High-throughput identification of novel phage-encoded bacterial defense mechanisms against protozoan predation

Bacteria are the backbone of natural communities. A single gram of soil may contain up to 10 billion cells.

Two types of bacterial predators are also abundant: protozoa, single-celled eukaryotes that phagocytize bacteria, and bacteriophages (phages), viruses that only infect bacteria. Each day, up to 20% of the global bacterial population is killed by these two predators. This puts pressure on bacteria to adapt defense strategies, but also on each predator to out-compete the other.

However, the interplay between bacteria, phages, and protozoa is rarely studied.Certain phages can integrate into bacterial genomes, becoming prophages, and encode beneficial traits for host fitness, like protection from other phages.

My hypothesis is that prophages encode defense mechanisms that may interfere with protozoan predation and protect the bacterial host, thereby indirectly benefiting the prophage.

To investigate this, I will develop a high-throughput quantitative fluorescence predation assay, using the amoeba Dictyostelium discoideum as the model protozoan.

Changes in abundance of fluorescently tagged bacteria with and without defense functions will report the strength of predation defense in a large bacterial isolate panel harbouring thousands of prophages. The assay will be scaled up with robotics platforms. Gain of function screens will be used to link defense phenotype with individual genes.

After prioritizing the defense hits for novel biology, the major stages of predation that are inhibited can be observed by real time tracking of interactions between fluorescently tagged cells. Biochemical and genetic follow-ups will pinpoint the exact mechanisms of defense. Proteomic, transcriptomic, and lipidomic methods will assess changes in bacterial and protozoan physiology.

Overall, this proposal addresses an important blind-spot in the interactions of bacteria, protozoa, and phages and will reveal new aspects on how predatory interactions shape microbial ecosystems.