Membrane vesicle-mediated bacterial communication

Abstract

The classical quorum-sensing (QS) model is based on the assumption that diffusible signaling molecules accumulate in the culture medium until they reach a critical concentration upon which expression of target genes is triggered. Here we demonstrate that the hydrophobic signal N-hexadecanoyl-L-homoserine lactone, which is produced by Paracoccus sp., is released from cells by the aid of membrane vesicles (MVs). Packed into MVs, the signal is not only solubilized in an aqueous environment but is also delivered with varying propensities to different bacteria. We propose a novel MV-based mechanism for binary trafficking of hydrophobic signal molecules, which may be particularly relevant for bacteria that live in open aqueous environments.

Figure 1

C16-HSL are associated with MVs in P. denitrificans Pd1222. (a) TEM image of MVs isolated from Pd1222. (b) C. violaceum VIR24 assay for the detection of C16-HSL. MVs isolated from Pd1222 wild-type or a pdnI mutant were analyzed. The purple pigment is indicative of the presence of C16-HSL. (c) C16-HSL inhibits Pd1222 aggregation. Arrows indicate cell aggregates of the pdnI mutant attached to the tube surface. A total of 5 μM C16-HSL or an equivalent amount of C16-HSL associated with MVs was added. The same amount of MVs derived from the wild-type or the pdnI mutant was added. (d) MV addition to a Pd1222 AHL reporter strain. A total of 5 μM C16-HSL or an equivalent amount of C16-HSL associated with MVs was added to a culture of P. denitrificans Pd1222ΔpdnI/pPLlas. Gfp expression in this strain is dependent on C16-HSL. n=3; mean±s.d. Unpaired t-test with Welch's correction. ns, not significant; ***P<0.001. (e) Quantification of C16-HSL and their localization. C16-HSL was quantified from each fractions with or without ethyl acetate extractions before measurement with UHPLC-qToF-MS. n=3; mean±s.e. (f) MV induction by MMC. MMC was added at an OD₆₀₀ of 0.5, following incubation for 5 h. MV production was measured by staining with the membrane-specific dye FM4–64. n=3; mean±s.d. Significant differences with the control were determined by two-way ANOVA with Dunnett's multiple comparisons post test. ns, not significant; ****P<0.0001. (g) C16-HSL concentration in the supernatant. MVs were induced by adding MMC as mentioned before. AHL concentration in the supernatant was measured using C. violaceum VIR24/pPROBE-vioA. Relative values are shown. n=3; mean±s.d. Significant differences were determined by two-way ANOVA with Dunnett's multiple comparisons post test. ns, not significant; ****P<0.0001.

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.