A single-cell imaging screen reveals multiple effects of secreted small molecules on bacteria

Abstract

Bacteria cells exist in close proximity to other cells of both the same and different species. Bacteria secrete a large number of different chemical species, and the local concentrations of these compounds at the surfaces of nearby cells may reach very high levels. It is fascinating to imagine how individual cells might sense and respond to the complex mix of signals at their surface. However, it is difficult to measure exactly what the local environmental composition looks like, or what the effects of individual compounds on nearby cells are. Here, an electron microscopy imaging screen was designed that would detect morphological changes induced by secreted small molecules. This differs from conventional approaches by detecting structural changes in individual cells rather than gene expression or growth rate changes at the population level. For example, one of the changes detected here was an increase in outer membrane vesicle production, which does not necessarily correspond to a change in gene expression. This initial study focussed on Pseudomonas aeruginosa, Escherichia coli, and Burkholderia dolosa, and revealed an intriguing range of effects of secreted small molecules on cells both within and between species.

Keywords: Cell-cell communication; Pseudomonas aeruginosa; electron microscopy; secondary metabolites; small molecules.

Figure 1

Figure showing experimental outline. (1) Pseudomonas aeruginosa was grown to stationary phase. (2) compounds were extracted from the supernatant and separated using reverse-phase chromatography. (3) Eluted fractions were added to growing Escherichia coli, Burkholderia dolosa or P. aeruginosa cells and grown on a electron microscope slide. Samples were fixed and (4) imaged by transmission electron microscopy.

Figure 2

Electron microscopy images showing upregulation of OMV production in response to addition of certain purified compounds. The target cell and compound fraction is shown in each panel. Bacteria cell and flagella are indicated in the untreated panels for clarity. Block arrows indicate representative outer membrane vesicles. Scale bar = 500 nm.

Figure 3

Quantification of outer membrane vesicle production. Graph showing the number of vesicles detected in electron microscopy images, shown as vesicles per μm². Average numbers (from at least three separate images) are shown below the graph, and error bars indicate the standard deviation. From left to right, bars represent vesicles produced by Pseudomonas aeruginosa, Escherichia coli, and Burkholderia dolosa in response to the fractions shown below. *indicates data not available.

Figure 4

Electron microscopy images showing production of secreted protein in response to addition of certain purified compounds. Arrows highlight representative examples of protein (P). Scale bar = 100 nm (A, B), 500 nm (C) or 50 nm (D).

Figure 5

Electron microscopy images showing blebbing of the outer membrane of P. aeruginosa, into structures described here as volcanoes and indicated by arrows (V). These were formed in response to certain purified compounds, indicated on the panels. Scale bar = 100 nm.

Figure 6

Electron microscopy images showing the formation of structures in E. coli and B. dolosa described here as ghosts. These formed in response to certain purified compounds which are noted on each panel. Scale bar = 500 nm.