title: A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system.
abstract: The Dot/Icm system is a type IVb secretion system used by Legionella pneumophila to modulate vesicular transport in both protozoan and mammalian host cells. It has been shown that proteins and processes that are highly conserved in all eukaryotic cells are targets for some of the proteins injected by the Dot/Icm system. For example, the Legionella protein RalF was shown previously to be a Dot/Icm substrate that functions as a guanine nucleotide exchange factor (GEF) for the Arf family of eukaryotic small GTP-binding proteins. Here we show that ectopic production of the RalF protein in Saccharomyces cerevisiae interferes with yeast growth. Inhibition of yeast growth was found to be dependent on the ability of RalF to function as an Arf-GEF in vivo. The possibility that other Dot/Icm substrate proteins would have the capacity to interfere with yeast growth was used as a rationale to screen plasmid libraries containing random fragments of Legionella chromosomal DNA positioned downstream of a galactose-inducible promoter. This screen identified Legionella proteins that conferred a conditional growth defect when overproduced by yeast cultured in the presence of galactose. Most of the Legionella proteins identified were determined to be substrates of the Dot/Icm system. This screen led to the identification of a new Dot/Icm substrate protein that was called YlfA, for yeast lethal factor A. A paralogue of YlfA was identified on an unlinked region of the Legionella chromosome and this protein was also translocated by the Dot/Icm system. It was determined that a hydrophobic region near the N-terminus of the YlfA protein and an adjacent region predicted to form a coiled-coil domain were necessary for a biological activity that interfered with yeast growth. The YlfA protein did not decorate the Legionella-containing vacuole during the first 7 h of infection but could be observed on the endoplasmic reticulum (ER)-derived replicative vacuole and on punctate structures throughout the host cell at later stages. Ectopic production of YlfA in mammalian cells revealed that the N-terminal hydrophobic domain in YlfA was able to localize the protein to early secretory organelles, including endoplasmic reticulum. These studies show that yeast genetics can be exploited to identify and characterize proteins that are injected into host cells by bacterial pathogens that utilize type IV secretion systems for pathogenesis.
authors.affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536, USA.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Campodonico
authors.forename: Laurent
authors.initials: L
authors.lastname: Chesnel
authors.forename: Craig R
authors.initials: CR
authors.lastname: Roy
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology*; D016466:CHO Cells; D006224:Cricetinae; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D016952:Legionella pneumophila / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; C497516:RalF protein, Legionella pneumophila