title: Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation.
abstract: Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors.
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Rodríguez-Escudero
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), Madrid, Spain.
authors.forename: Víctor J
authors.initials: VJ
authors.lastname: Cid
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Molina
authors.affiliation: Mikrobiologisches Institut-Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Schulze-Luehrmann
authors.affiliation: Mikrobiologisches Institut-Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Lührmann
authors.affiliation: Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRyCIS), Madrid, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Rodríguez-Escudero
mesh_terms: D017209:Apoptosis; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D016997:Coxiella burnetii / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D018384:Oxidative Stress; D066329:Protein Aggregates; D011778:Q Fever / Q000235:genetics / Q000378:metabolism / Q000382:microbiology*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D001426:Bacterial Proteins; D066329:Protein Aggregates
references.citation: Cell Rep. 2012 Oct 25;2(4):738-47
references.pmid: 23022486
references.citation: Adv Exp Med Biol. 2012;984:249-71
references.pmid: 22711636
references.citation: Nat Rev Microbiol. 2013 Aug;11(8):561-73
references.pmid: 23797173
references.citation: J Bacteriol. 2013 Sep;195(17):3914-24
references.pmid: 23813730
references.citation: Infect Immun. 2014 Sep;82(9):3740-52
references.pmid: 24958706
references.citation: Cell Microbiol. 2013 Apr;15(4):675-87
references.pmid: 23126667
references.citation: J Mol Biol. 2015 Apr 10;427(7):1564-74
references.pmid: 25681695
references.citation: J Vis Exp. 2015;(100):e52903
references.pmid: 26168006
references.citation: Cell Microbiol. 2015 Nov;17(11):1640-52
references.pmid: 25996657
references.citation: J Biol Chem. 2000 Jan 14;275(2):1511-9
references.pmid: 10625705
references.citation: Lancet Infect Dis. 2003 Nov;3(11):709-21
references.pmid: 14592601
references.citation: Eukaryot Cell. 2004 Jun;3(3):695-704
references.pmid: 15189990
references.citation: Cell Microbiol. 2004 Aug;6(8):743-51
references.pmid: 15236641
references.citation: Eukaryot Cell. 2004 Aug;3(4):827-34
references.pmid: 15302815
references.citation: Acta Virol. 1991 Nov;35(6):503-10
references.pmid: 1687632
references.citation: Yeast. 1993 Jul;9(7):715-22
references.pmid: 8368005
references.citation: J Cell Biol. 1995 Mar;128(5):779-92
references.pmid: 7533169
references.citation: J Biol Chem. 1997 Feb 14;272(7):4444-50
references.pmid: 9020168
references.citation: Mol Microbiol. 1997 Feb;23(3):431-44
references.pmid: 9044278
references.citation: J Cell Biol. 1997 Nov 3;139(3):729-34
references.pmid: 9348289
references.citation: J Biol Chem. 1997 Nov 7;272(45):28557-62
references.pmid: 9353319
references.citation: Cell Microbiol. 2005 Oct;7(10):1432-46
references.pmid: 16153243
references.citation: Mol Microbiol. 2006 Apr;60(1):51-66
references.pmid: 16556220
references.citation: Microbiology. 2006 Nov;152(Pt 11):3437-52
references.pmid: 17074912
references.citation: Cell Microbiol. 2007 Apr;9(4):829-40
references.pmid: 17381428
references.citation: Science. 2008 Jun 20;320(5883):1651-4
references.pmid: 18566289
references.citation: Cell Host Microbe. 2008 Jul 17;4(1):8-15
references.pmid: 18621006
references.citation: Nature. 2008 Aug 28;454(7208):1088-95
references.pmid: 18756251
references.citation: Nat Methods. 2008 Dec;5(12):1031-7
references.pmid: 18978785
references.citation: Curr Opin Microbiol. 2009 Feb;12(1):74-80
references.pmid: 19144560
references.citation: J Biol Chem. 2009 May 15;284(20):13373-83
references.pmid: 19307184
references.citation: J Bacteriol. 2009 Jul;191(13):4232-42
references.pmid: 19411324
references.citation: Cell Death Differ. 2010 May;17(5):737-45
references.pmid: 19911009
references.citation: Annu Rev Cell Dev Biol. 2010;26:261-83
references.pmid: 20929312
references.citation: Proc Natl Acad Sci U S A. 2010 Nov 2;107(44):18997-9001
references.pmid: 20944063
references.citation: Infect Immun. 2011 Jan;79(1):402-13
references.pmid: 20937765
references.citation: Nature. 2011 Apr 28;472(7344):476-80
references.pmid: 21525932
references.citation: Mol Microbiol. 2011 Jun;80(5):1220-40
references.pmid: 21435037
references.citation: PLoS Pathog. 2011 May;7(5):e1002056
references.pmid: 21637816
references.citation: EMBO J. 2011 Jul 20;30(14):2779-92
references.pmid: 21673659
references.citation: MBio. 2011;2(4):e00175-11
references.pmid: 21862628
references.citation: PLoS One. 2013;8(1):e54566
references.pmid: 23349930