Figure 2
Pd1222 derived MVs traffic C16-HSL signaling. (a) Attachment of Pd1222 MVs to P. denitrificans Pd1222 or P. aeruginosa PAO1. Cells of P. denitrificans Pd1222 or P. aeruginosa PAO1 were mixed with FM4–64 labeled MVs and fusion of MVs to bacterial cells was quantified by measuring red fluorescence, that is, cells that had fused with the labelled MVs. n=3; mean±s.d. (b) MVs show higher affinity to P. denitrificans Pd1222 than P. aeruginosa PAO1 cells in a mixed culture. Pd1222 and PAO1 cells were mixed 1:1 and incubated in PBS for 3 h in the presence of FM4–64-labeled MVs. Pd1222 and PAO1 cells are marked with eGFP in the upper and lower panel, respectively. Upon fusion of labeled MVs with a bacterial cell, it becomes red fluorescent. In the upper panel the red fluorescence (that is the MV target cell) co-localizes with the green fluorescence of the marked Pd1222 cells, indicating that these cells are the preferred targets of the MVs. By contrast, red fluorescence does not co-localize with the green fluorescence of the labeled PAO1 cells (lower panel), indicating that the MVs have a very low affinity for these cells. However, in the lower panel, the red fluorescence co-localizes with the unmarked Pd1222 cells as indicated by the white arrows. (c and d) MVs traffic C16-HSL signals. 5 μM C16-HSL or an equivalent amount of C16-HSL associated with MVs were added to P. aeruginosa PAO1ΔlasIΔrhlI/pPROBE-vioA-cviR (c) or P. denitrificans Pd1222ΔpdnI/pPLlas (d). While free C16-HSL induces both biosensors, MV-associated AHLs only induce the Pd1222 biosensor, as the MVs show little affinity for PAO1 cells. n=3; mean±s.d.
