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Targeted and Unbiased Screen for Genetic Suppressors of the Legionella pneumophila effector protein LegC7

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Targeted and Unbiased Screen for Genetic Suppressors of the Legionella pneumophila effector protein LegC7

  1. 1. A targeted and an unbiased screen for genetic suppressors of the Legionella pneumophila effector protein LegC7 Chetan N. Hebbale*, Kevin M. O’Brien*, and Vincent J. Starai*† Departments of *Microbiology and †Infectious Diseases, University of Georgia, Athens, GA 30602. Background This work is made possible by the National Science Foundation grant number 1062589, the Starai Lab grant from National Institutes of Health-Allergy and Infectious Disease (R01-Al 100913), and the University of Georgia Microbiology and Infectious Diseases Departments. Personal thanks to Vinny Starai and Kevin O’Brien. Acknowledgments University of Georgia Athens, GA Microbiology Department Conclusions pYES2/NTC Method – Unbiased Screen Abstract Legionella pneumophila is a Gram-negative bacterium that causes a severe form of pneumonia known as Legionnaires’ disease. During infection, L. pneumophila secretes nearly 300 effector proteins into host cells in order to evade lysosomal degradation by modulating vesicle trafficking pathways. One of these effector proteins, LegC7, has been shown to be toxic upon expression in the budding yeast, Saccharomyces cerevisiae. Upon LegC7 expression, S. cerevisiae accumulates membranous structures reminiscent of so called “class E” compartments, which result from defects in multivesicular body function. The proteins which comprise the Class E VPS family are members of the endosomal sorting complex required for transport proteins (ESCRT) which are responsible for recognizing, sequestering and packaging membrane proteins into vesicles for vacuolar degradation. Because the Class E phenotype was produced in yeast during LegC7 expression, we hypothesize that LegC7 interacts with one or more of the yeast Class E gene products. We therefore continued a targeted screen of the yeast Class E genes by transforming a plasmid encoding LEGC7 into yeast strains with deletions of vps23, vps28, snf7, or bro1 and found that deletion of these genes did not suppress LegC7 toxicity. We then performed an unbiased screen in an attempt to find genetic suppressors of LegC7 toxicity using ethyl methanesulfonate (EMS) mutagenesis to isolate a strain that exhibits a toxicity reversal due to a genomic mutation. We will sequence the genome from this strain to identify the gene products that LegC7 might require for toxicity. Future Directions Class E Vacuolar Protein Sorting Multivesicular Body: receives and sorts ubiquitinated membrane proteins from Golgi or plasma membrane to the vacuole ESCRT (endosomal sorting complex required for transport): proteins found on MVB that identify membrane proteins bound for destruction and establish intraluminal vesicles containing these proteins Class E Mutants Screened: VPS23, VPS28, SNF7, BRO1 Western Blot: LegC7 • LegC7 is highly toxic when expressed in Saccharomyces cerevisiae • Causes an accumulation of vesicular material and aberrantly secretes CPY Invertase – resembles Class E VPS Mutants • pYES2/NTC (pYES) plasmid allows for selection and expression • pGAL1 : Promoter that induces LegC7 expression if galactose is present • URA3: Involved in the biosynthesis of uracil. BY4742 yeast will not grow in CSM-uracil media without pYES being present – ensures the plasmid enters yeast Results – Targeted Screen Current Class E VPS Mutants: SEY6210 vps23∆ + LEGC7 + vps28∆ vps28∆ + LEGC7 + snf7∆ snf7∆ + LEGC7 + bro1∆ bro1∆ + LEGC7 + SEY6210 +LEGC7+ vps23∆ Targeted screen yielded no new genetic suppressors of LegC7 BY4742 BY4742 + LEGC7+ vps27∆ vps27∆ + LEGC7 + Previous Class E VPS Mutants: 2% Glucose 2% Galactose Results – Unbiased Screen Legionella pneumophila • Causative agent of Legionnaire's disease • Obligate intracellular pathogen, utilizes a type IV secretion system to inject effector proteins that manipulate host physiology Deletion of vps27 resulted in a partial toxicity reversal of LegC7 EMS Mutant Strains BY4742 β - EMS BY4742 β–EMS plasmid LEGC7+ α - EMS 2% Galactose BY4742 α–EMS plasmid BY4742 ε – EMS plasmid BY4742 +LEGC7+ γ – EMS plasmid Δ – EMS plasmid 2% Galactose • Wild type yeast (BY4742) are treated with ethyl methanesulfonate (EMS) - inducing random point mutations throughout the genome • LEGC7 plasmid is transformed into mutated yeast cells and plated onto selective media containing galactose, any colonies that grow are selected for further screening • Growth could be due to a plasmid mutation or a genomic mutation EMS Mutagenesis • LEGC7 plasmid is extracted from mutated yeast cells Yeast Plasmid Prep • Extracted LEGC7 plasmid is transformed into E.coli to amplify copy number • LEGC7 plasmid is then purified from the E.coli Bacterial Transformation and Mini-Prep • LEGC7 plasmid is transformed into BY4742 and spot plated to see if the LEGC7 plasmid is still inhibitory Yeast Transformation and Spot Plate • EMS Mutants are grown up in media containing galactose • Proteins are separated using SDS PAGE and transferred to nitrocellulose membrane • Blot is probed with α-LegC7 serum • Demonstrates if LegC7 is being expressed by plasmids that are no longer inhibitory Known LegC7 ε EMS γ EMS Δ EMS • Because there is no expression of LegC7 from non- inhibitory plasmids possible genetic mutations are: 1) Mutation in the GAL1 promoter region 2) Mutation resulting in a stop codon in LEGC7 ORF θ EMS α LegC7 deFelipe 2009 https://science.nichd.nih.gov/conf luence/display/machner/Home GFP-Leg C7 Localization GFP- LegC7 FM4-64 Deletion of Class E genes does not affect LegC7 localization Hansen, Annu. Rev. Cell Dev. Biol. 2012. vacuole Class E compartments vps4∆ + LEGC7+ did4∆ + LEGC7+ ESCRT – 0 Vps27,Hse1 ESCRT – I Vps23, Vps28,Srn2, Mvb12 ESCRT – II Vps36, Snf8, Vps25 ESCRT – III Vps20, Snf7, Vps24, Did4, Bro1 Class E VPS Mutants 1) Deletion of vps23, vps28, snf7 and bro1 do not result in a toxicity reversal of LegC7 2) Deletion of vps27 is unique among the ESCRT pathway as it is the only one that shows a reversal phenotype 3) Deletion of representative class E genes do not affect LegC7 localization at the vacuole EMS Mutagenesis 1) EMS strains α and β exhibited a toxicity reversal of LegC7 likely due to a genomic mutation 2) EMS strains γ, Δ, ε and θ exhibited a toxicity reversal of LegC7 due to a plasmid mutation Genomic Mutation Sequence: • EMS strains α and β will have the whole genome sequenced and screened for specific genetic mutations • After mutations are identified they will be reconstructed in a clean background and have LegC7 transformed to see if there is a toxicity reversal Over-Expression Screen: • Perform an overexpression screen to locate any genes that when overexpressed result in a decrease of LegC7 toxicity in order to find a biochemical interacting partner with LegC7 2% Galactose

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