The Yeast Saccharomyces cerevisiae: A Versatile Model System for the Identification and Characterization of Bacterial Virulence Proteins
2008; Cell Press; Volume: 4; Issue: 1 Linguagem: Inglês
10.1016/j.chom.2008.06.004
ISSN1934-6069
AutoresKeri A. Siggers, Cammie F. Lesser,
Tópico(s)Plant-Microbe Interactions and Immunity
ResumoMicrobial pathogens utilize complex secretion systems to deliver proteins into host cells. These effector proteins target and usurp host cell processes to promote infection and cause disease. While secretion systems are conserved, each pathogen delivers its own unique set of effectors. The identification and characterization of these effector proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. Model systems including yeast, worms, flies, and fish are being used to circumvent these issues. This technical review details the versatility and utility of yeast Saccharomyces cerevisiae as a system to identify and characterize bacterial effectors. Microbial pathogens utilize complex secretion systems to deliver proteins into host cells. These effector proteins target and usurp host cell processes to promote infection and cause disease. While secretion systems are conserved, each pathogen delivers its own unique set of effectors. The identification and characterization of these effector proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. Model systems including yeast, worms, flies, and fish are being used to circumvent these issues. This technical review details the versatility and utility of yeast Saccharomyces cerevisiae as a system to identify and characterize bacterial effectors. There are many advantages to working with the yeast, S. cerevisiae. It is easy to grow in the laboratory, genetically tractable, and has been used as a model system for studying eukaryotic cellular processes for over 50 years. These studies have provided insights into fundamental eukaryotic processes, including transcription, translation, RNA processing, cell signaling, cytoskeletal dynamics, and vesicle trafficking. Presently, over 75% of yeast ORFs have known or predicted functions, and much of this information is easily accessible in a variety of databases on the world wide web (see Table 1 for a listing of the sites).Table 1Useful Databases and ResourcesSaccharomyces Genome Database (SGD)http://www.yeastgenome.orgSGD is an organized collection of genetic and molecular biological information about annotated yeast ORFsThe Yeast Proteome Database (YPD)https://www.proteome.com/proteomeThis is a commercial comprehensive database of information regarding annotated yeast ORFsComprehensive Yeast Genome Database (CYGD-MIPS)http://mips.gsf.de/genre/proj/yeast/index.jspCYGD presents information on the molecular structure and functional network of S. cerevisiaeBiomolecular Interaction Network Database (BIND)http://bind.caBIND is a database designed to store full descriptions of interactions, molecular complexes and pathwaysYeast GFP Fusion Localization Databasehttp://yeastgfp.ucsf.eduThis database is a repository for localization of GFP fusion proteins in yeastYeast Protein Localization Database (YPL)http://ypl.uni-graz.at/pages/home.htmlThis database is a repository for global analyses of localization studies in yeastVirtual Library—Yeasthttp://www.yeastgenome.org/VL-yeast.htmlSource for general information regarding yeast as an experimental modelOpen Biosystemshttp://www.openbiosystems.com/GeneExpression/YeastCommercial source for yeast deletion and overexpressor strain collectionsEuroscarf: European Saccharomyces Cerevisiae Archive for Functional Analyseshttp://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.htmlSource of yeast deletion stains as well as other useful yeast strains and expression plasmidsInvitrogenhttp://clones.invitrogen.com/cloneinfo.php?clone=yeastgfpCommercial source for both yeast deletion strain and yeast GFP clone collections Open table in a new tab Many tools and resources are available for designing and executing both genome-wide (discovery-driven) and smaller-scale (hypothesis-driven) experiments. In addition to comprehensive yeast DNA (DeRisi et al., 1997DeRisi J.L. Iyer V.R. Brown P.O. Exploring the metabolic and genetic control of gene expression on a genomic scale.Science. 1997; 278: 680-686Crossref PubMed Scopus (3603) Google Scholar) and protein microarrays (Zhu et al., 2000Zhu H. Klemic J.F. Chang S. Bertone P. Casamayor A. Klemic K.G. Smith D. Gerstein M. Reed M.A. Snyder M. Analysis of yeast protein kinases using protein chips.Nat. Genet. 2000; 26: 283-289Crossref PubMed Scopus (717) Google Scholar), several isogenic strain collections are available where each strain carries a genetically altered version of one of the ∼6200 annotated ORFs. These alterations include targeted gene deletions for use in phenotypic assays (Giaever et al., 2002Giaever G. Chu A.M. Ni L. Connelly C. Riles L. Veronneau S. Dow S. Lucau-Danila A. Anderson K. Andre B. et al.Functional profiling of the Saccharomyces cerevisiae genome.Nature. 2002; 418: 387-391Crossref PubMed Scopus (3015) Google Scholar), fusions of each ORF to GFP for subcellular localization studies (Huh et al., 2003Huh W.K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Global analysis of protein localization in budding yeast.Nature. 2003; 425: 686-691Crossref PubMed Scopus (3118) Google Scholar), tandem affinity tags for protein expression and coimmunoprecipitation assays (Ghaemmaghami et al., 2003Ghaemmaghami S. Huh W.K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Global analysis of protein expression in yeast.Nature. 2003; 425: 737-741Crossref PubMed Scopus (2883) Google Scholar), and fusions to the GAL4-binding domain for two-hybrid assays (Uetz et al., 2000Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. et al.A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.Nature. 2000; 403: 623-627Crossref PubMed Scopus (3808) Google Scholar). Strain collections that conditionally overexpress each of the annotated yeast ORFs also exist (Gelperin et al., 2005Gelperin D.M. White M.A. Wilkinson M.L. Kon Y. Kung L.A. Wise K.J. Lopez-Hoyo N. Jiang L. Piccirillo S. Yu H. et al.Biochemical and genetic analysis of the yeast proteome with a movable ORF collection.Genes Dev. 2005; 19: 2816-2826Crossref PubMed Scopus (375) Google Scholar, Sopko et al., 2006Sopko R. Huang D. Preston N. Chua G. Papp B. Kafadar K. Snyder M. Oliver S.G. Cyert M. Hughes T.R. et al.Mapping pathways and phenotypes by systematic gene overexpression.Mol. Cell. 2006; 21: 319-330Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). The utilization of yeast in the study of pathogenic microbes relies on the observation that bacterial effector proteins often target eukaryotic cellular processes conserved between yeast and mammals. Currently, Agrobacterium tumefaciens, a plant pathogen, is the only pathogen known to be capable of delivering proteins directly through the yeast cell wall into the cytoplasm via its specialized type IV secretion system (Piers et al., 1996Piers K.L. Heath J.D. Liang X. Stephens K.M. Nester E.W. Agrobacterium tumefaciens-mediated transformation of yeast.Proc. Natl. Acad. Sci. USA. 1996; 93: 1613-1618Crossref PubMed Scopus (144) Google Scholar). Thus, rather than studying effector proteins in the context of an infection, individual effector proteins are expressed de novo in yeast. For this reason, the yeast system is particularly applicable for studying proteins thought to act within host cells. In addition, since this system only requires DNA, it provides a valuable resource for studying effector proteins from pathogens that are difficult to grow or genetically manipulate. Expression of effector proteins can lead to a variety of discernable phenotypes in yeast (discussed below) that can lead to testable hypotheses regarding their functions and/or their roles in pathogenesis. Once generated, hypotheses can be pursued in yeast as well as in physiologic models of disease. There is increasing evidence that yeast growth inhibition due to the expression of bacterial proteins is a sensitive and specific indicator of the activity of effector proteins that perturb conserved cellular processes. Effector proteins from both plant and animal pathogens—including Pseudomonas syringae (Munkvold et al., 2008Munkvold K.R. Martin M.E. Bronstein P.A. Collmer A. A survey of the Pseudomonas syringae pv. tomato DC3000 type III secretion system effector repertoire reveals several effectors that are deleterious when expressed in Saccharomyces cerevisiae.Mol. Plant Microbe Interact. 2008; 21: 490-502Crossref PubMed Scopus (39) Google Scholar), Pseudomonas aeruginosa (Rabin and Hauser, 2003Rabin S.D. Hauser A.R. Pseudomonas aeruginosa ExoU, a toxin transported by the type III secretion system, kills Saccharomyces cerevisiae.Infect. Immun. 2003; 71: 4144-4150Crossref PubMed Scopus (45) Google Scholar, Sato et al., 2003Sato H. Frank D.W. Hillard C.J. Feix J.B. Pankhaniya R.R. Moriyama K. Finck-Barbancon V. Buchaklian A. Lei M. Long R.M. et al.The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU.EMBO J. 2003; 22: 2959-2969Crossref PubMed Scopus (253) Google Scholar, Stirling and Evans, 2006Stirling F.R. Evans T.J. Effects of the type III secreted pseudomonal toxin ExoS in the yeast Saccharomyces cerevisiae.Microbiology. 2006; 152: 2273-2285Crossref PubMed Scopus (11) Google Scholar), Shigella flexneri (Alto et al., 2006Alto N.M. Shao F. Lazar C.S. Brost R.L. Chua G. Mattoo S. McMahon S.A. Ghosh P. Hughes T.R. Boone C. et al.Identification of a bacterial type III effector family with G protein mimicry functions.Cell. 2006; 124: 133-145Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, Slagowski et al., 2008Slagowski N.L. Kramer R.W. Morrison M.F. LaBaer J. Lesser C.F. A functional genomic yeast screen to identify pathogenic bacterial proteins.PLoS Pathog. 2008; 4: e9Crossref PubMed Scopus (50) Google Scholar), Salmonella typhimurium (Aleman et al., 2005Aleman A. Rodriguez-Escudero I. Mallo G.V. Cid V.J. Molina M. Rotger R. The amino-terminal non-catalytic region of Salmonella typhimurium SigD affects actin organization in yeast and mammalian cells.Cell. Microbiol. 2005; 7: 1432-1446Crossref PubMed Scopus (21) Google Scholar, Lesser and Miller, 2001Lesser C.F. Miller S.I. Expression of microbial virulence proteins in Saccharomyces cerevisiae models mammalian infection.EMBO J. 2001; 20: 1840-1849Crossref PubMed Scopus (79) Google Scholar, Rodriguez-Pachon et al., 2002Rodriguez-Pachon J.M. Martin H. North G. Rotger R. Nombela C. Molina M. A novel connection between the yeast Cdc42 GTPase and the Slt2-mediated cell integrity pathway identified through the effect of secreted Salmonella GTPase modulators.J. Biol. Chem. 2002; 277: 27094-27102Crossref PubMed Scopus (25) Google Scholar), Legionella pneumophila (Campodonico et al., 2005Campodonico E.M. Chesnel L. Roy C.R. A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system.Mol. Microbiol. 2005; 56: 918-933Crossref PubMed Scopus (109) Google Scholar, Derre and Isberg, 2005Derre I. Isberg R.R. LidA, a translocated substrate of the Legionella pneumophila type IV secretion system, interferes with the early secretory pathway.Infect. Immun. 2005; 73: 4370-4380Crossref PubMed Scopus (73) Google Scholar), Chlamydia trachomatis (Sisko et al., 2006Sisko J.L. Spaeth K. Kumar Y. Valdivia R.H. Multifunctional analysis of Chlamydia-specific genes in a yeast expression system.Mol. Microbiol. 2006; 60: 51-66Crossref PubMed Scopus (79) Google Scholar), enteropathogenic E. coli (Hardwidge et al., 2005Hardwidge P.R. Deng W. Vallance B.A. Rodriguez-Escudero I. Cid V.J. Molina M. Finlay B.B. Modulation of host cytoskeleton function by the enteropathogenic Escherichia coli and Citrobacter rodentium effector protein EspG.Infect. Immun. 2005; 73: 2586-2594Crossref PubMed Scopus (56) Google Scholar, Rodriguez-Escudero et al., 2005Rodriguez-Escudero I. Hardwidge P.R. Nombela C. Cid V.J. Finlay B.B. Molina M. Enteropathogenic Escherichia coli type III effectors alter cytoskeletal function and signalling in Saccharomyces cerevisiae.Microbiology. 2005; 151: 2933-2945Crossref PubMed Scopus (21) Google Scholar), and Yersinia species (Lesser and Miller, 2001Lesser C.F. Miller S.I. Expression of microbial virulence proteins in Saccharomyces cerevisiae models mammalian infection.EMBO J. 2001; 20: 1840-1849Crossref PubMed Scopus (79) Google Scholar, Nejedlik et al., 2004Nejedlik L. Pierfelice T. Geiser J.R. Actin distribution is disrupted upon expression of Yersinia YopO/YpkA in yeast.Yeast. 2004; 21: 759-768Crossref PubMed Scopus (22) Google Scholar, Von Pawel-Rammingen et al., 2000Von Pawel-Rammingen U. Telepnev M.V. Schmidt G. Aktories K. Wolf-Watz H. Rosqvist R. GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway: a mechanism for disruption of actin microfilament structure.Mol. Microbiol. 2000; 36: 737-748Crossref PubMed Scopus (262) Google Scholar)—have been observed to inhibit growth when expressed in yeast. In contrast, expression of very few nontranslocated proteins affects yeast growth (Campodonico et al., 2005Campodonico E.M. Chesnel L. Roy C.R. A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system.Mol. Microbiol. 2005; 56: 918-933Crossref PubMed Scopus (109) Google Scholar, Slagowski et al., 2008Slagowski N.L. Kramer R.W. Morrison M.F. LaBaer J. Lesser C.F. A functional genomic yeast screen to identify pathogenic bacterial proteins.PLoS Pathog. 2008; 4: e9Crossref PubMed Scopus (50) Google Scholar). Since a priori it is not known whether expression of the proteins will be toxic to yeast, it is best to first express an effector protein under the control of an inducible promotor. This is most commonly accomplished by use of the GAL1/10 promotor, a strong promotor whose activity is regulated by the carbon source in the media. However, this promotor is slightly leaky under repressing conditions, and expression of extremely toxic effector proteins can be difficult in this system (Slagowski et al., 2008Slagowski N.L. Kramer R.W. Morrison M.F. LaBaer J. Lesser C.F. A functional genomic yeast screen to identify pathogenic bacterial proteins.PLoS Pathog. 2008; 4: e9Crossref PubMed Scopus (50) Google Scholar, Stirling and Evans, 2006Stirling F.R. Evans T.J. Effects of the type III secreted pseudomonal toxin ExoS in the yeast Saccharomyces cerevisiae.Microbiology. 2006; 152: 2273-2285Crossref PubMed Scopus (11) Google Scholar). Alternatively, effector proteins can be placed under the control of the weaker MET3 (Von Pawel-Rammingen et al., 2000Von Pawel-Rammingen U. Telepnev M.V. Schmidt G. Aktories K. Wolf-Watz H. Rosqvist R. GAP activity of the Yersinia YopE cytotoxin specifically targets the Rho pathway: a mechanism for disruption of actin microfilament structure.Mol. Microbiol. 2000; 36: 737-748Crossref PubMed Scopus (262) Google Scholar) and CUP1 (Arnoldo et al., 2008Arnoldo A. Curak J. Kittanakom S. Chevelev I. Lee V.T. Sahebol-Amri M. Koscik B. Ljuma L. Roy P.J. Bedalov A. et al.Identification of small molecule inhibitors of Pseudomonas aeruginosa Exoenzyme S using a yeast phenotypic screen.PLoS Genet. 2008; 4: e1000005Crossref PubMed Scopus (71) Google Scholar) promotors. In these cases, expression is controlled by the presence of methionine or copper in the media, respectively. Another option is the tightly controlled tetracycline-responsive tetO promotor (Belli et al., 1998Belli G. Gari E. Piedrafita L. Aldea M. Herrero E. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast.Nucleic Acids Res. 1998; 26: 942-947Crossref PubMed Scopus (216) Google Scholar, Skrzypek et al., 2003Skrzypek E. Myers-Morales T. Whiteheart S.W. Straley S.C. Application of a Saccharomyces cerevisiae model to study requirements for trafficking of Yersinia pestis YopM in eucaryotic cells.Infect. Immun. 2003; 71: 937-947Crossref PubMed Scopus (35) Google Scholar). This is not an endogenous yeast promotor, and modified yeast strains that encode a tetR repressor must be used to tightly control expression. The copy number of the genes encoding the effector proteins will also influence protein levels in yeast. It is easiest to either encode the effector protein on centromere-containing (copy number 1–3) or 2 micron (copy number 40–60) plasmids, although targeted homologous recombination can be used to introduce a single copy of a gene into the yeast genome. Studies with Shigella effector proteins suggest that the sensitivity and specificity of growth inhibition as an indicator of effector proteins is optimized when the proteins are expressed from low copy-number plasmids (Slagowski et al., 2008Slagowski N.L. Kramer R.W. Morrison M.F. LaBaer J. Lesser C.F. A functional genomic yeast screen to identify pathogenic bacterial proteins.PLoS Pathog. 2008; 4: e9Crossref PubMed Scopus (50) Google Scholar). However, it is possible that effector proteins from other pathogens will not be as well expressed in yeast, and in these cases it might prove fruitful to express the effector proteins from high copy-number plasmids. Another variable to consider when expressing effector proteins in yeast is the addition of an epitope tag. Evidence exists that fusion of effector proteins to GFP can influence growth inhibition due to their expression (Slagowski et al., 2008Slagowski N.L. Kramer R.W. Morrison M.F. LaBaer J. Lesser C.F. A functional genomic yeast screen to identify pathogenic bacterial proteins.PLoS Pathog. 2008; 4: e9Crossref PubMed Scopus (50) Google Scholar). In the majority of cases, fusion to GFP results in increased growth inhibition, presumably due to increased expression and/or stability of the effector proteins (March et al., 2003March J.C. Rao G. Bentley W.E. Biotechnological applications of green fluorescent protein.Appl. Microbiol. Biotechnol. 2003; 62: 303-315Crossref PubMed Scopus (116) Google Scholar). However, there are also examples of where fusion to GFP decreased toxicity of the effector proteins, presumably due to steric interference. If the location of the secretion signal of the effector protein is known, fusing to GFP to this domain is presumably less likely to interfere with the activity of the effector protein, since this domain is thought to be unstructured. There are numerous options available for monitoring yeast growth inhibition due to expression of an effector protein. One relatively simple assay for detecting qualitative differences in growth is to plate serial dilutions of saturated yeast cultures on inducing media (Lesser and Miller, 2001Lesser C.F. Miller S.I. Expression of microbial virulence proteins in Saccharomyces cerevisiae models mammalian infection.EMBO J. 2001; 20: 1840-1849Crossref PubMed Scopus (79) Google Scholar, Sisko et al., 2006Sisko J.L. Spaeth K. Kumar Y. Valdivia R.H. Multifunctional analysis of Chlamydia-specific genes in a yeast expression system.Mol. Microbiol. 2006; 60: 51-66Crossref PubMed Scopus (79) Google Scholar). Quantitative measurements of growth inhibition can be achieved by measuring the optical density of liquid cultures in conventional growth assays or in 96-well liquid growth assays (Slagowski et al., 2008Slagowski N.L. Kramer R.W. Morrison M.F. LaBaer J. Lesser C.F. A functional genomic yeast screen to identify pathogenic bacterial proteins.PLoS Pathog. 2008; 4: e9Crossref PubMed Scopus (50) Google Scholar). Similarly, growth can be monitored using dyes that monitor cellular respiration (Sisko et al., 2006Sisko J.L. Spaeth K. Kumar Y. Valdivia R.H. Multifunctional analysis of Chlamydia-specific genes in a yeast expression system.Mol. Microbiol. 2006; 60: 51-66Crossref PubMed Scopus (79) Google Scholar). In addition, growth on solid media can be quantified (Dudley et al., 2005Dudley A.M. Janse D.M. Tanay A. Shamir R. Church G.M. A global view of pleiotropy and phenotypically derived gene function in yeast.Mol. Syst. Biol. 2005; 1 (2005.0001)Crossref PubMed Scopus (211) Google Scholar). Growth is usually measured under standard laboratory conditions. However, effector proteins targeting cellular processes that are not normally rate-limiting for growth will not be detected under these conditions. To address this, "stressors" can be introduced into the growth media at doses that do not perturb growth of wild-type yeast (Sisko et al., 2006Sisko J.L. Spaeth K. Kumar Y. Valdivia R.H. Multifunctional analysis of Chlamydia-specific genes in a yeast expression system.Mol. Microbiol. 2006; 60: 51-66Crossref PubMed Scopus (79) Google Scholar, Slagowski et al., 2008Slagowski N.L. Kramer R.W. Morrison M.F. LaBaer J. Lesser C.F. A functional genomic yeast screen to identify pathogenic bacterial proteins.PLoS Pathog. 2008; 4: e9Crossref PubMed Scopus (50) Google Scholar). For example, expression of Shigella OspB only inhibits growth when caffeine is added to the media (Slagowski et al., 2008Slagowski N.L. Kramer R.W. Morrison M.F. LaBaer J. Lesser C.F. A functional genomic yeast screen to identify pathogenic bacterial proteins.PLoS Pathog. 2008; 4: e9Crossref PubMed Scopus (50) Google Scholar). In addition to identifying additional candidate effector proteins, conditional sensitivity to a particular stressor can provide clues as to the cellular pathway targeted by the effector protein. For example, sensitivity to high salt can be due to targeting of a variety of cellular processes including mitogen-activated protein kinase (MAPK) signaling pathways, while sensitivity to nocodazole suggests a perturbation in microtubules. There are several potential situations that might limit yeast as a model system to study specific effector proteins. For example, since the effector proteins are normally delivered as preformed toxins directly into host cells, proteins that require bacteria-specific modifications might not function in yeast. Similarly, the topology of effector proteins that are directly inserted into host cell membranes might not be maintained in yeast. To circumvent this latter issue, an option is to express soluble domains of effector proteins. This strategy proved fruitful in studying the Chlamydia trachomatis Inc proteins, a subset of Chlamydia effector proteins that are membrane associated (Sisko et al., 2006Sisko J.L. Spaeth K. Kumar Y. Valdivia R.H. Multifunctional analysis of Chlamydia-specific genes in a yeast expression system.Mol. Microbiol. 2006; 60: 51-66Crossref PubMed Scopus (79) Google Scholar). There is growing evidence to suggest that yeast growth inhibition is a sensitive and specific reporter of effector proteins. A survey of the behavior of effector proteins and bacterial-confined proteins encoded on the Shigella virulence plasmid revealed that almost half of twenty effector proteins inhibit yeast growth when expressed from a low copy-number plasmid. A molecular mechanism was already known for seven of these proteins, five of which target conserved cellular processes. Notably, expression of only these five effectors inhibited yeast growth. Expression of the other two, which interact with proteins not found in yeast, did not affect yeast growth. Expression of only 1 of 20 bacterial-confined proteins, a bacterial toxin, severely inhibited growth. Notably, coexpression of the bacterial antitoxin suppressed yeast toxicity (Slagowski et al., 2008Slagowski N.L. Kramer R.W. Morrison M.F. LaBaer J. Lesser C.F. A functional genomic yeast screen to identify pathogenic bacterial proteins.PLoS Pathog. 2008; 4: e9Crossref PubMed Scopus (50) Google Scholar). Other bacterial toxin-antitoxin systems have also been observed to behave similarly in yeast (Picardeau et al., 2003Picardeau M. Le Dantec C. Richard G.F. Saint Girons I. The spirochetal chpK-chromosomal toxin-antitoxin locus induces growth inhibition of yeast and mycobacteria.FEMS Microbiol. Lett. 2003; 229: 277-281Crossref PubMed Scopus (18) Google Scholar, Kristoffersen et al., 2000Kristoffersen P. Jensen G.B. Gerdes K. Piskur J. Bacterial toxin-antitoxin gene system as containment control in yeast cells.Appl. Environ. Microbiol. 2000; 66: 5524-5526Crossref PubMed Scopus (51) Google Scholar). Presumably, these toxins target cellular processes conserved among prokaryotes and yeast. Since some bacterial housekeeping proteins are involved in cellular processes conserved from yeast to prokaryotes, their expression also can interfere with yeast growth. For example, expression of Legionella sterol desaturase, a homolog of S. cerevisiae ERG25, an essential protein involved in ergosterol biosynthesis, presumably inhibits yeast growth by perturbing membrane synthesis (Campodonico et al., 2005Campodonico E.M. Chesnel L. Roy C.R. A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system.Mol. Microbiol. 2005; 56: 918-933Crossref PubMed Scopus (109) Google Scholar). However, several high-throughput studies suggest that yeast growth inhibition due to the expression of bacterial housekeeping proteins is rare. For example, only 2 of 371 essential Pseudomonas aeruginosa proteins severely inhibit yeast growth (Arnoldo et al., 2008Arnoldo A. Curak J. Kittanakom S. Chevelev I. Lee V.T. Sahebol-Amri M. Koscik B. Ljuma L. Roy P.J. Bedalov A. et al.Identification of small molecule inhibitors of Pseudomonas aeruginosa Exoenzyme S using a yeast phenotypic screen.PLoS Genet. 2008; 4: e1000005Crossref PubMed Scopus (71) Google Scholar). Similarly, a screen that covered ∼60% of the Legionella pneumophilia genome identified only six bacterial-confined proteins that inhibit yeast growth (Campodonico et al., 2005Campodonico E.M. Chesnel L. Roy C.R. A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system.Mol. Microbiol. 2005; 56: 918-933Crossref PubMed Scopus (109) Google Scholar). And, lastly, expression of only 3 of ∼1100 Francisella tularenesis proteins, two-thirds of the Francisella proteome, reproducibly but minimally, inhibit growth (Slagowski et al., 2008Slagowski N.L. Kramer R.W. Morrison M.F. LaBaer J. Lesser C.F. A functional genomic yeast screen to identify pathogenic bacterial proteins.PLoS Pathog. 2008; 4: e9Crossref PubMed Scopus (50) Google Scholar). In the case of the Pseudomonas and Legionella screens the proteins were expressed from a high copy-number plasmid while the Francisella proteins were expressed from a low copy-number plasmid. Thus, growth inhibition due to expression of bacterial-confined proteins appears to be rare, and when it does occur it appears that it is often to due to conserved targeting of cellular processes and not a nonspecific effect due to overexpression of a heterologous protein in yeast. Accumulating evidence suggests that subcellular localization patterns of effector proteins expressed de novo in yeast accurately reflect their localization when injected into host cells during the course of an infection. This includes localization to the plasma membrane, nucleus, and the actin cytoskeleton (Benabdillah et al., 2004Benabdillah R. Mota L.J. Lutzelschwab S. Demoinet E. Cornelis G.R. Identification of a nuclear targeting signal in YopM from Yersinia spp.Microb. Pathog. 2004; 36: 247-261Crossref PubMed Scopus (65) Google Scholar, Lesser and Miller, 2001Lesser C.F. Miller S.I. Expression of microbial virulence proteins in Saccharomyces cerevisiae models mammalian infection.EMBO J. 2001; 20: 1840-1849Crossref PubMed Scopus (79) Google Scholar, Sisko et al., 2006Sisko J.L. Spaeth K. Kumar Y. Valdivia R.H. Multifunctional analysis of Chlamydia-specific genes in a yeast expression system.Mol. Microbiol. 2006; 60: 51-66Crossref PubMed Scopus (79) Google Scholar, Skrzypek et al., 2003Skrzypek E. Myers-Morales T. Whiteheart S.W. Straley S.C. Application of a Saccharomyces cerevisiae model to study requirements for trafficking of Yersinia pestis YopM in eucaryotic cells.Infect. Immun. 2003; 71: 937-947Crossref PubMed Scopus (35) Google Scholar). This targeting presumably reflects conserved interactions with eukaryotic structures and/or proteins. However, we have observed that bacterial proteins that normally never contact host cells often localize to specific yeast cellular compartments, including the nucleus and endoplasmic reticulum, presumably due to fortuitous sequences encoded within the bacteria proteins (data not shown). Thus, localization to specific yeast subcellular compartments is not as sensitive or specific an indicator of effector proteins as growth inhibition. The simplest way to determine the localization of an effector protein in yeast is to fuse the protein to a fluorescent tag such as GFP. Alternatively, one can conduct indirect immunofluorescence studies on fixed cells. To determine whether an effector protein localizes to specific yeast subcellular compartments, cells expressing the protein can be stained with DNA-binding dyes like DAPI (nucleus), fluorescently labeled rhodamine (actin), Mitotracker (mitochondria), or antibody markers for particular compartments. In addition, yeast reporter strains that constitutively express a series of mRFP fusion proteins that localize to specific structures are available and can be used in colocalization studies (Table 2) (Huh et al., 2003Huh W.K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Global analysis of protein localization in budding yeast.Nature. 2003; 425: 686-691Crossref PubMed Scopus (3118) Google Scholar).Table 2mRFP Reporter Strains for Colocalization StudiesStrainLocalizationmRFP-Sac6actinmRFP-Cop1early GolgimRFP-Snf7endosomemRFP-Sec13ER to Golgi vesiclemRFP-An
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