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• W2947200411 abstract "A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-d-Galp-(1→3)-β-d-Galf-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat-unit structure to the nonreducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-d-Galp-(1→3)-β-d-Galp-(1→] or O2c [→3)-β-d-GlcpNAc-(1→5)-β-d-Galf-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1 antigen polymerization and forming a complex with the O2a glycosyltransferase WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp–dependent GT-A galactosyltransferase adding β-(1→3)–linked d-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)–linked d-Galp. These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc transferase and noted that WbmW represents a UDP-Galf–dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf. Our results provide mechanistic and conceptual insights into an important strategy for polysaccharide antigen diversification in bacteria. A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-d-Galp-(1→3)-β-d-Galf-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat-unit structure to the nonreducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-d-Galp-(1→3)-β-d-Galp-(1→] or O2c [→3)-β-d-GlcpNAc-(1→5)-β-d-Galf-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1 antigen polymerization and forming a complex with the O2a glycosyltransferase WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp–dependent GT-A galactosyltransferase adding β-(1→3)–linked d-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)–linked d-Galp. These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc transferase and noted that WbmW represents a UDP-Galf–dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf. Our results provide mechanistic and conceptual insights into an important strategy for polysaccharide antigen diversification in bacteria. Antibiotic-resistant bacterial pathogens are a leading concern in modern healthcare, leading the World Health Organization and Centers for Disease Control and Prevention to create lists of priority pathogens (1World Health Organization Global Priority List of Antibiotic-resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics. World Health Organization, Geneva, Switzerland2017Google Scholar, 2Centers for Disease Control and Prevention (2013) Antibiotic Resistance Threats, Centers for Disease Control and Prevention, Atlanta, GAGoogle Scholar). Klebsiella pneumoniae is a high-profile target due to the global dissemination of isolates producing carbapenemases and extended spectrum β-lactamases (3Pitout J.D. Nordmann P. Poirel L. Carbapenemase-producing Klebsiella pneumoniae, a key pathogen set for global nosocomial dominance.Antimicrob. Agents Chemother. 2015; 59 (26169401): 5873-588410.1128/AAC.01019-15Crossref PubMed Scopus (522) Google Scholar). This organism is a Gram-negative nosocomial pathogen, causing urinary tract infections, bacteremia, and sepsis (4Paczosa M.K. Mecsas J. Klebsiella pneumoniae: going on the offense with a strong defense.Microbiol. Mol. Biol. Rev. 2016; 80 (27307579): 629-66110.1128/MMBR.00078-15Crossref PubMed Scopus (793) Google Scholar). Immunocompromised individuals are particularly at risk, and rapid-acting and highly-specific therapeutics are required for post-infection treatments. K. pneumoniae isolates are typically enveloped in a layer of capsular polysaccharide, with varied structures defining more than 80 serotypes (5Pan Y.-J. Lin T.-L. Chen C.-T. Chen Y.-Y. Hsieh P.-F. Hsu C.-R. Wu M.-C. Wang J.-T. Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp.Sci. Rep. 2015; 5 (26493302): 1557310.1038/srep15573Crossref PubMed Scopus (129) Google Scholar). The polysaccharide (O) antigens attached to the lipid A–core oligosaccharide component of lipopolysaccharides are less diverse. Two studies proposed nine O-antigen serotypes in K. pneumoniae with slight differences in the included serotypes (6Trautmann M. Ruhnke M. Rukavina T. Held T.K. Cross A.S. Marre R. Whitfield C. O-antigen seroepidemiology of Klebsiella clinical isolates and implications for immunoprophylaxis of Klebsiella infections.Clin. Diagn. Lab. Immunol. 1997; 4 (9302204): 550-555Crossref PubMed Google Scholar, 7Hansen D.S. Mestre F. Alberti S. Hernández-Allés S. Alvarez D. Doménech-Sánchez A. Gil J. Merino S. Tomás J.M. Benedć V.J. Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries.J. Clin. Microbiol. 1999; 37 (9854064): 56-62Crossref PubMed Google Scholar), and recent structural (8Stojkovic K. Szijártó V. Kaszowska M. Niedziela T. Hartl K. Nagy G. Lukasiewicz J. Identification of d-Galactan-III as part of the lipopolysaccharide of Klebsiella pneumoniae serotype O1.Front. Microbiol. 2017; 8 (28487676): 68410.3389/fmicb.2017.00684Crossref PubMed Scopus (15) Google Scholar, 9Guachalla L.M. Stojkovic K. Hartl K. Kaszowska M. Kumar Y. Wahl B. Paprotka T. Nagy E. Lukasiewicz J. Nagy G. Szijártó V. Discovery of monoclonal antibodies cross-reactive to novel subserotypes of K. pneumoniae O3.Sci. Rep. 2017; 7 (28747785): 663510.1038/s41598-017-06682-2Crossref PubMed Scopus (17) Google Scholar) and genomic data (10Follador R. Heinz E. Wyres K.L. Ellington M.J. Kowarik M. Holt K.E. Thomson N.R. The diversity of Klebsiella pneumoniae surface polysaccharides.Microb. Genom. 2016; 2 (28348868): e00007310.1099/mgen.0.000073PubMed Google Scholar) suggest more diversity. However, in an investigation of 500 isolates from a diverse collection, 93% were assigned to six known serotypes (of which 83% were O1, O2, or O3) based on sequences of their O-antigen polysaccharide (OPS) 5The abbreviations used are: OPSO-antigen polysaccharideGTglycosyltransferaseHMWhigh-molecular weightLPSlipopolysaccharideUnd-PPundecaprenyl diphosphateABCATP-binding cassetteEICextracted ion chromatogramNi-NTAnickel-nitrilotriacetic acidPDBProtein Data BankBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolGalpgalactopyranoseGalfgalactofuranosePSpolysaccharideX-gal5-bromo-4-chloro indolyl galactopyranosideKdo3-deoxy-d-manno-oct-2-ulosonic acidIPTGisopropyl 1-thio-β-d-galactopyranoside. genetic loci (10Follador R. Heinz E. Wyres K.L. Ellington M.J. Kowarik M. Holt K.E. Thomson N.R. The diversity of Klebsiella pneumoniae surface polysaccharides.Microb. Genom. 2016; 2 (28348868): e00007310.1099/mgen.0.000073PubMed Google Scholar). Because of the relatively limited range of O-antigen structures in clinical isolates, they have been considered as feasible targets for immunotherapies for infection treatment and have recently been shown to be protective in animal models by passive immunization through administration of O-antigen–specific antibodies (11Szijártó V. Guachalla L.M. Hartl K. Varga C. Badarau A. Mirkina I. Visram Z.C. Stulik L. Power C.A. Nagy E. Nagy G. Endotoxin neutralization by an O-antigen specific monoclonal antibody: a potential novel therapeutic approach against Klebsiella pneumoniae ST258.Virulence. 2017; 8 (28103139): 1203-121510.1080/21505594.2017.1279778Crossref PubMed Scopus (30) Google Scholar, 12Hegerle N. Choi M. Sinclair J. Amin M.N. Ollivault-Shiflett M. Curtis B. Laufer R.S. Shridhar S. Brammer J. Toapanta F.R. Holder I.A. Pasetti M.F. Lees A. Tennant S.M. Cross A.S. Simon R. Development of a broad spectrum glycoconjugate vaccine to prevent wound and disseminated infections with Klebsiella pneumoniae and Pseudomonas aeruginosa.PLoS ONE. 2018; 13 (30188914): e020314310.1371/journal.pone.0203143Crossref PubMed Scopus (44) Google Scholar). O-antigen polysaccharide glycosyltransferase high-molecular weight lipopolysaccharide undecaprenyl diphosphate ATP-binding cassette extracted ion chromatogram nickel-nitrilotriacetic acid Protein Data Bank 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol galactopyranose galactofuranose polysaccharide 5-bromo-4-chloro indolyl galactopyranoside 3-deoxy-d-manno-oct-2-ulosonic acid isopropyl 1-thio-β-d-galactopyranoside. In addition to their potential value in immunotherapeutic strategies, K. pneumoniae OPSs have provided important prototypes for elucidating the key concepts in bacterial polysaccharide biosynthesis and export by members of the ATP-binding cassette (ABC) transporter family (13Liston S.D. Mann E. Whitfield C. Glycolipid substrates for ABC transporters required for the assembly of bacterial cell-envelope and cell-surface glycoconjugates.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2017; 1862 (27793707): 1394-140310.1016/j.bbalip.2016.10.008Crossref PubMed Scopus (27) Google Scholar, 14Greenfield L.K. Whitfield C. Synthesis of lipopolysaccharide O-antigens by ABC transporter-dependent pathways.Carbohydr. Res. 2012; 356 (22475157): 12-2410.1016/j.carres.2012.02.027Crossref PubMed Scopus (125) Google Scholar). These OPSs also offer insight into strategies used by bacteria in diversification of polysaccharide antigens. These processes create related structures that complicate serotype distinction among the clinically prevalent O1/O2 group (Fig. 1). They share a common repeat unit called O2a (sometimes referred to as d-Galactan I) (15Bronner D. Clarke B.R. Whitfield C. Identification of an ATP-binding cassette transport system required for translocation of lipopolysaccharide O-antigen side-chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1.Mol. Microbiol. 1994; 14 (7533882): 505-51910.1111/j.1365-2958.1994.tb02185.xCrossref PubMed Scopus (84) Google Scholar) as part of their structures. The O2a repeat unit is composed of alternating α-(1→3)–linked galactopyranose (Galp) and β-(1→3)–linked galactofuranose (Galf) residues (16Whitfield C. Richards J.C. Perry M.B. Clarke B.R. MacLean L.L. Expression of two structurally distinct d-galactan O-antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1.J. Bacteriol. 1991; 173 (1704883): 1420-143110.1128/jb.173.4.1420-1431.1991Crossref PubMed Google Scholar, 17Kol O. Wieruszeski J.M. Strecker G. Fournet B. Zalisz R. Smets P. Structure of the O-specific polysaccharide chain of Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide. A complementary elucidation.Carbohydr. Res. 1992; 236 (1291059): 339-34410.1016/0008-6215(92)85028-XCrossref PubMed Scopus (36) Google Scholar). O2a antigen biosynthesis is directed by the rfb chromosomal locus (18Clarke B.R. Whitfield C. Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the d-galactan I O polysaccharide.J. Bacteriol. 1992; 174 (1378055): 4614-462110.1128/jb.174.14.4614-4621.1992Crossref PubMed Google Scholar), encoding six gene products that are necessary for the biosynthesis and transport of the O2a repeat unit (18Clarke B.R. Whitfield C. Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the d-galactan I O polysaccharide.J. Bacteriol. 1992; 174 (1378055): 4614-462110.1128/jb.174.14.4614-4621.1992Crossref PubMed Google Scholar). Wzm and Wzt make up the ABC transporter required for transport of the completed undecaprenyl diphosphate (Und-PP)-linked OPS (18Clarke B.R. Whitfield C. Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the d-galactan I O polysaccharide.J. Bacteriol. 1992; 174 (1378055): 4614-462110.1128/jb.174.14.4614-4621.1992Crossref PubMed Google Scholar). The remaining four proteins (Glf, WbbM, WbbN, and WbbO) are responsible for the biosynthesis of the O2a polysaccharide (Fig. 1) (18Clarke B.R. Whitfield C. Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the d-galactan I O polysaccharide.J. Bacteriol. 1992; 174 (1378055): 4614-462110.1128/jb.174.14.4614-4621.1992Crossref PubMed Google Scholar19Clarke B.R. Bronner D. Keenleyside W.J. Severn W.B. Richards J.C. Whitfield C. Role of Rfe and RfbF in the initiation of biosynthesis of d-galactan I, the lipopolysaccharide O-antigen from Klebsiella pneumoniae serotype O1.J. Bacteriol. 1995; 177 (7559323): 5411-541810.1128/jb.177.19.5411-5418.1995Crossref PubMed Google Scholar, 20Guan S. Clarke A.J. Whitfield C. Functional analysis of the galactosyltransferases required for biosynthesis of d-galactan I, a component of the lipopolysaccharide O1 antigen of Klebsiella pneumoniae.J. Bacteriol. 2001; 183 (11344139): 3318-332710.1128/JB.183.11.3318-3327.2001Crossref PubMed Scopus (52) Google Scholar21Kos V. Cuthbertson L. Whitfield C. The Klebsiella pneumoniae O2a antigen defines a second mechanism for O-antigen ATP-binding cassette transporters.J. Biol. Chem. 2009; 284 (19036729): 2947-295610.1074/jbc.M807213200Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Glf is a UDP-galactopyranose mutase that produces UDP-Galf from UDP-Galp (22Köplin R. Brisson J.-R. Whitfield C. UDP-galactofuranose precursor required for formation of the lipopolysaccharide O-antigen of Klebsiella pneumoniae serotype O1 is synthesized by the product of the rfbDKPO1 gene.J. Biol. Chem. 1997; 272 (9020123): 4121-412810.1074/jbc.272.7.4121Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), and WbbN, WbbO, and WbbM are glycosyltransferases (GTs) (19Clarke B.R. Bronner D. Keenleyside W.J. Severn W.B. Richards J.C. Whitfield C. Role of Rfe and RfbF in the initiation of biosynthesis of d-galactan I, the lipopolysaccharide O-antigen from Klebsiella pneumoniae serotype O1.J. Bacteriol. 1995; 177 (7559323): 5411-541810.1128/jb.177.19.5411-5418.1995Crossref PubMed Google Scholar, 20Guan S. Clarke A.J. Whitfield C. Functional analysis of the galactosyltransferases required for biosynthesis of d-galactan I, a component of the lipopolysaccharide O1 antigen of Klebsiella pneumoniae.J. Bacteriol. 2001; 183 (11344139): 3318-332710.1128/JB.183.11.3318-3327.2001Crossref PubMed Scopus (52) Google Scholar, 23Kos V. Whitfield C. A membrane-located glycosyltransferase complex required for biosynthesis of the d-galactan I lipopolysaccharide O-antigen in Klebsiella pneumoniae.J. Biol. Chem. 2010; 285 (20410291): 19668-1968710.1074/jbc.M110.122598Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). After its biosynthesis, the Und-PP–linked OPS is exported to the periplasm by the ABC transporter, ligated to lipid A–core oligosaccharide, and translocated to the outer leaflet of the outer membrane (24Whitfield C. Trent M.S. Biosynthesis and export of bacterial lipopolysaccharides.Annu. Rev. Biochem. 2014; 83 (24580642): 99-12810.1146/annurev-biochem-060713-035600Crossref PubMed Scopus (436) Google Scholar, 25Okuda S. Sherman D.J. Silhavy T.J. Ruiz N. Kahne D. Lipopolysaccharide transport and assembly at the outer membrane: the PEZ model.Nat. Rev. Microbiol. 2016; 14 (27026255): 337-34510.1038/nrmicro.2016.25Crossref PubMed Scopus (238) Google Scholar). The O2 serotype is actually made up of a collection of four sub-serotypes that, along with O1, represent different modified versions of O2a. The O8 serotype has the same repeat-unit structure as O2a, but is nonstoichiometrically O-acetylated (26Kelly R.F. Severn W.B. Richards J.C. Perry M.B. MacLean L.L. Tomás J.M. Merino S. Whitfield C. Structural variation in the O-specific polysaccharides of Klebsiella pneumoniae serotype O1 and O8 lipopolysaccharide: evidence for clonal diversity in rfb genes.Mol. Microbiol. 1993; 10 (7526122): 615-62510.1111/j.1365-2958.1993.tb00933.xCrossref PubMed Scopus (42) Google Scholar). The O2afg and O2aeh serotypes modify the O2a repeat unit by side-chain addition of (1→4)- or (1→2)–linked Galp residues, respectively (Fig. 1) (27Kelly R.F. Perry M.B. MacLean L.L. Whitfield C. Structures of the O-antigens of Klebsiella serotypes 02 (2a,2e), 02 (2a,2e,2h), and 02 (2a,2f,2g), members of a family of related d-galactan O-antigens in Klebsiella spp.Innate Immun. 1995; 2: 131-14010.1177/096805199500200208Google Scholar). The side-chain modification is catalyzed by a system similar to bacteriophage-mediated OPS glucosylation in Salmonella and Shigella, and it is directed by a set of three genes, denoted gmlABC responsible for the (1→4)-linkage in O2afg and gmlABD responsible for the (1→2)- linkage in O2aeh (8Stojkovic K. Szijártó V. Kaszowska M. Niedziela T. Hartl K. Nagy G. Lukasiewicz J. Identification of d-Galactan-III as part of the lipopolysaccharide of Klebsiella pneumoniae serotype O1.Front. Microbiol. 2017; 8 (28487676): 68410.3389/fmicb.2017.00684Crossref PubMed Scopus (15) Google Scholar, 28Clarke B.R. Ovchinnikova O.G. Kelly S.D. Williamson M.L. Butler J.E. Liu B. Wang L. Gou X. Follador R. Lowary T.L. Whitfield C. Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae.J. Biol. Chem. 2018; 293 (29602878): 4666-467910.1074/jbc.RA117.000646Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). NMR spectroscopic investigations of the OPS structure revealed that the O1 antigen is covalently attached to the nonreducing terminus of O2a (16Whitfield C. Richards J.C. Perry M.B. Clarke B.R. MacLean L.L. Expression of two structurally distinct d-galactan O-antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1.J. Bacteriol. 1991; 173 (1704883): 1420-143110.1128/jb.173.4.1420-1431.1991Crossref PubMed Google Scholar, 17Kol O. Wieruszeski J.M. Strecker G. Fournet B. Zalisz R. Smets P. Structure of the O-specific polysaccharide chain of Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide. A complementary elucidation.Carbohydr. Res. 1992; 236 (1291059): 339-34410.1016/0008-6215(92)85028-XCrossref PubMed Scopus (36) Google Scholar) or O2afg (8Stojkovic K. Szijártó V. Kaszowska M. Niedziela T. Hartl K. Nagy G. Lukasiewicz J. Identification of d-Galactan-III as part of the lipopolysaccharide of Klebsiella pneumoniae serotype O1.Front. Microbiol. 2017; 8 (28487676): 68410.3389/fmicb.2017.00684Crossref PubMed Scopus (15) Google Scholar) and is composed of a [→3)-α-d-Galp-(1→3)-β-d-Galp-(1→] disaccharide repeat-unit structure (Fig. 1). A gene (wbbY) outside the rfb region has been reported as being responsible for production of the O1 antigen (28Clarke B.R. Ovchinnikova O.G. Kelly S.D. Williamson M.L. Butler J.E. Liu B. Wang L. Gou X. Follador R. Lowary T.L. Whitfield C. Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae.J. Biol. Chem. 2018; 293 (29602878): 4666-467910.1074/jbc.RA117.000646Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 29Hsieh P.-F. Wu M.-C. Yang F.-L. Chen C.-T. Lou T.-C. Chen Y.-Y. Wu S.-H. Sheu J.-C. Wang J.-T. d-Galactan II is an immunodominant antigen in O1 lipopolysaccharide and affects virulence in Klebsiella pneumoniae: implication in vaccine design.Front. Microbiol. 2014; 5 (25477867): 60810.3389/fmicb.2014.00608Crossref PubMed Scopus (24) Google Scholar), but the role of the WbbY gene product and the possible interplay between the O1 and O2a biosynthesis machinery is unknown. The O2c antigen is also extended from the O2a antigen and possesses a [→3)-β-d-GlcpNAc-(1→5)-β-d-Galf-(1→] repeat unit (Fig. 1). Its production depends on an unlinked genetic locus denoted wbmVWX (27Kelly R.F. Perry M.B. MacLean L.L. Whitfield C. Structures of the O-antigens of Klebsiella serotypes 02 (2a,2e), 02 (2a,2e,2h), and 02 (2a,2f,2g), members of a family of related d-galactan O-antigens in Klebsiella spp.Innate Immun. 1995; 2: 131-14010.1177/096805199500200208Google Scholar, 28Clarke B.R. Ovchinnikova O.G. Kelly S.D. Williamson M.L. Butler J.E. Liu B. Wang L. Gou X. Follador R. Lowary T.L. Whitfield C. Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae.J. Biol. Chem. 2018; 293 (29602878): 4666-467910.1074/jbc.RA117.000646Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), but precise functions for these genes have not been resolved. Although the minimum requirements for O1 and O2c production have been established, the biochemical function of WbbY and the individual roles of WbmVWX remain unclear. Here, we unequivocally establish the activities of the two GT catalytic modules of WbbY in O1 biosynthesis, and we assign the GT activities of WbmV and WbmW, which are sufficient for O2ac biosynthesis. Completing the characterization of O2a modifications provides a fundamental understanding of the minimal genetic and biosynthetic requirements for clinically-important glycans. It also offers the first enzymatic insight into a novel strategy for diversification of polysaccharide antigens. All available structural data are consistent with the O1 glycan being linked to the nonreducing end of the underlying OPS (e.g. O2a or O2afg) (Fig. 1) (8Stojkovic K. Szijártó V. Kaszowska M. Niedziela T. Hartl K. Nagy G. Lukasiewicz J. Identification of d-Galactan-III as part of the lipopolysaccharide of Klebsiella pneumoniae serotype O1.Front. Microbiol. 2017; 8 (28487676): 68410.3389/fmicb.2017.00684Crossref PubMed Scopus (15) Google Scholar, 17Kol O. Wieruszeski J.M. Strecker G. Fournet B. Zalisz R. Smets P. Structure of the O-specific polysaccharide chain of Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide. A complementary elucidation.Carbohydr. Res. 1992; 236 (1291059): 339-34410.1016/0008-6215(92)85028-XCrossref PubMed Scopus (36) Google Scholar), rather than being directly linked to lipid A–core oligosaccharide. Biosynthesis of the O1 antigen should therefore require the activities of each of the O2a assembly and export components in addition to WbbY. However, the shared linkages in the O2a and O1 antigens opened the possibility that WbbY makes one of the O2a GTs expendable. This was examined using a genetic approach in a recombinant Escherichia coli CWG286 host, which has its own OPS biosynthesis locus deleted, but it can assemble and export the O2a antigen from plasmid-encoded genes (22Köplin R. Brisson J.-R. Whitfield C. UDP-galactofuranose precursor required for formation of the lipopolysaccharide O-antigen of Klebsiella pneumoniae serotype O1 is synthesized by the product of the rfbDKPO1 gene.J. Biol. Chem. 1997; 272 (9020123): 4121-412810.1074/jbc.272.7.4121Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 23Kos V. Whitfield C. A membrane-located glycosyltransferase complex required for biosynthesis of the d-galactan I lipopolysaccharide O-antigen in Klebsiella pneumoniae.J. Biol. Chem. 2010; 285 (20410291): 19668-1968710.1074/jbc.M110.122598Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 28Clarke B.R. Ovchinnikova O.G. Kelly S.D. Williamson M.L. Butler J.E. Liu B. Wang L. Gou X. Follador R. Lowary T.L. Whitfield C. Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae.J. Biol. Chem. 2018; 293 (29602878): 4666-467910.1074/jbc.RA117.000646Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The LPS products were examined in silver-stained SDS-PAGE and the corresponding immunoblots using polyclonal anti-O2a and monoclonal anti-O1 antibodies (Fig. 2). Plasmids containing individual mutations in each of the O2a biosynthesis genes were introduced together with a plasmid expressing wbbY into E. coli CWG286. When wbbY and a functional rfb2a gene cluster were introduced, E. coli GWG286 produced HMW OPS that was reactive only with a mAb specific for O1 PS (Fig. 2, A–C). In the absence of transport (Δwzm-wzt), no OPS-containing LPS species were visible by silver staining, but the anti-O1 antibodies detected OPS. This material is consistent with Und-PP–OPS retained in the cytoplasm, and the higher apparent molecular mass (compared with the corresponding LPS-linked form) is consistent with previous results indicating that active export is required to control O2a glycan chain length (21Kos V. Cuthbertson L. Whitfield C. The Klebsiella pneumoniae O2a antigen defines a second mechanism for O-antigen ATP-binding cassette transporters.J. Biol. Chem. 2009; 284 (19036729): 2947-295610.1074/jbc.M807213200Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Any of the deletions that abrogated O2a biosynthesis also eliminated O1 production. These results indicated that the O1 antigen is assembled on an acceptor composed of at least one O2a repeat unit. Although the results above indicated that O2a production is essential for O1 production, they do not distinguish between a model where one or more of the O2a GTs participate in polymerization of the O1 repeat unit itself, as opposed to a role confined to forming an acceptor for O1 extension. This was investigated by in vitro biosynthesis experiments. His6–WbbY was overexpressed in E. coli K-12. Attempts to purify full-length protein failed due to extensive and unresolvable degradation during protein extraction and purification. Therefore, membrane fractions were isolated and used as a source of enzyme. The two potential O2a acceptors for initiating O1 PS synthesis were synthesized as disaccharides (1 and 2) attached to fluorescein-containing aglycones for detection (Fig. 3 and Schemes S2 and S3). Robust polymerization was observed only with 2, which has a terminal Galp residue. A trace of activity was seen with 1. The product of WbbY polymerization on 2 was purified, size-fractionated, and analyzed by NMR spectroscopy (Table 1 and Fig. S1). This confirmed that the glycan synthesized by WbbY in vitro was structurally identical to authentic O1 PS from K. pneumoniae (16Whitfield C. Richards J.C. Perry M.B. Clarke B.R. MacLean L.L. Expression of two structurally distinct d-galactan O-antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1.J. Bacteriol. 1991; 173 (1704883): 1420-143110.1128/jb.173.4.1420-1431.1991Crossref PubMed Google Scholar, 17Kol O. Wieruszeski J.M. Strecker G. Fournet B. Zalisz R. Smets P. Structure of the O-specific polysaccharide chain of Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide. A complementary elucidation.Carbohydr. Res. 1992; 236 (1291059): 339-34410.1016/0008-6215(92)85028-XCrossref PubMed Scopus (36) Google Scholar). Furthermore, these results indicated that WbbY alone was sufficient for polymerization of the O1 antigen and that it is initiated on a terminal Galp residue in the O2a antigen.Table 11H and 13C NMR chemical shifts of the in vitro synthesized O1 and O2c polymers (δ, ppm)Sugar residueH-1, C-1H-2, C-2H-3, C-3H-4, C-4H-5, C-5H-6 (6a,b), C-6O1→3)-α-d-Galp-(1→A5.19, 96.74.07, 68.74.16, 80.44.29, 70.54.24, 71.93.75, 62.3→3)-β-d-Galp-(1→B4.71, 105.53.78, 71.03.80, 78.64.20, 66.23.70, 76.13.78, 3.82, 62.3→3)-α-d-Galp-(1→P5.07, 100.94.00, 68.64.00, 80.54.25,→3)-β-d-Galf-(1→F4.97, 108.94.22, 80.54.04, 86.24.14, 83.43.86, 72.33.67, 64.2O2c→5)-β-d-Galf-(1→A5.00, 109.54.00, 82.34.26, 77.14.14, 82.53.98, 79.03.70, 62.5→3)-β-d-GlcpNAc-(1→B4.72, 101.93.82, 56.43.63, 82.53.49, 69.83.48, 76.73.77, 3.93 62.1 Open table in a new tab A query of the conserved domain database (30Marchler-Bauer A. Bo Y. Han L. He J. Lanczycki C.J. Lu S. Chitsaz F. Derbyshire M.K. Geer R.C. Gonzales N.R. Gwadz M. Hurwitz D.I. Lu F. Marchler G.H. Song J.S. et al.CDD/SPARCLE: functional classification of proteins via subfamily domain architectures.Nucleic Acids Res. 2017; 45 (27899674): D200-D20310.1093/nar/gkw1129Crossref PubMed Scopus (1681) Google Scholar) revealed that WbbY contains two putative domains, each with homology to distinct GT superfamilies, GT-B and GT-A, respectively (Fig. 4A) (31Lairson L.L. Henrissat B. Davies G.J. Withers S.G. Glycosyltransferases: structures, functions, and mechanisms.Annu. Rev. Biochem. 2008; 77 (18518825): 521-55510.1146/annurev.biochem.76.061005.092322Crossref PubMed Scopus (1326) Google Scholar). This arrangement implied that WbbY was bifunctional, consistent with its capacity to polymerize a glycan containing alternating α-(1→3)-Galp and β-(1→3)-Galp residues (Figure 1, Figure 3). The Phyre2 server (32Kelley L.A. Mezuli" @default.
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• W2947200411 title "Klebsiella pneumoniae O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification" @default.
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