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To date, several studies have identified\n              <jats:italic>L. pneumophila<\/jats:italic>\n              effectors that indirectly and directly regulate the activity of other effectors, providing an additional layer of regulatory complexity. Among these are \u201cmetaeffectors,\u201d a special class of effectors that regulate the activity of other effectors once inside the host. A defining feature of metaeffectors is direct, physical interaction with a target effector. Metaeffector identification, to date, has depended on phenotypes in heterologous systems and experimental serendipity. Using a multiplexed, recombinant barcode-based yeast two-hybrid technology we screened for protein\u2013protein interactions among all\n              <jats:italic>L. pneumophila<\/jats:italic>\n              effectors and 28 components of the Dot\/Icm type IV secretion system (&gt;167,000 protein combinations). Of the 52 protein interactions identified by this approach, 44 are novel protein interactions, including 10 novel effector\u2013effector interactions (doubling the number of known effector\u2013effector interactions).\n            <\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>IMPORTANCE<\/jats:title>\n            <jats:p>\n              Secreted bacterial effector proteins are typically viewed as modulators of host activity, entering the host cytosol to physically interact with and modify the activity of one or more host proteins in support of infection. A growing body of evidence suggests that a subset of effectors primarily function to modify the activities of other effectors inside the host. These \u201ceffectors of effectors\u201d or metaeffectors are often identified through experimental serendipity during the study of canonical effector function against the host. We previously performed the first global effector-wide genetic interaction screen for metaeffectors within the arsenal of\n              <jats:italic>Legionella pneumophila<\/jats:italic>\n              , an intracellular bacterial pathogen with over 300 effectors. Here, using a high-throughput, scalable methodology, we present the first global interaction network of physical interactions between\n              <jats:italic>L. pneumophila<\/jats:italic>\n              effectors. This data set serves as a complementary resource to identify and understand both the scope and nature of non-canonical effector activity within this important human pathogen.\n            <\/jats:p>\n          <\/jats:sec>","DOI":"10.1128\/msystems.01004-24","type":"journal-article","created":{"date-parts":[[2024,11,11]],"date-time":"2024-11-11T14:00:29Z","timestamp":1731333629000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":10,"title":["A comprehensive two-hybrid analysis to explore the\n            <i>Legionella pneumophila<\/i>\n            effector\u2013effector interactome"],"prefix":"10.1128","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1401-9178","authenticated-orcid":false,"given":"Harley O'Connor","family":"Mount","sequence":"first","affiliation":[{"name":"Department of Molecular Genetics, University of Toronto","place":["Toronto, 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