A nanoluciferase-encoded bacteriophage illuminates viral infection dynamics of Pseudomonas aeruginosa cells

Abstract

Bacteriophages (phages) are increasingly considered for both treatment and early detection of bacterial pathogens given their specificity and rapid infection kinetics. Here, we exploit an engineered phage expressing nanoluciferase to detect signals associated with Pseudomonas aeruginosa lysis spanning single cells to populations. Using several P. aeruginosa strains we found that the latent period, burst size, fraction of infected cells, and efficiency of plating inferred from fluorescent light intensity signals were consistent with inferences from conventional population assays. Notably, imaging-based traits were obtained in minutes to hours in contrast to the use of overnight plaques, which opens the possibility to study infection dynamics in spatial and/or temporal contexts where plaque development is infeasible. These findings support the use of engineered phages to study infection kinetics of virus-cell interactions in complex environments and potentially accelerate the determination of viral host range in therapeutically relevant contexts.

Keywords: bacterial detection; host range; infection kinetics; luminescence; microscopy; one step growth; phage therapy.

Figure 1

NanoLUZ19v phage produces a bioluminescent signal that reflects infection dynamics at the population scale level. (A) Time series luminescence records using a microplate reader when strains PAO1, PAK, CHA, and RP73 are independently infected by nanoLUZ19v at phage:bacteria ratio of 1:1 in presence of 1/50 dilution of the substrate (upper right corner zoomed in on the first 60 min), N = 3. Vertical dotted lines with digits above indicate the average time range (in min) where the signal increased above background. (B) One-step growth curves of nanoLUZ19v phage on the same strains as in panel A (n = 2). Vertical dotted lines with digits above indicate the average latent period (in min). (C–F) Luminescence and absorbance (600 nm) records in parallel under the same conditions as (A) for nanoLuz19v infecting (C) PAO1, (D) PAK, (E) CHA, and (F) RP73. N = 3 for (C–F). The maximal intensity recorded by the plate reader at a ratio of phage:bacteria of 1:1 (n = 3) is plotted versus: (G) the burst size as derived from one-step growth curves of nanoLUZ19v phage on the four indicated strains (n = 2); (H) the EOP (number of plaques formed on lawn of each strain compared to PAO1 (n = 4)). Average and standard deviation are plotted for both X and Y variables. The specific R² value is displayed on each panel.

Figure 2

Single-cell images illuminate phage infection dynamics. Representative microscopy images of the bioluminescent signal produced by nanoLUZ19v phage-infected cells of P. aeruginosa strains PAO1, PAK, CHA, and RP73 (phage:bacteria ratio of 0.1:1) are shown over time (from top to bottom). Magnification 625×. Samples were taken from a liquid culture at 0, 15, 60, 120, and 180 min post phage infection, mixed with luciferase substrate and agarose and subsequently imaged using a fluorescent spinning-disk microscope. Strains PAO1 and PAK express constitutively the GFP. The 527 nm emission filter was used to detect the fluorescence originating from the GFP produced by the bacteria and is presented in the images in green. The 455 nm emission filter was used to detect luminescence produced by the phage nanoLUZ19v, presented in the images in red. For strains CHA and RP73 only luminescence could be recorded (at t = 0 the signal was not distinguishable from noise). Cells producing both fluorescence and bioluminescence signals appear yellow/orange depending on the relative intensity of these two signals (see zoom in the panel PAO1 T = 60 min). The average number of cells per field of view with standard deviation (N = 3) of luminescence producing cells is indicated in the top right corner of each image.