HIGH-THROUGHPUT YEAST SCREENING REVEALS A NEW DIMENSION OF INTRACELLULAR PATHOGENESIS. Malene L. Urbanus2, Harley O’Connor Mount1, Eleanor A. Latomanski3, Dylan Valleau4, Frederick P. Roth1,5,6, Hayley J. Newton3, Alexei Savchenko4,7, Alexander W. Ensminger1,2 1. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; 2. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada; 3. Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia; 4. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada; 5. Lunenfeld‐Tanenbaum Research Institute Mt. Sinai Hospital, Toronto, Ontario, Canada; 6. Donnelly Centre, University of Toronto, Toronto, Ontario, Canada; 7. Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada. WHAT IS A METAEFFECTOR? USING YEAST TO FIND THEM. WHAT’S NEXT? Metaeffectors directly target other effectors HOST TARGET indirect Effectors target host proteins Legionella direct Effectors are proteins delivered to host cells via dedicated bacterial translocation machineries. The intracellular bacterial pathogen, Legionella pneumophila, uses a Type IV translocation system to deliver over 300 effectors to the eukaryotic host cell (~10% of its proteome). The canonical role of effectors is to modify host targets. Metaeffectors (or effectors of effectors) directly modify other translocated bacterial proteins rather than the host. Yin-Yang effectors behave like canonical effectors, but indirectly interact through counteracting modulation of a shared host target. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 20 40 60 80 100 L. pneumophila infects a broad range of eukaryotic hosts (from protozoa to humans) and modulates conserved eukaryotic pathways. Because they target conserved pathways, 37% of L. pneumophila effectors cause a yeast growth defect of ≥20%) when heterologously expressed. Normalized spot sizes of effector-expressing yeast clones grown at 1,536 spot density. Yin-Yang effectors Query (MATα, URA3) Array (MATa, HIS3) Diploid (MATa/α, HIS3, URA3) x sidD (lpg2465) sidM/drrA (lpg2464) query screen Induce expression. Image diploid spot size: ceg3 (lpg0080) 0 1000 2000 3000 4000 0 1000 2000 3000 4000 spot size (px), replicate 2 spot size (px), replicate 1 lpg0081 legA8/ankX (lpg0695) lem3 (lpg0696) 0 1000 2000 3000 4000 spot size (px), replicate 2 0 1000 2000 3000 4000 spot size (px), replicate 1 0 1000 2000 3000 4000 0 1000 2000 3000 4000 spot size (px), replicate 2 spot size (px), replicate 1 sdeB (lpg2156) sdjA* (lpg2508) sidJ (lpg2155) sdbB (lpg2482) sdbC (lpg2391) 0 1000 2000 3000 4000 0 1000 2000 3000 4000 spot size (px), replicate 2 spot size (px), replicate 1 0 1000 2000 3000 4000 0 1000 2000 3000 4000 spot size (px), array + query spot size (px), single IDTS lpg1148 legC3 (lpg1701) 0 1000 2000 3000 4000 0 1000 2000 3000 4000 spot size (px), replicate 2 spot size (px), replicate 1 ceg6/pkn5 (lpg0208) ravG (lpg0210) ravJ (lpg0944) legL1 (lpg0945) 0 1000 2000 3000 4000 0 1000 2000 3000 4000 spot size (px), replicate 2 spot size (px), replicate 1 legC7/ylfA lpg2298) mavE (lpg2344) 0 1000 2000 3000 4000 0 1000 2000 3000 4000 spot size (px), replicate 2 spot size (px), replicate 1 lem26 (lpg2523) mavL (lpg2526) 0 1000 2000 3000 4000 0 1000 2000 3000 4000 spot size (px), replicate 2 spot size (px), replicate 1 0 1000 2000 3000 4000 0 1000 2000 3000 4000 spot size (px), replicate 2 spot size (px), replicate 1 sidM/drrA (lpg2464) sidD (lpg2465) sidL/ceg14 (lpg0437) 0 1000 2000 3000 4000 0 1000 2000 3000 4000 spot size (px), replicate 2 spot size (px), replicate 1 legA11/ankJ (lpg0436) 0 1000 2000 3000 4000 0 1000 2000 3000 4000 spot size (px), replicate 2 spot size (px), replicate 1 sdeC (lpg2153) sdjA* (lpg2508) sidJ (lpg2155) 0 1000 2000 3000 4000 spot size (px), replicate 1 mavQ (lpg