cholerae.
(A) Growth curves of A1552 in normal LB (square), LB supplemented with 12.5 µg/ml of PmB (diamond) and LB supplemented with 12.5 µg/ml of LL-37 (triangles). (B) Electron micrographs of OMVs from A1552 control cultures without AMPs (panel a) or cultures with a sub-lethal concentration of LL-37 (pannel b) or PmB (pannel c). Bars: 50 nm.
Figure 2.
Analyses of OMVs from AMP treated V. cholerae.
SDS-PAGE (A), immunoblot analyses using anti-OmpU (B) and anti-Bap1 antiserum (C) and quantification of OMV yield by analyzing LPS content by ELISA (D) of OMVs samples from control A1552 cultures (lane 1) or A1552 cultures with a sub-lethal concentration of LL-37 (lane 2) or PmB (lane 3).
Figure 3.
Altered AMP-protective effect of OMVs from PmB treated V. cholerae.
Protective effect against LL-37 of OMVs from wild type A1552 grown with and without AMPs (A) and the Δbap1 mutant (B). Results are shown in µg/ml and represent the MIC values for LL-37 on A1552 measured after 1 h incubation of LL-37 with OMVs from A1552 grown in presence or absence of AMPs, followed by 16 h incubation with A1552 bacteria.
Figure 4.
Immunoblot detection of OmpT and Bap1 in OMV samples and in total cell extracts from whole cell bacteria.
(A) Detection of Bap1 in OMVs from A1552 grown with PmB (lane 1), OMVs from A1552 grown without OMVs(lane 2), OMVs from ΔompT mutant grown with PmB (lane 3) and OMVs from ΔtoxR mutant grown with PmB (lane 4). (B) Detection of OmpT in OMVs from A1552 grown without PmB (lane 1), OMVs from A1552 grown with PmB (lane 2), whole cell extract of A1552 grown without PmB (lane 3) and whole cell extract of A1552 grown with PmB (lane 4). (C) Detection of OmpT and Bap1 in control-OMVs isolated from ΔompT/pBR-ompT (lane 1), in PmB-OMVs isolated from ΔompT/pBR-ompT (lane 2), incontrol-OMVs from ΔompT/pBR-ompT* (LDV mutant) (lane 3) and PmB-OMVs from ΔompT/pBR-ompT* (LDV mutant) (lane 4).
Figure 5.
Detection of LL-37 binding to V. cholerae OMVs.
(A) Fluorescence microscopy analysis of LL-37 binding to OMVs. Control-OMVs from A1552 grown without AMPs (a, b and c), A1552 OMVs grown with PmB (d, e and f), Δbap1 OMVs grown with PmB (g, h and i) and ΔompT OMVs grown with PmB (j, k, and l) were stained using PKH26 Red Fluorescent dye (a, d, g and j) and LL-37 was detected using anti-LL-37 and FITC-labelled (green) anti-rabbit antibodies (b, e, h and k). Merged images are shown in frames c, f, i and l. (B) Immunoblot quantification of LL-37 binding to control-OMVs from A1552, PmB-OMVs from A1552, PmB-OMVs from Δbap1 and PmB-OMVs from ΔompT.
Figure 6.
Immuno-gold labeling and electron microscopic analysis of LL-37 binding to OMVs.
The following samples were analyzed: a) Control-OMVs from A1552 grown without AMPs, b) OMVs from A1552 grown with PmB, c) OMVs from the Δbap1 mutant grown without PmB), d) OMVs from the Δbap1 mutant grown with PmB, e) OMVs from the ΔompT mutant grown without PmB, f) OMVs from the ΔompT mutant grown with PmB. Arrows in panel b) indicate examples of gold labeling. Horizontal bars represent 100 nm.
Figure 7.
Effect of PmB treatment on LL-37 binding to V. cholerae OMVs.
Immunoblot detection of LL-37 after 1 h, 3 h and 6 h of incubation with OMVs from A1552 grown without and with sub-lethal concentration of PmB.
Figure 8.
Model for the role of OMVs from V. cholerae grown with sub-lethal concentration of PmB in cross-resistance mechanism against LL-37 in V. cholerae.
(A) In the absence of PmB, OMVs cannot protect A1552 against a lethal concentration of LL-37, leading to bacterial death. (B) Growing A1552 with a sub-lethal concentration of PmB can induce the release of OMVs able to bind Bap1 through the LDV domain of OmpT. Bap1 then serves as an adapter protein between LL-37 and the OmpT on the surface of the OMVs. The concentration of free LL-37 was thereby reduced to a sub-lethal concentration, leading to the apparent resistance and survival of V. cholerae. OM: outer membrane, PG: peptioglycan, IM: inner membrane.
Table 1.
Strains and plasmids used in this study.
Table 2.
Primers used in this study.