Tracking tripartite interaction dynamics: isolation, integration, and influence of bacteriophages in the Paraburkholderia-Dictyostelium discoideum symbiosis system

Abstract

Introduction: Bacteriophages influence interactions between bacterial symbionts and their hosts by exerting parasitic pressure on symbiont populations and facilitating bacterial evolution through selection, gene exchange, and prophage integration. Host organisms also modulate phage-bacteria interactions, with host-specific contexts potentially limiting or promoting phage access to bacterial symbionts or driving alternative phenotypic or evolutionary outcomes.

Methods: To better elucidate tripartite phage-bacteria-host interactions in real-time, we expanded the Dictyostelium discoideum-Paraburkholderia symbiosis system to include Paraburkholderia-specific phages. We isolated six environmental Paraburkholderia phages from soil samples using a multi-host enrichment approach. We also identified a functional prophage from monocultures of one of the Paraburkholderia symbiont strains implemented in the enrichment approach. These phages were evaluated across all three amoeba-associated Paraburkholderia symbiont species. Finally, we treated Paraburkholderia infected amoeba lines with select phage isolates and assessed their effects on symbiont prevalence and host fitness.

Results: The isolated phages exhibited diverse plaquing characteristics and virion morphologies, collectively targeting Paraburkholderia strains belonging to each of the amoeba-symbiotic species. Following amoeba treatment experiments, we observed that phage application in some cases reduced symbiont infection prevalence and alleviated host fitness impacts, while in others, no significant effects were noted. Notably, phages were able to persist within the symbiont-infected amoeba populations over multiple culture transfers, indicating potential long-term interactions.

Discussion: These findings highlight the variability of phage-symbiont interactions within a host environment and underscore the complex nature of phage treatment outcomes. The observed variability lays the foundation for future studies exploring the long-term dynamics of tripartite systems, suggesting potential mechanisms that may shape differential phage treatment outcomes and presenting valuable avenues for future investigation.

Keywords: Dictyostelium discoideum; Paraburkholderia; amoeba; bacteriophage; symbiosis.

Figure 1

Phage screening. Tile plots representing lawn clearance from pre- and post-enriched filtrates generated from (a) Texas (TX) and (b) South Carolina (SC) soil samples, and of (c) culture filtrates from mixed- and mono-cultures representative of the enrichment conditions. The Y axis represents bacterial strains tested against the filtrate types indicated on the X axis. Left-hand vertical lines correspond to symbiont strains of D. discoideum, right-hand asterisks indicate each strain used in the multi-host enrichment condition. Observation of lysis zones were qualitatively scaled from no change (dark purple) to a full clearance (light yellow) in lawn thickness. See Supplementary Table 1 for a full list of bacterial strains.

Figure 2

Host range and plaque clearance patterns. Tile plot representing lawn clearance values for the final phage isolates (x axis) when spot-tested on bacterial lawns (y axis). Test strains are arranged vertically according to phylogenetic relatedness based on aligned atpD gene sequences (Supplementary Table 1).

Figure 3

Plaque morphology and plating efficiency. Representative plaques (a) and efficiency of plating (EOP %) (b) for the environmental phage isolates on select bacterial host strains. Efficiency of plaquing is represented as the percentage of PFU/ml generated from test strains relative to PFU/ml generated from corresponding reference strains for each phage isolate (Pb433 for Bonzo and Paranha, Pa145 for Balex and Scuba, and Pa317 for Bagra and Scugar). Scale bar is representative for all images in a.

Figure 4

Phage virion morphology. Representative negatively stained transmission electron micrographs from high-titer filtrates of purified environmental phage isolates. Scale bars are representative for all images.

Figure 5

Bonzo stability in amoeba culture conditions. Total Bonzo PFU’s recovered following the indicated incubation periods on agar medium alone (media plate, left), or with D. discoideum amoeba that were either uninfected (+ amoeba, middle), or infected with Pb433 (+ Pb433, right). For amoeba co-culture conditions, transfer + 24 h represents re-culturing from D. discoideum spores developed from the initial plating conditions.

Figure 6

Phage treatment of Paraburkholderia infected amoeba. Representative confocal micrographs (z-projections) of sori contents (a) and box plots of total phage pfu’s, amoeba spores, and intracellular infection prevalence in spore populations (top, middle, and bottom panels, respectively) (b) from developed amoeba cultures (1 week incubation) for the indicated phage treatment and Paraburkholderia (rfp) infection combinations. Scale bar represents 5um. Plot points represent individual biological replicates from V12 (boxes) and QS18 (diamonds) amoeba lines (n ≥ 4 per strain). Asterisks represent statistically significant differences between indicated groups (**p-value < 0.01, ***p < 0.001).

Figure 7

Tracking phage and symbiont infection parameters over multiple social cycles. Box plots of total phage PFU’s, amoeba spores, and intracellular infection prevalence from spore populations (top, middle, and bottom panels, respectively) after plating phage treated amoeba spores for the indicated infected amoeba lines, sampling after one social cycle (1), and serially transferring spores from harvested fruiting bodies from the prior cycle to complete a second (2), and third (3), round of development. For the second (2) cycle, data was collected for intracellular infection prevalence (bottom panel) but not for total PFU and spore production (top panels). Plot points represent individual biological replicates from QS18 amoeba lines (n = 3).