A Systematic Proteomic Analysis of Listeria monocytogenes House-keeping Protein Secretion Systems
2014; Elsevier BV; Volume: 13; Issue: 11 Linguagem: Inglês
10.1074/mcp.m114.041327
ISSN1535-9484
AutoresSven Halbedel, Swantje Reiß, Birgit Hahn, Dirk Albrecht, Gopala Krishna Mannala, Trinad Chakraborty, Torsten Hain, Susanne Engelmann, Antje Flieger,
Tópico(s)Essential Oils and Antimicrobial Activity
ResumoListeria monocytogenes is a firmicute bacterium causing serious infections in humans upon consumption of contaminated food. Most of its virulence factors are secretory proteins either released to the medium or attached to the bacterial surface. L. monocytogenes encodes at least six different protein secretion pathways. Although great efforts have been made in the past to predict secretory proteins and their secretion routes using bioinformatics, experimental evidence is lacking for most secretion systems. Therefore, we constructed mutants in the main housekeeping protein secretion systems, which are the Sec-dependent transport, the YidC membrane insertases SpoIIIJ and YqjG, as well as the twin-arginine pathway, and analyzed their secretion and virulence defects. Our results demonstrate that Sec-dependent secretion and membrane insertion of proteins via YidC proteins are essential for viability of L. monocytogenes. Depletion of SecA or YidC activity severely affected protein secretion, whereas loss of the Tat-pathway was without any effect on secretion, viability, and virulence. Two-dimensional gel electrophoresis combined with protein identification by mass spectrometry revealed that secretion of many virulence factors and of enzymes synthesizing and degrading the cell wall depends on the SecA route. This finding was confirmed by SecA inhibition experiments using sodium azide. Analysis of secretion of substrates typically dependent on the accessory SecA2 ATPase in wild type and azide resistant mutants of L. monocytogenes revealed for the first time that SecA2-dependent protein secretion also requires the ATPase activity of the house-keeping SecA protein. Listeria monocytogenes is a firmicute bacterium causing serious infections in humans upon consumption of contaminated food. Most of its virulence factors are secretory proteins either released to the medium or attached to the bacterial surface. L. monocytogenes encodes at least six different protein secretion pathways. Although great efforts have been made in the past to predict secretory proteins and their secretion routes using bioinformatics, experimental evidence is lacking for most secretion systems. Therefore, we constructed mutants in the main housekeeping protein secretion systems, which are the Sec-dependent transport, the YidC membrane insertases SpoIIIJ and YqjG, as well as the twin-arginine pathway, and analyzed their secretion and virulence defects. Our results demonstrate that Sec-dependent secretion and membrane insertion of proteins via YidC proteins are essential for viability of L. monocytogenes. Depletion of SecA or YidC activity severely affected protein secretion, whereas loss of the Tat-pathway was without any effect on secretion, viability, and virulence. Two-dimensional gel electrophoresis combined with protein identification by mass spectrometry revealed that secretion of many virulence factors and of enzymes synthesizing and degrading the cell wall depends on the SecA route. This finding was confirmed by SecA inhibition experiments using sodium azide. Analysis of secretion of substrates typically dependent on the accessory SecA2 ATPase in wild type and azide resistant mutants of L. monocytogenes revealed for the first time that SecA2-dependent protein secretion also requires the ATPase activity of the house-keeping SecA protein. Listeria monocytogenes is a facultative pathogenic firmicute bacterium that is found frequently in nature where it lives as a saprophyte in the soil and on decaying plant material. Because of its ubiquitousness, it frequently enters the food chain giving raise to listeriosis outbreaks that often reveal a high rate of fatal cases in humans (1Freitag N.E. Port G.C. 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Simultaneous deficiency of both MurA and p60 proteins generates a rough phenotype in Listeria monocytogenes.J. Bacteriol. 2005; 187: 8385-8394Crossref PubMed Scopus (51) Google Scholar). SecA2 proteins are found in many Gram-positive bacteria in addition to their housekeeping SecA homologs. L. monocytogenes SecA2 has all key domains characteristically found in SecA proteins and shares 44% identity (62% similarity) with listerial SecA (25Lenz L.L. Portnoy D.A. Identification of a second Listeria secA gene associated with protein secretion and the rough phenotype.Mol. Microbiol. 2002; 45: 1043-1056Crossref PubMed Scopus (107) Google Scholar). SecA2 proteins typically serve the secretion of a limited number of proteins that often are linked to virulence (26Bensing B.A. Seepersaud R. Yen Y.T. Sullam P.M. Selective transport by SecA2: an expanding family of customized motor proteins.Biochim. Biophys. Acta. 2013; PubMed Google Scholar). However, presently it is not clear why exactly these substrates require SecA2 for their translocation (26Bensing B.A. Seepersaud R. Yen Y.T. Sullam P.M. Selective transport by SecA2: an expanding family of customized motor proteins.Biochim. Biophys. Acta. 2013; PubMed Google Scholar).Table IProtein secretion systems encoded by the L. monocytogenes EGD-e genome. Classification of secretion systems is according to a genome survey published by Desvaux and Hébraud (8Desvaux M. Hebraud M. The protein secretion systems in Listeria: inside out bacterial virulence.FEMS Microbiol. Rev. 2006; 30: 774-805Crossref PubMed Scopus (88) Google Scholar)Transport systemComponentsGenesSubstratesReferencesSec system• Pore componentsSecYlmo2612Sec substratesSecElmo0245SecGlmo2451• Associated proteinsSecDFlmo1527(40Burg-Golani T. Pozniak Y. Rabinovich L. Sigal N. Nir Paz R. Herskovits A.A. Membrane chaperone SecDF plays a role in the secretion of Listeria monocytogenes major virulence factors.J. Bacteriol. 2013; 195: 5262-5272Crossref PubMed Scopus (26) Google Scholar)YajClmo1529• General ATPaseSecAlmo2510(43Monk I.R. Gahan C.G. Hill C. Tools for functional postgenomic analysis of Listeria monocytogenes.Appl. Environ. Microbiol. 2008; 74: 3921-3934Crossref PubMed Scopus (164) Google Scholar)• Accessory ATPaseSecA2lmo0583Specific Sec substrates(20Lenz L.L. Mohammadi S. Geissler A. Portnoy D.A. SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12432-12437Crossref PubMed Scopus (235) Google Scholar, 22Renier S. Chambon C. Viala D. Chagnot C. Hebraud M. Desvaux M. Exoproteomic analysis of the SecA2-dependent secretion in Listeria monocytogenes EGD-e.J. 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Food Microbiol. 2011; 148: 177-183PubMed Google Scholar)FliOlmo0682FliPQRlmo0676-0678• ATPase complexFliHlmo0715FliIlmo0716(77Bigot A. Pagniez H. Botton E. Frehel C. Dubail I. Jacquet C. Charbit A. Raynaud C. Role of FliF and FliI of Listeria monocytogenes in flagellar assembly and pathogenicity.Infect. Immun. 2005; 73: 5530-5539Crossref PubMed Scopus (55) Google Scholar)Fimbrial exporter• ATPaseComGAlmo1347Prepilins ComGC-GG(36Rabinovich L. Sigal N. Borovok I. Nir-Paz R. Herskovits A.A. Prophage excision activates Listeria competence genes that promote phagosomal escape and virulence.Cell. 2012; 150: 792-802Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar)• pore componentComGBlmo1346• prepilin peptidaseComClmo1550Holins• TcdE typeLmo0128lmo0128Phage endolysins• ϕA118 typeLmo2279lmo2279(78Vukov N. Moll I. Blasi U. Scherer S. Loessner M.J. Functional regulation of the Listeria monocytogenes bacteriophage A118 holin by an intragenic inhibitor lacking the first transmembrane domain.Mol. Microbiol. 2003; 48: 173-186Crossref PubMed Scopus (19) Google Scholar)WXG100 system• pore componentsLmo0057-60lmo0057-60WXG100 proteins(38Way S.S. Wilson C.B. The Mycobacterium tuberculosis ESAT-6 homolog in Listeria monocytogenes is dispensable for growth in vitroin vivo.Infect. Immun. 2005; 73: 6151-6153Crossref PubMed Scopus (28) Google Scholar)• ATPaseYukABlmo0061a There is no lmo number available for the 4.5S RNA gene, which is located between the lmo2710 and lmo2711 open reading frames. Open table in a new tab For membrane insertion, transmembrane segments of integral membrane proteins are laterally released from the SecYEG pore in a process involving the YidC membrane insertases (27Wang P. Dalbey R.E. Inserting membrane proteins: the YidC/Oxa1/Alb3 machinery in bacteria, mitochondria, and chloroplasts.Biochim. Biophys. Acta. 2011; 1808: 866-875Crossref PubMed Scopus (76) Google Scholar). For some substrates, however, YidC proteins can even act as Sec-independent membrane insertases (28Serek J. Bauer-Manz G. Struhalla G. van den Berg L. Kiefer D. Dalbey R. Kuhn A. Escherichia coli YidC is a membrane insertase for Sec-independent proteins.EMBO J. 2004; 23: 294-301Crossref PubMed Scopus (170) Google Scholar). Firmicute bacteria such as Bacillus subtilis or L. monocytogenes contain two YidC homologs, SpoIIIJ and YqjG (27Wang P. Dalbey R.E. Inserting membrane proteins: the YidC/Oxa1/Alb3 machinery in bacteria, mitochondria, and chloroplasts.Biochim. Biophys. Acta. 2011; 1808: 866-875Crossref PubMed Scopus (76) Google Scholar, 29Dalbey R.E. Kuhn A. Zhu L. Kiefer D. The membrane insertase YidC.Biochim. Biophys. Acta. 2014; 1843: 1489-1496Crossref PubMed Scopus (68) Google Scholar). Both proteins have distinct as well as overlapping substrate spectra, at least in B. subtilis (30Murakami T. Haga K. 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YlxM is a newly identified accessory protein that influences the function of signal recognition particle pathway components in Streptococcus mutans.J. Bacteriol. 2014; 196: 2043-2052Crossref PubMed Scopus (12) Google Scholar) and the SRP 4.5S RNA. The SRP guides the ribosome-nascent chain complexes to the SecA-SecYEG translocon via the transmembrane SRP receptor FtsY (33Driessen A.J. Nouwen N. Protein translocation across the bacterial cytoplasmic membrane.Annu. Rev. Biochem. 2008; 77: 643-667Crossref PubMed Scopus (471) Google Scholar, 34Grudnik P. Bange G. Sinning I. Protein targeting by the signal recognition particle.Biol. Chem. 2009; 390: 775-782Crossref PubMed Scopus (121) Google Scholar). Although Sec substrates are translocated in the unfolded state, substrates of the twin-arginine transport (Tat) are secreted as folded proteins that even can be complexed with cofactors (35Goosens V.J. Monteferrante C.G. van Dijl J.M. The Tat system of Gram-positive bacteria.Biochim. Biophys. Acta. 2014; 1843: 1698-1706Crossref PubMed Scopus (62) Google Scholar). The L. monocytogenes Tat system is composed of the polytopic transmembrane protein TatC required for substrate recognition and the small membrane spanning TatA protein (8Desvaux M. Hebraud M. The protein secretion systems in Listeria: inside out bacterial virulence.FEMS Microbiol. Rev. 2006; 30: 774-805Crossref PubMed Scopus (88) Google Scholar), forming the pore through which protein transport is driven by proton motive force (35Goosens V.J. Monteferrante C.G. van Dijl J.M. The Tat system of Gram-positive bacteria.Biochim. Biophys. Acta. 2014; 1843: 1698-1706Crossref PubMed Scopus (62) Google Scholar). Next to these systems, four additional, but more specialized protein secretion pathways are present in the L. monocytogenes genome: The flagellum exporter for translocation of flagellar proteins, the fimbriae exporter for secretion of pseudo-pilus subunits, which contribute to phagosomal escape (36Rabinovich L. Sigal N. Borovok I. Nir-Paz R. Herskovits A.A. Prophage excision activates Listeria competence genes that promote phagosomal escape and virulence.Cell. 2012; 150: 792-802Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), two holins for secretion of phage endolysins, as well as the WXG100 system supposed to mediate secretion of short, ∼100 amino acids long substrate proteins with the typical WXG motif (Table I) (8Desvaux M. Hebraud M. The protein secretion systems in Listeria: inside out bacterial virulence.FEMS Microbiol. Rev. 2006; 30: 774-805Crossref PubMed Scopus (88) Google Scholar). signal recognition particle brain heart infusion polyvinylidene fluoride signal-to-noise. signal recognition particle brain heart infusion polyvinylidene fluoride signal-to-noise. The contribution of most L. monocytogenes secretion systems to general protein secretion has not been studied experimentally. Whereas SecA2-dependent protein secretion significantly contributes to L. monocytogenes virulence (20Lenz L.L. Mohammadi S. Geissler A. Portnoy D.A. SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12432-12437Crossref PubMed Scopus (235) Google Scholar, 37Halbedel S. Hahn B. Daniel R.A. Flieger A. DivIVA affects secretion of virulence-related autolysins in Listeria monocytogenes.Mol. Microbiol. 2012; 83: 821-839Crossref PubMed Scopus (44) Google Scholar), the Tat as well as the WXG100 system seemed to be dispensable for listerial pathogenicity (38Way S.S. Wilson C.B. The Mycobacterium tuberculosis ESAT-6 homolog in Listeria monocytogenes is dispensable for growth in vitroin vivo.Infect. Immun. 2005; 73: 6151-6153Crossref PubMed Scopus (28) Google Scholar, 39Machado H. Lourenco A. Carvalho F. Cabanes D. Kallipolitis B.H. Brito L. The Tat pathway is prevalent in Listeria monocytogenes lineage II and is not required for infection and spread in host cells.J. Mol. Microbiol. Biotechnol. 2013; 23: 209-218Crossref PubMed Scopus (2) Google Scholar). However, a recent investigation of L. monocytogenes SecDF showed that the Sec system contributes to virulence factor secretion (40Burg-Golani T. Pozniak Y. Rabinovich L. Sigal N. Nir Paz R. Herskovits A.A. Membrane chaperone SecDF plays a role in the secretion of Listeria monocytogenes major virulence factors.J. Bacteriol. 2013; 195: 5262-5272Crossref PubMed Scopus (26) Google Scholar). According to in silico predictions using the presence of signal peptides, trans-membrane domains, and surface retention signals as typical characteristics of secreted proteins, roughly one third of all 2853 L. monocytogenes proteins (41Glaser P. Frangeul L. Buchrieser C. Rusniok C. Amend A. Baquero F. Berche P. Bloecker H. Brandt P. Chakraborty T. Charbit A. Chetouani F. Couve E. de Daruvar A. Dehoux P. Domann E. Dominguez-Bernal G. Duchaud E. Durant L. Dussurget O. Entian K.D. Fsihi H. Garcia-del Portillo F. Garrido P. Gautier L. Goebel W. Gomez-Lopez N. Hain T. Hauf J. Jackson D. Jones L.M. Kaerst U. Kreft J. Kuhn M. Kunst F. Kurapkat G. Madueno E. Maitournam A. Vicente J.M. Ng E. Nedjari H. Nordsiek G. Novella S. de Pablos B. Perez-Diaz J.C. Purcell R. Remmel B. Rose M. Schlueter T. Simoes N. Tierrez A. Vazquez-Boland J.A. Voss H. Wehland J. Cossart P. Comparative genomics of Listeria species.Science. 2001; 294: 849-852Crossref PubMed Scopus (34) Google Scholar) is either inserted into the cytoplasmic membrane (686), or translocated across the membrane to be released into the extracellular space (80Osborne A.R. Clemons Jr., W.M. Rapoport T.A. A large conformational change of the translocation ATPase SecA.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10937-10942Crossref PubMed Scopus (125) Google Scholar), or to be presented on the surface of the cell (143) (17Renier S. Micheau P. Talon R. Hebraud M. Desvaux M. Subcellular localization of extracytoplasmic proteins in monoderm bacteria: rational secretomics-based strategy for genomic and proteomic analyses.PLoS One. 2012; 7: e42982Crossref PubMed Scopus (32) Google Scholar). This approach has also facilitated an assignment of all secreted proteins to the different protein secretion mechanisms (17Renier S. Micheau P. Talon R. Hebraud M. Desvaux M. Subcellular localization of extracytoplasmic proteins in monoderm bacteria: rational secretomics-based strategy for genomic and proteomic analyses.PLoS One. 2012; 7: e42982Crossref PubMed Scopus (32) Google Scholar). However, the only secretion system for which these predictions have been validated by experimental data is the SecA2-dependent translocation route. Here, we studied the different protein secretion systems of L. monocytogenes. Mutant strains lacking or conditionally expressing the SecA ATPase, the YidC membrane insertases SpoIIIJ and YqjG, as well as the Tat system components TatAC were characterized with regard to in vitro and in vivo growth and possible secretion defects. Mutant strains devoid of the fliI (flagellar export), yukA (WXG100), and spoIIIAH genes (encoding a ring-forming membrane embedded protein possibly involved in protein secretion, see below) were also analyzed. Although the three latter mutants did not show any phenotypic differences compared with wild type, SecA and the YidC proteins were clearly required for viability and contribute to bulk protein secretion under standard laboratory conditions. Proteomic analyses of secretion patterns and construction of epitope tagged substrate proteins for analysis of their secretion by Western blotting enabled us to assign secretion substrates of the SecA-, the YidC-, and the TatAC-dependent protein secretion routes. Table II lists all bacterial strains used in this study. Cells of L. monocytogenes were routinely cultivated on brain heart infusion (BHI) agar or in BHI broth. Where necessary, antibiotics and supplements were added at the following concentrations: erythromycin (5 μg/ml), kanamycin (50 μg/ml), X-Gal (100 μg/ml), and IPTG (0.005–1 mm). For all cloning procedures Escherichia coli TOP10 was used as the standard plasmid host (42Sambrook J. Fritsch E.F. Maniatis T. Molecular cloning : a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar).Table IIPlasmids and strains used in this studyNameRelevant characteristicsSourceaThe arrow (→) stands for a transformation event and the double arrow (⇆) indicates gene deletions obtained by chromosomal insertion and subsequent excision of pMAD plasmid derivatives (see experimental procedures for details)./referencePlasmidspAUL-Aerm lacZα(79Chakraborty T. Leimeister-Wächter M. Domann E. Hartl M. Goebel W. Nichterlein T. Notermans S. Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene.J. Bacteriol,. 1992; 174: 568-574Crossref PubMed Scopus (316) Google Scholar)pET19b-secAbla lacI his6-secA(80Osborne A.R. Clemons Jr., W.M. Rapoport T.A. A large conformational change of the translocation ATPase SecA.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10937-10942Crossref PubMed Scopus (125) Google Scholar)pIMK2Phelp neo(43Monk I.R. Gahan C.G. Hill C. Tools for functional postgenomic analysis of Listeria monocytogenes.Appl. Environ. Microbiol. 2008; 74: 3921-3934Crossref PubMed Scopus (164) Google Scholar)pIMK3Phelp-lacO lacI neo(43Monk I.R. Gahan C.G. Hill C. Tools for functional postgenomic analysis of Listeria monocytogenes.Appl. Environ. Microbiol. 2008; 74
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