A comprehensive two-hybrid analysis to explore the L. pneumophila effector-effector interactome

Legionella pneumophila uses over 300 translocated effector proteins to rewire host cells during infection and create a replicative niche for intracellular growth. To date, several studies have identified L. pneumophila effectors that indirectly and directly regulate the activity of other effectors, providing an additional layer of regulatory complexity. L. pneumophila has the largest contingent of a new class of effectors, so called “metaeffectors” that directly regulate the activity of other effectors in the host. A defining quality of metaeffectors is the direct, physical interaction of the metaeffector with its cognate target effector. Metaeffectors identification to date has depended on phenotypes in heterologous systems, experimental serendipity and they represent only one class of physical interactions between effectors. Using a multiplexed, sequence-based yeast two-hybrid technology we screened the entire L. pneumophila effector proteome and components of the Dot/Icm type IV secretion system for protein-protein interactions (>167,000 protein combinations). Our screen captured 52 protein interactions, including 8 known and 44 novel protein interactions. Most notably, we identified ten novel effector-effector interactions, doubling the number of known effector-effector interactions.