Legionella pneumophila; bacterial effector; host–pathogen; metaeffector; yeast two-hybrid; Bacterial Proteins; Type IV Secretion Systems; Humans; Host-Pathogen Interactions; Protein Interaction Maps; Protein Interaction Mapping; Legionnaires' Disease/microbiology; Legionnaires' Disease/metabolism; Type IV Secretion Systems/metabolism; Type IV Secretion Systems/genetics; Legionella pneumophila/genetics; Legionella pneumophila/metabolism; Two-Hybrid System Techniques; Bacterial Proteins/metabolism; Bacterial Proteins/genetics; Legionnaires' Disease; Microbiology; Physiology; Biochemistry; Ecology, Evolution, Behavior and Systematics; Modeling and Simulation; Molecular Biology; Genetics; Computer Science Applications
Abstract :
[en] [en] UNLABELLED: 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. Among these are "metaeffectors," 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-protein interactions among all L. pneumophila effectors and 28 components of the Dot/Icm type IV secretion system (>167,000 protein combinations). Of the 52 protein interactions identified by this approach, 44 are novel protein interactions, including 10 novel effector-effector interactions (doubling the number of known effector-effector interactions).
IMPORTANCE: 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 "effectors of effectors" 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 Legionella pneumophila, 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 L. pneumophila 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.
Disciplines :
Life sciences: Multidisciplinary, general & others
Author, co-author :
Mount, Harley O'Connor ; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
Urbanus, Malene L ; Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
Sheykhkarimli, Dayag ; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada ; Lunenfeld-Tanenbaum Research Institute, Sinai Health, Toronto, Ontario, Canada
Coté, Atina G ; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada ; Lunenfeld-Tanenbaum Research Institute, Sinai Health, Toronto, Ontario, Canada
Laval, Florent ; Université de Liège - ULiège > TERRA Research Centre > Microbial technologies ; Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, Massachusetts, USA ; Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA ; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
Coppin, Georges ; Université de Liège - ULiège > GIGA ; Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, Massachusetts, USA ; Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA ; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
Kishore, Nishka ; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada ; Lunenfeld-Tanenbaum Research Institute, Sinai Health, Toronto, Ontario, Canada
Li, Roujia; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada ; Lunenfeld-Tanenbaum Research Institute, Sinai Health, Toronto, Ontario, Canada
Spirohn-Fitzgerald, Kerstin ; Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, Massachusetts, USA ; Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA ; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
Petersen, Morgan O ; Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada ; Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
Knapp, Jennifer J ; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada ; Lunenfeld-Tanenbaum Research Institute, Sinai Health, Toronto, Ontario, Canada
Kim, Dae-Kyum ; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada ; Lunenfeld-Tanenbaum Research Institute, Sinai Health, Toronto, Ontario, Canada
Calderwood, Michael A ; Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, Massachusetts, USA ; Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA ; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA
Vidal, Marc ; Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, Massachusetts, USA ; Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA
Roth, Frederick P ; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada ; Donnelly Centre, University of Toronto, Toronto, Ontario, Canada ; Lunenfeld-Tanenbaum Research Institute, Sinai Health, Toronto, Ontario, Canada ; Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
Ensminger, Alexander W ; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada ; Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
NSERC - Natural Sciences and Engineering Research Council CIHR - Canadian Institutes of Health Research WBI - Wallonie-Bruxelles International F.R.S.-FNRS - Fonds de la Recherche Scientifique
Funding text :
H.O.M. was supported by a CGS-D fellowship from the Natural Sciences and Engineering Research Council of Canada. This work was supported by a project grant (AWE) from the Canadian Institutes of Health Research (PJT-162256). This work was supported by a Wallonie-Brussels International-World Excellence Fellowship (F.L. and G.C.), a Fonds de la Recherche Scientifique (FRS-FNRS)-T\u00E9l\u00E9vie Grant (FC31747, Cr\u00E9dit n\u00B0 7459421F) (F.L. and J.-C.T.), the Fondation L\u00E9on Fredericq (F.L. and J.-C.T.), a University of Li\u00E8ge mobility grant (F.L.), an FRS-FNRS Mobility and Congress funding (no. 40020393) (F.L.), a Jos\u00E9e and Jean Schmets Prize (F.L.), a Herman-van Beneden Prize (F.L.), and an FRS-FNRS-Fund for Research Training in Industry and Agriculture grant (FC31543, Cr\u00E9dit n\u00B0 1E00419F) (G.C.). M.V. is a Chercheur Qualifi\u00E9 Honoraire, and J.-C.T. is a Ma\u00EEtre de Recherche from the FRS-FNRS (Wallonia-Brussels Federation, Belgium). D.S., A.G.C., N.K., R.L., J.J.K., D.-K.K., and F.P.R. were supported by a Canadian Institutes of Health Research Foundation grant (FDN159926). We thank members of the Roth Lab: Marinella Gebbia for assistance with library construction and Jochen Weile, Natascha van Lieshout, Anjali Gopal, and Nozomu Yachie for bioinformatic advice. Finally, we thank members of the Ensminger Lab: Veronique Cartier-Archambault and Guangqi Zhou for help with cloning; Beth Nicholson, Jordan Lin, and John McPherson for their suggestions and careful reading of the manuscript. M.L.U. and A.W.E. conceived and designed the screen for effector\u2013effector interactions. H.O.M. constructed the library and performed the inducible BFG-Y2H screen with assistance and training from A.G.C., D.S., and M.L.U. Large-scale gateway cloning was performed by M.L.U., A.G.C., N.K., and R.L., with subsequent robotic cherry-picking by A.G.C., N.K., R.L., D.K., and J.K. Library sequencing was performed by H.O.M., D.S., A.G.C., N.K., and R.L. H.O.M. and M.L.U. analyzed the data with assistance from D.S. M.L.U. performed the Y2H confirmation experiments, M.O.P. assisted M.L.U. with data analysis and network visualization. F.L. performed the yN2H validation experiment with assistance from K.S.F. G.C. analyzed the yN2H data. M.C., J.C.T., M.V., F.P.R., and A.W.E. provided project supervision and advice. M.L.U. and A.W.E. prepared the manuscript with input from other authors. Canadian Government | Natural Sciences and Engineering Research Council of Canada (NSERC) Harley O'Connor Mount Canadian Government | Canadian Institutes of Health Research (CIHR) PJT-162256 Alexander W. Ensminger Wallonie-Bruxelles International (WBI) Florent Laval Georges Coppin Fonds De La Recherche Scientifique - FNRS (FNRS) FC31747 Florent Laval Jean-Claude Twizere Fonds De La Recherche Scientifique - FNRS (FNRS) FC31543 Georges Coppin Canadian Government | Canadian Institutes of Health Research (CIHR) FDN159926 Frederick P. Roth
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