Pseudomonas aeruginosa rhamnolipid micelles deliver toxic metabolites and antibiotics into Staphylococcus aureus

Abstract

Efficient delivery of toxic compounds to bacterial competitors is essential during interspecies microbial warfare. Rhamnolipids (RLPs) are glycolipids produced by Pseudomonas and Burkholderia species involved in solubilization and uptake of environmental aliphatic hydrocarbons and perform as biosurfactants for swarming motility. Here, we show that RLPs produced by Pseudomonas aeruginosa associate to form micelles. Using high-resolution microscopy, we found that RLP micelles serve as carriers for self-produced toxic compounds, which they deliver to Staphylococcus aureus cells, thereby enhancing and accelerating S. aureus killing. RLPs also potentiated the activity of lincosamide antibiotics, suggesting that RLP micelles may transport not only self-produced but also heterologous compounds to target competing bacterial species.

Keywords: Lipid; Microbiology.

Graphical abstract

Figure 1

P. aeruginosa ΔwspF supernatant contains rhamnolipid micelles (A) Electron microscopy imaging of RLP micelles present in the ΔwspF supernatant (left panel) and formed by purified RLPs (right panel). (B) Schematic representation of RLP micelles staining with Nile Red (NR). (C) Critical micellar concentration (CMC) was determined by adding purified RLPs to either medium or to RLP-deficient ΔwspFΔrhlA supernatant. (D) Detection of RLP micelles by NR staining and fluorescence measurement in supernatants of P. aeruginosa wild type and mutants. Student test (∗∗∗∗, p value <0.0001). The scale bar indicates 1 μm. Six-thirteen images per condition, showing similar heterogeneous size distributions of RLP micelles, were analyzed.

Figure 2

RLP micelles are essential for initial S. aureus killing activity of ΔwspF mutant supernatants (A) Scheme of isolation for crude and ultracentrifuged supernatants, and ultracentrifuged pellets. (B–G) Killing curves of S. aureus exposed to crude or ultracentrifuged supernatants, or resuspended ultracentrifuged pellets produced by PA14 wild type (B), ΔwspF mutant (C), and ΔwspFΔrhlA mutant (D). ΔwspFΔrhlA mutant crude or ultracentrifuged supernatants, or resuspended ultracentrifuged pellets complemented with commercial rhamnolipids (E), RLP C₁₀C₁₀ (F) and RLP C₁₂C₁₂ (G). Parts of the data (E–G) are imported from (C) for comparison. Additional data and statistical details are given in Figure S1.

Figure 3

Interaction between RLPs and S. aureus membrane (A–C) Merged STED nanoscopy image of S. aureus cells. Peptidoglycans were visualised using Abberior dye STAR 488 (artificially in green) labeled vancomycin (also shown in B). RLPs were labeled with the dye Abberior STAR 555 (artificially in red) (also shown in C). (D) Plot profile (FIJI software) of the corresponding green and red lines shown in (B) and (C). Green arrows show the presence of the cell wall, while red arrows show the localization of RLPs. (E and F) Surface profiles (FIJI software) of respectively vancomycin (E) and RLPs (F).

Figure 4

Rhamnolipid micelles serve as a vehicle for pyochelin Confocal microscopy was performed on S. aureus Newman cells incubated in the presence of pyochelin labeled with Aberrior dye 580 (artificially colored in magenta) in the presence of RLP concentrations below (50 μg/mL) or above (250 μg/mL) the CMC. S. aureus peptidoglycan layer was visualized using Aberrior 488 labeled vancomycin (artificially colored in yellow). Cells were observed under a Zeiss confocal microscope LSM800. The scale bar corresponds to 5 μm and applies to all images.

Figure 5

Rhamnolipids accelerate the killing of S. aureus by lincosamides (A) S. aureus Newman killing kinetics. (B) S. aureus COL killing kinetics. RLPs and lincosamides (CLINDA, clindamycin; LINCO, lincomycin) were added at t = 0 and CFU counts were determined at indicated time points. Values are the average of triplicate determinations. Error bars are standard deviations.