Structural and Functional Characterization of Three DsbA Paralogues from Salmonella enterica Serovar Typhimurium
2010; Elsevier BV; Volume: 285; Issue: 24 Linguagem: Inglês
10.1074/jbc.m110.101360
ISSN1083-351X
AutoresBegoña Heras, Makrina Totsika, Russell Jarrott, Stephen R. Shouldice, Gregor Gunčar, Maud E. S. Achard, Timothy J. Wells, M. Pilar Argente, Alastair G. McEwan, Mark A. Schembri,
Tópico(s)Salmonella and Campylobacter epidemiology
ResumoIn prototypic Escherichia coli K-12 the introduction of disulfide bonds into folding proteins is mediated by the Dsb family of enzymes, primarily through the actions of the highly oxidizing protein EcDsbA. Homologues of the Dsb catalysts are found in most bacteria. Interestingly, pathogens have developed distinct Dsb machineries that play a pivotal role in the biogenesis of virulence factors, hence contributing to their pathogenicity. Salmonella enterica serovar (sv.) Typhimurium encodes an extended number of sulfhydryl oxidases, namely SeDsbA, SeDsbL, and SeSrgA. Here we report a comprehensive analysis of the sv. Typhimurium thiol oxidative system through the structural and functional characterization of the three Salmonella DsbA paralogues. The three proteins share low sequence identity, which results in several unique three-dimensional characteristics, principally in areas involved in substrate binding and disulfide catalysis. Furthermore, the Salmonella DsbA-like proteins also have different redox properties. Whereas functional characterization revealed some degree of redundancy, the properties of SeDsbA, SeDsbL, and SeSrgA and their expression pattern in sv. Typhimurium indicate a diverse role for these enzymes in virulence. In prototypic Escherichia coli K-12 the introduction of disulfide bonds into folding proteins is mediated by the Dsb family of enzymes, primarily through the actions of the highly oxidizing protein EcDsbA. Homologues of the Dsb catalysts are found in most bacteria. Interestingly, pathogens have developed distinct Dsb machineries that play a pivotal role in the biogenesis of virulence factors, hence contributing to their pathogenicity. Salmonella enterica serovar (sv.) Typhimurium encodes an extended number of sulfhydryl oxidases, namely SeDsbA, SeDsbL, and SeSrgA. Here we report a comprehensive analysis of the sv. Typhimurium thiol oxidative system through the structural and functional characterization of the three Salmonella DsbA paralogues. The three proteins share low sequence identity, which results in several unique three-dimensional characteristics, principally in areas involved in substrate binding and disulfide catalysis. Furthermore, the Salmonella DsbA-like proteins also have different redox properties. Whereas functional characterization revealed some degree of redundancy, the properties of SeDsbA, SeDsbL, and SeSrgA and their expression pattern in sv. Typhimurium indicate a diverse role for these enzymes in virulence. IntroductionThioredoxin (TRX) 2The abbreviations used are: TRXthioredoxinAssTarylsulfate sulfotransferaseDsbdisulfide bondDTTdithiothreitolIPTGisopropyl β-d-1-thiogalactopyranosideLICligation-independent cloningMBPmaltose-binding proteinPEGpolyethylene glycolSADsingle-wavelength anomalous dispersionSPI2Salmonella Pathogenicity Island 2SPI1Salmonella Pathogenicity Island 1SeMetselenomethionineT3SSType III secretion systemsTEVtobacco etch virusr.m.s.d.root mean square deviationFITCfluorescein isothiocyanateMES4-morpholineethanesulfonic acidsv.serovar. -like oxidoreductases play a major role in controlling the redox environment of the cell. These enzymes catalyze thiol-disulfide oxidoreductase reactions that are important in enzyme catalysis and in the maintenance of the correct thiol redox state in proteins. Dsb (disulfide bond) proteins are a specific of TRX-like proteins that are essential for the oxidative folding of secreted proteins in many Gram-negative bacteria (1Kadokura H. Katzen F. Beckwith J. Annu. Rev. Biochem. 2003; 72: 111-135Crossref PubMed Scopus (444) Google Scholar, 2Masip L. Pan J.L. Haldar S. Penner-Hahn J.E. DeLisa M.P. Georgiou G. Bardwell J.C. Collet J.F. Science. 2004; 303: 1185-1189Crossref PubMed Scopus (69) Google Scholar, 3Inaba K. J. Biochem. 2009; 146: 591-597Crossref PubMed Scopus (65) Google Scholar). In Escherichia coli K-12 five different Dsb proteins catalyze the correct introduction of disulfide bonds in substrate proteins (4Bardwell J.C. Lee J.O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 1038-1042Crossref PubMed Scopus (357) Google Scholar). DsbA and DsbB (5Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 6Akiyama Y. Kamitani S. Kusukawa N. Ito K. J. Biol. Chem. 1992; 267: 22440-22445Abstract Full Text PDF PubMed Google Scholar) form the oxidase pathway that introduces disulfide bonds into target proteins, whereas DsbC, DsbG and DsbD (7Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) comprise the isomerase pathway which proofreads and corrects incorrect disulfide bonds in proteins with multiple cysteines. Like all TRX-related proteins, Dsb enzymes contain a conserved CXXC catalytic motif and a X-cisProline loop in their active site. The identity of the XX dipeptide located between the cysteines as well as the residue preceding the cisProline residue determines the properties and function of these enzymes; variation in these residues accounts for the wide range of redox activities described above (8Ren G. Stephan D. Xu Z. Zheng Y. Tang D. Harrison R.S. Kurz M. Jarrott R. Shouldice S.R. Hiniker A. Martin J.L. Heras B. Bardwell J.C. J. Biol. Chem. 2009; 284: 10150-10159Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 9Quan S. Schneider I. Pan J. Von Hacht A. Bardwell J.C. J. Biol. Chem. 2007; 282: 28823-28833Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 10Mössner E. Huber-Wunderlich M. Rietsch A. Beckwith J. Glockshuber R. Aslund F. J. Biol. Chem. 1999; 274: 25254-25259Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 11Huber-Wunderlich M. Glockshuber R. Fold. Des. 1998; 3: 161-171Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar).Until recently it was considered that the paradigm redox system present in E. coli K-12 was conserved in all bacteria. However, new studies on Dsb proteins in other bacteria show that these systems can vary considerably between different genera and even strains of the same species (12Dutton R.J. Boyd D. Berkmen M. Beckwith J. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 11933-11938Crossref PubMed Scopus (191) Google Scholar, 13Heras B. Shouldice S.R. Totsika M. Scanlon M.J. Schembri M.A. Martin J.L. Nat. Rev. Microbiol. 2009; 7: 215-225Crossref PubMed Scopus (219) Google Scholar). This is observed for many Gram-negative pathogenic bacteria, some of which encode multiple Dsb proteins (14Grimshaw J.P. Stirnimann C.U. Brozzo M.S. Malojcic G. Grütter M.G. Capitani G. Glockshuber R. J. Mol. Biol. 2008; 380: 667-680Crossref PubMed Scopus (64) Google Scholar, 15Lafaye C. Iwema T. Carpentier P. Jullian-Binard C. Kroll J.S. Collet J.F. Serre L. J. Mol. Biol. 2009; 392: 952-966Crossref PubMed Scopus (43) Google Scholar). Significantly, there is growing evidence that a range of virulence-related functions depend on the strict redox control carried out by Dsb enzymes. Thus, these catalysts are required for the correct folding (and therefore function) of many secreted or surface exposed virulence determinants. Examples include, the flagellar P-ring motor protein FlgI, the secretin component of Type III secretion systems (T3SS) and a range of toxins and secreted enzymes (13Heras B. Shouldice S.R. Totsika M. Scanlon M.J. Schembri M.A. Martin J.L. Nat. Rev. Microbiol. 2009; 7: 215-225Crossref PubMed Scopus (219) Google Scholar, 16Sinha S. Langford P.R. Kroll J.S. Microbiology. 2004; 150: 2993-3000Crossref PubMed Scopus (39) Google Scholar).The Gram-negative bacterium Salmonella enterica serovar (sv.) Typhimurium is an important causative agent of acute human gastroenteritis and can cause life-threatening bacteremia in immuno-compromised individuals (17Kankwatira A.M. Mwafulirwa G.A. Gordon M.A. Trop. Doct. 2004; 34: 198-200Crossref PubMed Scopus (41) Google Scholar). It is an intracellular pathogen that resides in macrophages. In addition to the oxidase and isomerase systems described above for E. coli K-12, sv. Typhimurium also has a DsbL/DsbI pair, similar to the uropathogenic E. coli (UPEC) CFT073 DsbL/DsbI system (14Grimshaw J.P. Stirnimann C.U. Brozzo M.S. Malojcic G. Grütter M.G. Capitani G. Glockshuber R. J. Mol. Biol. 2008; 380: 667-680Crossref PubMed Scopus (64) Google Scholar, 18Totsika M. Heras B. Wurpel D.J. Schembri M.A. J. Bacteriol. 2009; 191: 3901-3908Crossref PubMed Scopus (60) Google Scholar), and a virulence plasmid-encoded DsbA-like protein, called SrgA (19Bouwman C.W. Kohli M. Killoran A. Touchie G.A. Kadner R.J. Martin N.L. J. Bacteriol. 2003; 185: 991-1000Crossref PubMed Scopus (68) Google Scholar). The sv. Typhimurium DsbA paralogues, termed SeDsbA, SeDsbL, and SeSrgA, share low sequence identity with each other (between 18 and 34%). They each contain different residues in the CXXC redox active site as well as in the cisProline. These differences suggest that the three DsbA paralogues have different functions and most likely act on different target protein(s). In support of this notion, characterization of SeDsbA and SeSrgA showed that these two disulfide oxidoreductases have specificity for different substrates (20Turcot I. Ponnampalam T.V. Bouwman C.W. Martin N.L. Can. J. Microbiol. 2001; 47: 711-721Crossref PubMed Google Scholar). SeSrgA is required for the biogenesis of the major structural subunit of the plasmid-encoded fimbriae, PefA (19Bouwman C.W. Kohli M. Killoran A. Touchie G.A. Kadner R.J. Martin N.L. J. Bacteriol. 2003; 185: 991-1000Crossref PubMed Scopus (68) Google Scholar). Together with SeDsbA it also catalyzes the folding of the outer membrane secretin SpiA, a component of the T3SS encoded in Salmonella Pathogenicity Island 2 (SPI2) (21Miki T. Okada N. Danbara H. J. Biol. Chem. 2004; 279: 34631-34642Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). SeDsbA is required for the folding of FlgI (20Turcot I. Ponnampalam T.V. Bouwman C.W. Martin N.L. Can. J. Microbiol. 2001; 47: 711-721Crossref PubMed Google Scholar) and most likely many other periplasmic/secreted proteins. The change in the periplasmic redox state upon disruption of the dsbA gene also affects the expression of the SPI1 T3SS (22Lin D. Rao C.V. Slauch J.M. J. Bacteriol. 2008; 190: 87-97Crossref PubMed Scopus (79) Google Scholar). Although no substrate is known for SeDsbL, this protein shares 93% sequence identity with its E. coli CFT073 orthologue, and could most likely fold the periplasmic enzyme arylsulfate sulfotransferase (AssT) (14Grimshaw J.P. Stirnimann C.U. Brozzo M.S. Malojcic G. Grütter M.G. Capitani G. Glockshuber R. J. Mol. Biol. 2008; 380: 667-680Crossref PubMed Scopus (64) Google Scholar). Moreover, as in UPEC CFT073, the sv. Typhimurium assT gene is encoded immediately upstream of dsbL and dsbI. Despite these differences in substrate specificity, there is indication that some overlap also exists between the activities of SeDsbA, SeDsbL, and SeSrgA (21Miki T. Okada N. Danbara H. J. Biol. Chem. 2004; 279: 34631-34642Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar).The specificity of disulfide catalysis in Gram-negative pathogens with an extended collection of Dsb proteins remains mostly uncharacterized. To further investigate oxidative folding processes in these organisms we have focused on the three sv. Typhimurium DsbA paralogues. Here, we perform a structural, biochemical and functional characterization of SeDsbA, SeDsbL, and SeSrgA, which identifies important structural differences in areas surrounding the catalytic sites. These differences result in diverse redox properties and are likely to underline the distinct substrate specificities and redox functions.DISCUSSIONThe formation of disulfide bonds is a crucial step required for the correct folding of many secreted proteins such as bacterial virulence factors. In prokaryotes disulfide formation machineries show considerable diversity (12Dutton R.J. Boyd D. Berkmen M. Beckwith J. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 11933-11938Crossref PubMed Scopus (191) Google Scholar), particularly among Gram-negative microbes, which contain an extended arsenal of thiol-disulfide oxido-reductases (14Grimshaw J.P. Stirnimann C.U. Brozzo M.S. Malojcic G. Grütter M.G. Capitani G. Glockshuber R. J. Mol. Biol. 2008; 380: 667-680Crossref PubMed Scopus (64) Google Scholar, 15Lafaye C. Iwema T. Carpentier P. Jullian-Binard C. Kroll J.S. Collet J.F. Serre L. J. Mol. Biol. 2009; 392: 952-966Crossref PubMed Scopus (43) Google Scholar), underscoring the relevance of redox control in these organisms.In this study we have carried out a comprehensive analysis of the sv. Typhimurium oxidative pathway. Unlike prototypic E. coli K-12, which encodes a single thiol oxidase (EcDsbA), sv. Typhimurium possesses three DsbA homologues. Structural characterization of SeDsbA, SeDsbL, and SeSrgA has shown that they all have the canonical EcDsbA fold consisting of a TRX domain that incorporates a helical insertion. Despite this, the three DsbA paralogues share low sequence identity and it is therefore not surprising that they contain unique structural and enzymatic properties. SeDsbA retains all the surface features of the E. coli homologue (44Martin J.L. Bardwell J.C. Kuriyan J. Nature. 1993; 365: 464-468Crossref PubMed Scopus (350) Google Scholar), including a hydrophobic groove that binds the key partner protein DsbB (45Inaba K. Murakami S. Suzuki M. Nakagawa A. Yamashita E. Okada K. Ito K. Cell. 2006; 127: 789-801Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). SeDsbA is postulated to interact with unfolded peptides (44Martin J.L. Bardwell J.C. Kuriyan J. Nature. 1993; 365: 464-468Crossref PubMed Scopus (350) Google Scholar) and possesses a broad hydrophobic region surrounding the active site. SeSrgA and particularly SeDsbL differ from SeDsbA in that they have truncated peptide binding grooves and positively charged surfaces surrounding the active site (FIGURE 1, FIGURE 2, FIGURE 3) Additionally, SeDsbL incorporates long loops in the helical domain, including the one linking α3 and α4 (Fig. 2C), which is lined with negatively charged residues and generates an acidic protrusion that maps near the redox catalytic site (Fig. 3).SeSrgA, SeDsbA, and SeDsbL have markedly different oxidizing strengths (Fig. 4). SeSrgA, with an Eo′ of −154 mV, is one of the most reducing DsbA proteins characterized (Wolbachia pipientis α-DsbA1 being the most reducing one, Eo′ −163 mV (50Kurz M. Iturbe-Ormaetxe I. Jarrott R. Shouldice S.R. Wouters M.A. Frei P. Glockshuber R. Oneill S.L. Heras B. Martin J.L. Antioxid. Redox Signal. 2009; 11: 1485-1500Crossref PubMed Scopus (34) Google Scholar)). SeDsbA is as strong an oxidant as EcDsbA (Eo′ −126 mV and −122 mV, respectively (11Huber-Wunderlich M. Glockshuber R. Fold. Des. 1998; 3: 161-171Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar)), while SeDsbL (Eo′ −97 mV) is together with the three Neisserial DsbA homologues (redox potentials of −80 mV) (15Lafaye C. Iwema T. Carpentier P. Jullian-Binard C. Kroll J.S. Collet J.F. Serre L. J. Mol. Biol. 2009; 392: 952-966Crossref PubMed Scopus (43) Google Scholar) and DsbL from uropathogenic E. coli CFT073 (Eo′ −90 mV) (14Grimshaw J.P. Stirnimann C.U. Brozzo M.S. Malojcic G. Grütter M.G. Capitani G. Glockshuber R. J. Mol. Biol. 2008; 380: 667-680Crossref PubMed Scopus (64) Google Scholar) one of the most oxidizing proteins so far characterized. Moreover, the reactivity of the nucleophilic cysteine also varies across the three Salmonella proteins (Fig. 4). As expected from their intrinsic redox potentials, SeDsbA and SeDsbL contain very acidic cysteines (pKa values, 3.6 and 3.8, respectively) and the more reducing SeSrgA has a less reactive cysteine with a pKa similar to that of isomerases like DsbC (8Ren G. Stephan D. Xu Z. Zheng Y. Tang D. Harrison R.S. Kurz M. Jarrott R. Shouldice S.R. Hiniker A. Martin J.L. Heras B. Bardwell J.C. J. Biol. Chem. 2009; 284: 10150-10159Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) (pKa 4.7 and 4.6, respectively).The different redox properties of SeSrgA seem to be mainly dictated by the dipeptide in the active site. The histidine residue in the Cys-Pro-His-Cys motif of EcDsbA plays a key role in determining the oxidizing properties of this protein and substitution of this residue with a proline dramatically increases the pKa of the nucleophilic cysteine and reduces the oxidizing strength of EcDsbA (11Huber-Wunderlich M. Glockshuber R. Fold. Des. 1998; 3: 161-171Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In the same way, SeSrgA, which naturally has a proline instead of a histidine in the catalytic site (Cys-Pro-Pro-Cys), is more reducing than EcDsbA or SeDsbA and contains a much less reactive active site cysteine. In the case of SeDsbL, the cluster of positively charged residues surrounding the Cys-Pro-Phe-Cys active site likely stabilize the thiolate form of the protein which results in a decreased pKa of the nucleophilic active site cysteine and an increase in the redox potential.The structural differences and divergent redox properties suggest that the three DsbA proteins encoded by sv. Typhimurium may have different substrate specificities and redox function. Indeed, these proteins have distinct in vitro oxidoreductase activity; SeSrgA is a strong insulin reductase, with an activity similar to that of the disulfide isomerase EcDsbC, SeDsbA is as active as EcDsbA in this assay and SeDsbL did not show any insulin reductase activity (Fig. 4). Despite some similarities between the biochemical properties of SeSrgA and EcDsbC, in vitro we found no activity for disulfide bond isomerase in SeSrgA or in any of the Salmonella DsbA proteins (data not shown).To further investigate the functional properties of SeDsbA, SeDsbL, and SeSrgA, we examined their expression and target specificity in the context of a sv. Typhimurium background. We were able to detect expression of SeDsbA and SeSrgA, but not SeDsbL. The lack of SeDsbL expression is consistent with a previous report that examined SeDsbL expression in uropathogenic E. coli (18Totsika M. Heras B. Wurpel D.J. Schembri M.A. J. Bacteriol. 2009; 191: 3901-3908Crossref PubMed Scopus (60) Google Scholar). All three oxidoreductases were able to restore motility and Pef fimbriae production in the sv. Typhimurium triple mutant SL1344dsbA,dsbLI,srgA. In contrast, DsbL exhibited target specificity for AssT.In conclusion, although SeSrgA, SeDsbA, and SeDsbL share the same overall fold, structural differences between these proteins (mainly localized in the redox active site and peptide binding interface) imply that they may target different substrates and have distinct functionality. Biochemical characterization confirmed these postulations, since SeSrgA, SeDsbA, and SeDsbL have different redox strengths, the reactivity of their surface exposed cysteine varies and they show dissimilar oxido-reductase activity in vitro. Despite some degree of functional redundancy, SeDsbA, SeDsbL, and SeSrgA therefore represent thiol oxidases that cover a wide range of structural and biochemical properties to efficiently catalyze the oxidative folding of target proteins. During sv. Typhimurium infection, SeDsbA, SeDsbL, and SeSrgA are probably under differential temporal and spatial regulation and this, together with their different degree of target specificity, emphasizes the importance of this critical folding step in bacterial survival and virulence. IntroductionThioredoxin (TRX) 2The abbreviations used are: TRXthioredoxinAssTarylsulfate sulfotransferaseDsbdisulfide bondDTTdithiothreitolIPTGisopropyl β-d-1-thiogalactopyranosideLICligation-independent cloningMBPmaltose-binding proteinPEGpolyethylene glycolSADsingle-wavelength anomalous dispersionSPI2Salmonella Pathogenicity Island 2SPI1Salmonella Pathogenicity Island 1SeMetselenomethionineT3SSType III secretion systemsTEVtobacco etch virusr.m.s.d.root mean square deviationFITCfluorescein isothiocyanateMES4-morpholineethanesulfonic acidsv.serovar. -like oxidoreductases play a major role in controlling the redox environment of the cell. These enzymes catalyze thiol-disulfide oxidoreductase reactions that are important in enzyme catalysis and in the maintenance of the correct thiol redox state in proteins. Dsb (disulfide bond) proteins are a specific of TRX-like proteins that are essential for the oxidative folding of secreted proteins in many Gram-negative bacteria (1Kadokura H. Katzen F. Beckwith J. Annu. Rev. Biochem. 2003; 72: 111-135Crossref PubMed Scopus (444) Google Scholar, 2Masip L. Pan J.L. Haldar S. Penner-Hahn J.E. DeLisa M.P. Georgiou G. Bardwell J.C. Collet J.F. Science. 2004; 303: 1185-1189Crossref PubMed Scopus (69) Google Scholar, 3Inaba K. J. Biochem. 2009; 146: 591-597Crossref PubMed Scopus (65) Google Scholar). In Escherichia coli K-12 five different Dsb proteins catalyze the correct introduction of disulfide bonds in substrate proteins (4Bardwell J.C. Lee J.O. Jander G. Martin N. Belin D. Beckwith J. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 1038-1042Crossref PubMed Scopus (357) Google Scholar). DsbA and DsbB (5Bardwell J.C. McGovern K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 6Akiyama Y. Kamitani S. Kusukawa N. Ito K. J. Biol. Chem. 1992; 267: 22440-22445Abstract Full Text PDF PubMed Google Scholar) form the oxidase pathway that introduces disulfide bonds into target proteins, whereas DsbC, DsbG and DsbD (7Rietsch A. Belin D. Martin N. Beckwith J. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 13048-13053Crossref PubMed Scopus (242) Google Scholar) comprise the isomerase pathway which proofreads and corrects incorrect disulfide bonds in proteins with multiple cysteines. Like all TRX-related proteins, Dsb enzymes contain a conserved CXXC catalytic motif and a X-cisProline loop in their active site. The identity of the XX dipeptide located between the cysteines as well as the residue preceding the cisProline residue determines the properties and function of these enzymes; variation in these residues accounts for the wide range of redox activities described above (8Ren G. Stephan D. Xu Z. Zheng Y. Tang D. Harrison R.S. Kurz M. Jarrott R. Shouldice S.R. Hiniker A. Martin J.L. Heras B. Bardwell J.C. J. Biol. Chem. 2009; 284: 10150-10159Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 9Quan S. Schneider I. Pan J. Von Hacht A. Bardwell J.C. J. Biol. Chem. 2007; 282: 28823-28833Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 10Mössner E. Huber-Wunderlich M. Rietsch A. Beckwith J. Glockshuber R. Aslund F. J. Biol. Chem. 1999; 274: 25254-25259Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 11Huber-Wunderlich M. Glockshuber R. Fold. Des. 1998; 3: 161-171Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar).Until recently it was considered that the paradigm redox system present in E. coli K-12 was conserved in all bacteria. However, new studies on Dsb proteins in other bacteria show that these systems can vary considerably between different genera and even strains of the same species (12Dutton R.J. Boyd D. Berkmen M. Beckwith J. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 11933-11938Crossref PubMed Scopus (191) Google Scholar, 13Heras B. Shouldice S.R. Totsika M. Scanlon M.J. Schembri M.A. Martin J.L. Nat. Rev. Microbiol. 2009; 7: 215-225Crossref PubMed Scopus (219) Google Scholar). This is observed for many Gram-negative pathogenic bacteria, some of which encode multiple Dsb proteins (14Grimshaw J.P. Stirnimann C.U. Brozzo M.S. Malojcic G. Grütter M.G. Capitani G. Glockshuber R. J. Mol. Biol. 2008; 380: 667-680Crossref PubMed Scopus (64) Google Scholar, 15Lafaye C. Iwema T. Carpentier P. Jullian-Binard C. Kroll J.S. Collet J.F. Serre L. J. Mol. Biol. 2009; 392: 952-966Crossref PubMed Scopus (43) Google Scholar). Significantly, there is growing evidence that a range of virulence-related functions depend on the strict redox control carried out by Dsb enzymes. Thus, these catalysts are required for the correct folding (and therefore function) of many secreted or surface exposed virulence determinants. Examples include, the flagellar P-ring motor protein FlgI, the secretin component of Type III secretion systems (T3SS) and a range of toxins and secreted enzymes (13Heras B. Shouldice S.R. Totsika M. Scanlon M.J. Schembri M.A. Martin J.L. Nat. Rev. Microbiol. 2009; 7: 215-225Crossref PubMed Scopus (219) Google Scholar, 16Sinha S. Langford P.R. Kroll J.S. Microbiology. 2004; 150: 2993-3000Crossref PubMed Scopus (39) Google Scholar).The Gram-negative bacterium Salmonella enterica serovar (sv.) Typhimurium is an important causative agent of acute human gastroenteritis and can cause life-threatening bacteremia in immuno-compromised individuals (17Kankwatira A.M. Mwafulirwa G.A. Gordon M.A. Trop. Doct. 2004; 34: 198-200Crossref PubMed Scopus (41) Google Scholar). It is an intracellular pathogen that resides in macrophages. In addition to the oxidase and isomerase systems described above for E. coli K-12, sv. Typhimurium also has a DsbL/DsbI pair, similar to the uropathogenic E. coli (UPEC) CFT073 DsbL/DsbI system (14Grimshaw J.P. Stirnimann C.U. Brozzo M.S. Malojcic G. Grütter M.G. Capitani G. Glockshuber R. J. Mol. Biol. 2008; 380: 667-680Crossref PubMed Scopus (64) Google Scholar, 18Totsika M. Heras B. Wurpel D.J. Schembri M.A. J. Bacteriol. 2009; 191: 3901-3908Crossref PubMed Scopus (60) Google Scholar), and a virulence plasmid-encoded DsbA-like protein, called SrgA (19Bouwman C.W. Kohli M. Killoran A. Touchie G.A. Kadner R.J. Martin N.L. J. Bacteriol. 2003; 185: 991-1000Crossref PubMed Scopus (68) Google Scholar). The sv. Typhimurium DsbA paralogues, termed SeDsbA, SeDsbL, and SeSrgA, share low sequence identity with each other (between 18 and 34%). They each contain different residues in the CXXC redox active site as well as in the cisProline. These differences suggest that the three DsbA paralogues have different functions and most likely act on different target protein(s). In support of this notion, characterization of SeDsbA and SeSrgA showed that these two disulfide oxidoreductases have specificity for different substrates (20Turcot I. Ponnampalam T.V. Bouwman C.W. Martin N.L. Can. J. Microbiol. 2001; 47: 711-721Crossref PubMed Google Scholar). SeSrgA is required for the biogenesis of the major structural subunit of the plasmid-encoded fimbriae, PefA (19Bouwman C.W. Kohli M. Killoran A. Touchie G.A. Kadner R.J. Martin N.L. J. Bacteriol. 2003; 185: 991-1000Crossref PubMed Scopus (68) Google Scholar). Together with SeDsbA it also catalyzes the folding of the outer membrane secretin SpiA, a component of the T3SS encoded in Salmonella Pathogenicity Island 2 (SPI2) (21Miki T. Okada N. Danbara H. J. Biol. Chem. 2004; 279: 34631-34642Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). SeDsbA is required for the folding of FlgI (20Turcot I. Ponnampalam T.V. Bouwman C.W. Martin N.L. Can. J. Microbiol. 2001; 47: 711-721Crossref PubMed Google Scholar) and most likely many other periplasmic/secreted proteins. The change in the periplasmic redox state upon disruption of the dsbA gene also affects the expression of the SPI1 T3SS (22Lin D. Rao C.V. Slauch J.M. J. Bacteriol. 2008; 190: 87-97Crossref PubMed Scopus (79) Google Scholar). Although no substrate is known for SeDsbL, this protein shares 93% sequence identity with its E. coli CFT073 orthologue, and could most likely fold the periplasmic enzyme arylsulfate sulfotransferase (AssT) (14Grimshaw J.P. Stirnimann C.U. Brozzo M.S. Malojcic G. Grütter M.G. Capitani G. Glockshuber R. J. Mol. Biol. 2008; 380: 667-680Crossref PubMed Scopus (64) Google Scholar). Moreover, as in UPEC CFT073, the sv. Typhimurium assT gene is encoded immediately upstream of dsbL and dsbI. Despite these differences in substrate specificity, there is indication that some overlap also exists between the activities of SeDsbA, SeDsbL, and SeSrgA (21Miki T. Okada N. Danbara H. J. Biol. Chem. 2004; 279: 34631-34642Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar).The specificity of disulfide catalysis in Gram-negative pathogens with an extended collection of Dsb proteins remains mostly uncharacterized. To further investigate oxidative folding processes in these organisms we have focused on the three sv. Typhimurium DsbA paralogues. Here, we perform a structural, biochemical and functional characterization of SeDsbA, SeDsbL, and SeSrgA, which identifies important structural differences in areas surrounding the catalytic sites. These differences result in diverse redox properties and are likely to underline the distinct substrate specificities and redox functions.
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