Random encounters and amoeba locomotion drive the predation of Listeria monocytogenes by Acanthamoeba castellanii

Abstract

Predatory protozoa play an essential role in shaping microbial populations. Among these protozoa, Acanthamoeba are ubiquitous in the soil and aqueous environments inhabited by Listeria monocytogenes. Observations of predator-prey interactions between these two microorganisms revealed a predation strategy in which Acanthamoeba castellanii assemble L. monocytogenes in aggregates, termed backpacks, on their posterior. The rapid formation and specific location of backpacks led to the assumption that A. castellanii may recruit L. monocytogenes by releasing an attractant. However, this hypothesis has not been validated, and the mechanisms driving this process remained unknown. Here, we combined video microscopy, microfluidics, single-cell image analyses, and theoretical modeling to characterize predator-prey interactions of A. castellanii and L. monocytogenes and determined whether bacterial chemotaxis contributes to the backpack formation. Our results indicate that L. monocytogenes captures are not driven by chemotaxis. Instead, random encounters of bacteria with amoebae initialize bacterial capture and aggregation. This is supported by the strong correlation between experimentally derived capture rates and theoretical encounter models at the single-cell level. Observations of the spatial rearrangement of L. monocytogenes trapped by A. castellanii revealed that bacterial aggregation into backpacks is mainly driven by amoeboid locomotion. Overall, we show that two nonspecific, independent mechanisms, namely random encounters enhanced by bacterial motility and predator surface-bound locomotion, drive backpack formation, resulting in a bacterial aggregate on the amoeba ready for phagocytosis. Due to the prevalence of these two processes in the environment, we expect this strategy to be widespread among amoebae, contributing to their effectiveness as predators.

Keywords: Acanthamoeba; Listeria; capture dynamics; predation; random encounter.

Fig. 1.

Captured L. monocytogenes cells form backpack structures on the surface of A. castellanii trophozoites (SI Appendix, Visualization of Backpacks). (A–C) Confocal laser scanning microscopy images of GFP-expressing L. monocytogenes (strain Scott A::pPL2P_hyp gfp) in coculture with A. castellanii. Backpacks consisting of trapped L. monocytogenes cells are marked with white arrowheads. Images were taken 15 min (A), 30 min (B), or 1 h (C) after the start of coincubation. (D and E) SEM images of L. monocytogenes (strain Scott A wild type) backpacks, marked with white arrowheads, on the surface of A. castellanii trophozoites after 1 h of coincubation.

Fig. 2.

L. monocytogenes is not attracted by A. castellanii secretions. (A and B) Schematic representation of the LGG used for the chemotaxis assays (Materials and Methods). Outer channels allow the flow of a potential chemoattractant solution and PAS buffer, generating a gradient across the hydrogel walls and the central channel. The central channel, which has no flow, contains a suspension of L. monocytogenes. By quantifying the spatial distribution of Listeria cells across the width of the central channel (subdivided into five 200-µm-wide regions, R1 to R5, for analysis), the chemotactic response of the cells to the potential chemoattractant can be quantified. (C) Example image showing accumulation of L. monocytogenes cells close to the left hydrogel wall, saturated with 10% brain heart infusion (BHI) broth as the positive control, 5 min after cells were introduced into the central channel. (D–G) Relative cell concentrations of L. monocytogenes as a function of time for three regions within the central channel: R1 closest to the source channel containing the potential chemoattractant (orange), R3 along the middle of the central channel (purple), and R5 closest to the sink channel (blue). Solid colored lines show the average probability distributions over three replicate experiments, and shaded areas represent the SD. Relative cell concentrations over time are shown for experiments in which the chemoattractant channel contained (D) 10% BHI medium, (E) PAS buffer, (F) A. castellanii cells used directly after washing (Acanthamoeba), and (G) A. castellanii cells incubated overnight in PAS buffer after washing (Acanthamoeba overnight). Accumulation of L. monocytogenes toward the source channel was only observed in response to BHI broth used as a positive control.

Fig. 3.

Random encounters enhanced by bacterial motility govern the capture of L. monocytogenes by A. castellanii. (A) Schematic representation of the region of interest around an A. castellanii trophozoite, illustrating the image analysis masks used to quantify L. monocytogenes capture rates in a microfluidic arena (Movie S1). For each A. castellanii trophozoite, two masks were created: an Acanthamoeba mask (M_Ac), covering the cell and an interaction mask (M_int), which extended 4 µm out from the boundary of the Acanthamoeba mask. The number of captured bacteria over time was quantified by tracking individual L. monocytogenes cells and calculating the number of bacteria entering the interaction mask (red “In” arrow) minus the number of cells exiting from the interaction mask (blue “Out” arrow). (B) Example image showing an A. castellanii trophozoite overlaid with the two masks. The mask boundaries are indicated by white arrows. (C) Cumulative number of captured L. monocytogenes cells for each of nine monitored A. castellanii trophozoites (color-coded, from A to I), for one experiment at an initial concentration of 1.0 × 10⁷ CFU/mL L. monocytogenes. Colored lines represent the fitted exponential saturation function for each A. castellanii trophozoite (see Materials and Methods). Plots for each experiment are provided in SI Appendix, Fig. S1. (D) Mean L. monocytogenes swimming speed (v), mean A. castellanii trophozoite radius (r_A), and mean L. monocytogenes concentration (b_L), each as a function of time, estimated within the region of interest centered on each of the nine A. castellanii trophozoites shown in C (color-coded, from A to I). The time-averaged values of these parameters for each individual A. castellanii trophozoite were used to estimate their theoretical encounter rates using the equations in E. (E) Schematic representations and equations of the theoretical encounter rate models for a flux of particles that encounters a perfectly absorbing disk (orange) or halfsphere (purple) with radius (r_A). (F) Observed capture rates, γ (Eq. 2), of individual A. castellanii trophozoites plotted as a function of the theoretical encounter rates calculated using the model for a disk (orange circles) or for a half-sphere (purple triangles) that assume random bacterial encounter and perfect absorption (see Results). The two models were parameterized for each A. castellanii trophozoite using values for Listeria concentration, Listeria swimming speed, and A. castellanii radius quantified from individual experiments. Solid lines represent linear regressions for each model in the respective color (R² = 0.90 in both cases).

Fig. 4.

Captured bacteria are incorporated into a backpack upon reaching the rear of the A. castellanii trophozoite as the amoeba moves forward. (A) Time series monitoring the capture and relocation of fluorescent L. monocytogenes on the surface of a moving and a nonmoving A. castellanii trophozoite. Motile L. monocytogenes from all directions (green rods, marked with circles of color according to their time of arrival, in the order yellow, red, blue, purple, white, orange) were trapped on the surface of an A. castellanii trophozoite. A backpack, marked with a green arrowhead, was observed 8 min after the first capture by the moving Acanthamoeba trophozoite. In contrast, L. monocytogenes cells trapped on nonmoving Acanthamoeba trophozoites did not assemble into a backpack. The white cross indicates the position of the geometric center of the A. castellanii trophozoite, and the white line and dots indicate its trajectory as the trophozoite moves (Movies S2 and S3). (B) Schematic representation of our proposed model for backpack formation. The initial location of the anteriormost bacterium is marked with a blue bar. As the A. castellanii trophozoite moves on a surface, the bacteria (in green) remain stationary relative to the substratum until they reach the posterior of the trophozoite, where the bacteria are accumulated into a backpack. (C) Hypothetic model for backpack formation associated with the sol–gel–sol theory for the movement of amoebae on surfaces. Actin filaments are polymerized at the front of the trophozoite to form plasmagel and are subsequently depolymerized at the rear to form plasmasol that contains a pool of unpolymerized monomers. Bacteria (in green) captured on the surface of the trophozoite are deposited in a backpack at the region where plasmagel becomes plasmasol.