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Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae

Open AccessPublished:February 12, 2018DOI:https://doi.org/10.1074/jbc.RA117.000646
      Klebsiella pneumoniae is a major health threat. Vaccination and passive immunization are considered as alternative therapeutic strategies for managing Klebsiella infections. Lipopolysaccharide O antigens are attractive candidates because of the relatively small range of known O-antigen polysaccharide structures, but immunotherapeutic applications require a complete understanding of the structures found in clinical settings. Currently, the precise number of Klebsiella O antigens is unknown because available serological tests have limited resolution, and their association with defined chemical structures is sometimes uncertain. Molecular serotyping methods can evaluate clinical prevalence of O serotypes but require a full understanding of the genetic determinants for each O-antigen structure. This is problematic with Klebsiella pneumoniae because genes outside the main rfb (O-antigen biosynthesis) locus can have profound effects on the final structure. Here, we report two new loci encoding enzymes that modify a conserved polysaccharide backbone comprising disaccharide repeat units [→3)-α-d-Galp-(1→3)-β-d-Galf-(1→] (O2a antigen). We identified in serotype O2aeh a three-component system that modifies completed O2a glycan in the periplasm by adding 1,2-linked α-Galp side-group residues. In serotype O2ac, a polysaccharide comprising disaccharide repeat units [→5)-β-d-Galf-(1→3)-β-d-GlcpNAc-(1→] (O2c antigen) is attached to the non-reducing termini of O2a-antigen chains. O2c-polysaccharide synthesis is dependent on a locus encoding three glycosyltransferase enzymes. The authentic O2aeh and O2c antigens were recapitulated in recombinant Escherichia coli hosts to establish the essential gene set for their synthesis. These findings now provide a complete understanding of the molecular genetic basis for the known variations in Klebsiella O-antigen carbohydrate structures based on the O2a backbone.

      Introduction

      Klebsiella pneumoniae is a Gram-negative bacterium that colonizes the skin, mouth, gut mucosa, and oropharynx and is carried asymptomatically as part of the human microbiome (
      • Gorrie C.L.
      • Mirceta M.
      • Wick R.R.
      • Edwards D.J.
      • Thomson N.R.
      • Strugnell R.A.
      • Pratt N.F.
      • Garlick J.S.
      • Watson K.M.
      • Pilcher D.V.
      • McGloughlin S.A.
      • Spelman D.W.
      • Jenney A.W.J.
      • Holt K.E.
      Gastrointestinal carriage is a major reservoir of Klebsiella pneumoniae infection in intensive care patients.
      ). However, it is also an opportunistic pathogen causing a variety of infections, including bacteremia, urinary tract infections, and liver abcesses (
      • Paczosa M.K.
      • Mecsas J.
      Klebsiella pneumoniae: going on the offense with a strong defense.
      ). K. pneumoniae is prevalent in nosocomial infections, and immunocompromised individuals are particularly at risk (
      • Gorrie C.L.
      • Mirceta M.
      • Wick R.R.
      • Edwards D.J.
      • Thomson N.R.
      • Strugnell R.A.
      • Pratt N.F.
      • Garlick J.S.
      • Watson K.M.
      • Pilcher D.V.
      • McGloughlin S.A.
      • Spelman D.W.
      • Jenney A.W.J.
      • Holt K.E.
      Gastrointestinal carriage is a major reservoir of Klebsiella pneumoniae infection in intensive care patients.
      ,
      • Paczosa M.K.
      • Mecsas J.
      Klebsiella pneumoniae: going on the offense with a strong defense.
      ). The emergence of hypervirulent isolates and the rise in isolates resistant to most β-lactams, including carbapenems, make this organism a high-profile global health concern (
      • Boucher H.W.
      • Talbot G.H.
      • Bradley J.S.
      • Edwards J.E.
      • Gilbert D.
      • Rice L.B.
      • Scheld M.
      • Spellberg B.
      • Bartlett J.
      Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America.
      ,
      • Pitout J.D.D.
      • Nordmann P.
      • Poirel L.
      Carbapenemase-producing Klebsiella pneumoniae, a key pathogen set for global nosocomial dominance.
      ).
      Over the years, researchers have investigated potential immunotherapeutic strategies targeting K. pneumoniae cell-surface polysaccharides, with both vaccination and passive immunization approaches being considered. Capsular polysaccharide (K antigen) and lipopolysaccharide (LPS)
      The abbreviations used are: LPS
      lipopolysaccharide
      Galf
      galactofuranose
      Galp
      galactopyranose
      GlcpNAc
      N-acetylglucosamine
      OPS
      O polysaccharide
      Und
      undecaprenol
      ABC
      ATP-binding cassette
      Ara4N
      4-amino-4-deoxy-l-arabinose
      HSQC
      heteronuclear single quantum coherence
      HMBC
      heteronuclear multiple bond correlation
      ROESY
      rotating frame nuclear Overhauser effect spectroscopy
      TOCSY
      total correlation spectroscopy
      COSY
      correlation spectroscopy
      contig
      contiguous DNA sequence assembled from whole genome shotgun sequencing
      LB
      lysogeny broth.
      O antigen (O polysaccharide (OPS)) provide important virulence factors, where they typically protect the bacterium against components of the host immune defenses. A K antigen–based immunotherapeutic strategy is complicated by high structural diversity. Seventy-seven serologically distinct Klebsiella K antigens have been identified by conventional serological methods (
      • Ørskov I.
      • Ørskov F.
      Serotyping of Klebsiella.
      ). Many of the underlying polysaccharide structures are known, and the sequences of the corresponding genetic loci have been reported (
      • Pan 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.
      ). However, genome sequences from large collections of clinical isolates suggest a higher number (
      • Wyres K.L.
      • Wick R.R.
      • Gorrie C.
      • Jenney A.
      • Follador R.
      • Thomson N.R.
      • Holt K.E.
      Identification of Klebsiella capsule synthesis loci from whole genome data.
      ), and the polysaccharide structures correlated with most of these newly discovered loci are unknown.
      In contrast to K antigens, the diversity of OPS structures in the lipopolysaccharides of Klebsiella is limited. In a revised O-serotyping classification, nine serotypes (O1, O2ab (= O2a), O2a/O9 (= O2aeh), O3, O4, O5, O7, O11, and O12) were proposed (
      • Trautmann 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.
      ). A subsequent proposal also included nine O groups but with a slightly different composition (O1, O2, O2ac, O3, O4, O5, O7, O8, and O12) (
      • Hansen D.S.
      • Mestre F.
      • Alberti S.
      • Hernández-Allés S.
      • Alvarez D.
      • Doménech-Sánchez A.
      • Gil J.
      • Merino S.
      • Tomás J.M.
      • Benedí V.J.
      Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries.
      ). The repeat-unit structures of known Klebsiella OPSs are shown in Fig. 1 (
      • Kol 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.
      • Kelly 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.
      ,
      • Whitfield C.
      • Perry M.B.
      • MacLean L.L.
      • Yu S.H.
      Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).
      • Lindberg B.
      • Lönngren J.
      • Nimmich W.
      • Rudén U.
      Structural studies on the Klebsiella O group 7 lipopolysaccharide.
      ). Surveys of clinical isolates in these two studies revealed that 82 and 77% (respectively) were accounted for by serotypes O1, O2a, O3, and O5 (
      • Trautmann 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.
      ). However, as described below, the structural relationships between O1, O8, and the various O2 subtypes are complex, and the known structures in the O2 subgroups (Fig. 1) were not distinguished in the revised serological systems or in surveys of clinical isolates. Highlighting this deficiency, neither of these published studies distinguished serotype O2afg, which was recently found to be predominant in isolates of the globally disseminated multidrug-resistant ST258 clone (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ). Developing reagents for classical serological tests to accurately distinguish closely related O antigens can be challenging, and molecular serotyping methods are increasingly adopted. A recent survey of K. pneumoniae genome sequences discovered that the OPS-biosynthesis (rfb) genetic loci in 93% of the isolates could be assigned to six known serotypes (of which 83% were O1, O2, or O3) (
      • Follador R.
      • Heinz E.
      • Wyres K.L.
      • Ellington M.J.
      • Kowarik M.
      • Holt K.E.
      • Thomson N.R.
      The diversity of Klebsiella pneumoniae surface polysaccharides.
      ). Five novel rfb locus variants were identified, but it remains unclear whether these reflect new OPS structures. The rfb gene clusters provide a helpful starting point, but molecular serotyping is challenging when additional (unlinked) genes determine important structural elements and corresponding epitopes. This is the case with the prevalent galactose-based O1, O8, and O2 serotypes from Klebsiella, where the existing serology is particularly complex and includes several O2 subtypes whose precise epitopes have not been established.
      Figure thumbnail gr1
      Figure 1The known carbohydrate repeating unit structures of OPSs of K. pneumoniae. The sugars contained within the square brackets are from the repeat units of the OPS. The O1 and O2c antigens are polysaccharides attached to the non-reducing end of the O2a antigen in the serotypes O1/O8 and O2ac, respectively. A subset of O2afg serotypes can also contain the O1 antigen. Not shown here are non-stoichiometric O-acetyl groups that distinguish serotypes O1 and O8, for example.
      These OPSs all contain a shared backbone structural motif, the O2a antigen, composed of alternating α-d-Galp and β-d-Galf residues (also referred to as d-galactan I) (Fig. 1), whose synthesis is directed by genes in the rfb locus located adjacent to the hisI (histidine biosynthesis) gene on the K. pneumoniae genome (Fig. 2).(
      • Bronner 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.
      ). Six genes, wzm–wbbO, are necessary and sufficient for the production of the O2a antigen when expressed in Escherichia coli hosts (
      • Clarke 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.
      • Clarke 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.
      ,
      • Guan 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.
      • Kos V.
      • Whitfield C.
      A membrane-located glycosyltransferase complex required for biosynthesis of the d-galactan I lipopolysaccharide O antigen in Klebsiella pneumoniae.
      ). Hereafter, these six genes are collectively referred to as rfb2a. The O-antigen assembly process occurs at the cytoplasmic face of the inner cell membrane using undecaprenol-phosphate (Und-P) as an acceptor. Synthesis is initiated by transfer of GlcNAc-1-P to this lipid by WecA (
      • Clarke 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.
      ), a conserved phosphoglycosyltransferase enzyme, to form Und-PP–linked intermediates. The WbbM, WbbN, and WbbO proteins contain glycosyltransferase (GT) motifs, and all are required for O2a-antigen biosynthesis in vivo (
      • Clarke 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.
      ,
      • Guan 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.
      • Kos V.
      • Whitfield C.
      A membrane-located glycosyltransferase complex required for biosynthesis of the d-galactan I lipopolysaccharide O antigen in Klebsiella pneumoniae.
      ). The glf gene encodes UDP-galactopyranose mutase, which converts UDP-Galp to UDP-Galf (
      • Köplin 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.
      ). The ABC transporter (whose transmembrane and nucleotide-binding domains are encoded by wzm and wzt, respectively) exports the completed lipid-linked OPS to the periplasm (
      • Bronner 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.
      ), where it is ligated to lipid A core and translocated to the outer membrane (
      • Whitfield C.
      • Trent M.S.
      Biosynthesis and export of bacterial lipopolysaccharides.
      ). An additional open reading frame (orf7) at the 3′ end of the O-antigen biosynthesis operon encodes a predicted glycosyltransferase (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ), but it has no known function and is not required for expression of authentic O2a OPS in an E. coli host (
      • Bronner 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.
      ).
      Figure thumbnail gr2
      Figure 2Organization of the genetic determinants of O-antigen biosynthesis in K. pneumoniae serotypes derived from O2a. The O serotypes are indicated at the left along with the designations of the strains from which the DNA sequences were obtained. hisI defines the 3′ end of the O2a-antigen biosynthesis cluster (rfb region). The wzm-wbbO genes are necessary and sufficient for biosynthesis of the O2a antigen. The function of orf7 is unknown, and it is not necessary for biosynthesis of OPS in E. coli K-12. In serotype O2afg, the gmlABC cluster is located adjacent to the rfb region, whereas in O2aeh and O2ae, this cluster (gmlABD) is located next to the genomic proA locus. orf8 is present at the 3′ end of the rfb region in CWK53 and encodes a putative acetyltransferase. The O1 (wbbY) and O2c gene clusters (wbmVWX) are not linked to the rfb region, and they are flanked by transposase genes (indicated by tnp and the insertion element (IS) family to which they belong). wbbY can be present in strains expressing either the O2a or the O2afg (gmlABC) antigens, and an example of each is shown.
      The O2a-antigen structure was first identified in a subset of LPS molecules in serotype O1 (
      • Kol 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.
      ,
      • Whitfield 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.
      ) and later as the sole OPS in serotype O2a (
      • Whitfield C.
      • Perry M.B.
      • MacLean L.L.
      • Yu S.H.
      Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).
      ). The O1 antigen (also called d-galactan II) is composed only of d-Galp residues (
      • Kol 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.
      ,
      • Whitfield 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.
      ) and forms a structurally distinct polymeric domain linked to the non-reducing end of chains of O2a polysaccharide (
      • Kol 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.
      ,
      • Vinogradov E.
      • Frirdich E.
      • MacLean L.L.
      • Perry M.B.
      • Petersen B.O.
      • Duus J.Ø.
      • Whitfield C.
      Structures of lipopolysaccharides from Klebsiella pneumoniae: eluicidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains.
      ). The serotype O2ac antigen is also coexpressed with the O2a glycan and is proposed to possess a similar tandem arrangement (
      • Whitfield C.
      • Perry M.B.
      • MacLean L.L.
      • Yu S.H.
      Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).
      ,
      • Vinogradov E.
      • Frirdich E.
      • MacLean L.L.
      • Perry M.B.
      • Petersen B.O.
      • Duus J.Ø.
      • Whitfield C.
      Structures of lipopolysaccharides from Klebsiella pneumoniae: eluicidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains.
      ). Additional O2 variants are composed of an O2a-antigen backbone modified by 1→2- or 1→4-linked α-d-Galp side groups, forming serotypes O2aeh and O2afg, respectively (
      • Kelly 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.
      ). The O2afg structure is the predominant OPS in isolates of the ST258 clone (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ), and it can be further modified by the addition of the O1 antigen in about 40% of tested O2afg isolates (
      • Stojkovic 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.
      ). Further glycosylation of the O2a antigen in serotypes O1 (
      • McCallum K.L.
      • Schoenhals G.
      • Laakso D.
      • Clarke B.
      • Whitfield C.
      A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.
      ) and O2afg (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ) is essential for resistance to serum-mediated killing, providing an obvious selective advantage to isolates with modifications. Several of these glycans are also O-acetylated. For example, the carbohydrate backbones of the OPSs of serotype O1 and O8 are identical (and serologically cross-reactive), but they differ in partial O-acetylation of the O2a-antigen component of O8 (
      • Kelly 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.
      ). Also, the serotypes originally designated O9 and O2ae have essentially the same glycan structure as O2aeh, differing only in the frequency of side-branch addition (
      • MacLean L.L.
      • Whitfield C.
      • Perry M.B.
      Characterization of the polysaccharide antigen of Klebsiella pneumoniae O:9 lipopolysaccharide.
      ). Acetylation status does not seem to significantly alter the recognition of the OPSs by antibodies raised against the non-acetylated carbohydrate backbones (
      • Trautmann 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.
      ) and is probably not an important factor from an immunotherapy perspective.
      Immunotherapeutic approaches to treat Klebsiella infections require that antibodies against O antigen are protective in vivo. A monoclonal antibody against an epitope in O1 offered some protection at high dosage in an experimental infection model (
      • Rukavina T.
      • Tícac B.
      • Susa M.
      • Jendrike N.
      • Jonjíc S.
      • Lucin P.
      • Marre R.
      • Doríc M.
      • Trautmann M.
      Protective effect of antilipopolysaccharide monoclonal antibody in experimental Klebsiella infection.
      ). More recently, a humanized monoclonal recognizing the O2afg antigen in the ST258 clone was protective in a murine model of endotoxemia (
      • Szijártó 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.
      ). However, immunotherapeutic strategies require a complete understanding of the full range of OPS structures and their clinical prevalence. The objective of this study was to complete our understanding of the molecular basis for diversity in the carbohydrate structures of O1/O2 OPSs from the recognized serotypes. To this end, we identified new genetic loci that determine the O2aeh and O2c antigens and correlated OPS genetic complements to structures in the reference strains that were used to determine the OPS structures in the known serotypes.

      Results

      Biosynthesis of the K. pneumoniae O2afg antigen in the prototype strain, CWK55, is dependent on an rfb-linked gmlABC gene cluster

      In serotype O2afg, the products of three additional genes (gmlABC) located between the O2a-antigen biosynthesis operon and hisI (Fig. 2) are involved in the biosynthesis of the O antigen containing an α-(1→4)-linked Galp side group (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ,
      • Stojkovic 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.
      ). A plasmid containing the gmlABC genes (hereafter gmlABC2afg) from clinical isolate KP-27 converted a Klebsiella strain expressing the O2a antigen to the O2afg serotype (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ). To confirm that no additional Klebsiella genes were required for O2afg OPS biosynthesis and correlate these data with the isolate used to determine the structure of the O2afg polysaccharide (
      • Kelly 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.
      ), we reconstituted the system in E. coli K-12. The genomic region between wbbO and hisI, containing the putative gml2afg cluster, was amplified from CWK55, the prototype strain for the O2afg antigen (
      • Kelly 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.
      ). Sequence analysis revealed the presence of the gmlABC2afg cluster with the same organization as that described for the K. pneumoniae O2afg strain, NTUH-K2044 (GenBankTM accession number AP006725) (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ), and a BLAST comparison of the predicted protein sequences from these two isolates showed 100, 99, and 98% identity for GmlA2afg, B2afg, and C2afg, respectively. E. coli DH5α was co-transformed with pWQ393 (gmlABC2afg) and pWQ288 (rfb2a). The SDS-PAGE profile of LPS from DH5α [rfb2a, gmlABC2afg] revealed a typical LPS ladder pattern reflecting the distribution of OPS-substituted lipid A core (Fig. 3A). However, the individual bands in the ladder profile exhibited a band shift, relative to a control sample from DH5α [rfb2a], synthesizing only the O2a antigen (Fig. 3A), consistent with a structural modification of the O2a polysaccharide due to the activities of GmlABC2afg. In immunoblots, LPS from DH5α [rfb2a, gmlABC2afg] did not react to antiserum raised against a K. pneumoniae strain expressing the O2a antigen (Fig. 3B), but this LPS did react with O2afg-specific antiserum (Fig. 3C).
      Figure thumbnail gr3
      Figure 3SDS-PAGE and immunoblotting of LPS showing gmlABC2afg- and gmlABD2aeh-dependent seroconversion of the O2a antigen to the O2afg and O2aeh antigens. A and D, silver-stained SDS-PAGE of LPS in whole-cell lysates. The corresponding immunoblots were probed with antiserum specific for O2a (B and E), the O2afg OPS of CWK55 (C), or the O2aeh OPS of CWK53 (F). The rfb2a genes were expressed from pWQ288. The gmlABC2afg and gmlABD2aeh gene clusters were contained on pWQ393 and pWQ394, respectively. Some cross-reactivity was observed with the anti-O2a serum and unsubstituted lipid A-core molecules in CWK53 and CWK55. The orphan band in the last lane of E reflects an undigested protein antigen, which sometimes occurs in such lysates. In F, the higher reactivity of the wildtype CWK53 LPS (compared with the recombinant) could reflect an epitope(s) contributed by O-acetyl groups present in CWK53 but absent from the recombinant.
      To confirm the antigenic conversion of the O2a antigen to O2afg, LPS was purified from DH5α [rfb2a] and from DH5α [rfb2a, gmlABC2afg], and the chemical structures of the OPS fractions were determined by a combination of 1D and 2D 1H and 13C NMR spectroscopy (Fig. 4, Fig. S1, and Table 1). As expected, the OPS fraction from the control LPS of DH5α [rfb2a] gave a 13C NMR spectrum identical to that reported for the O2a antigen (
      • Whitfield C.
      • Perry M.B.
      • MacLean L.L.
      • Yu S.H.
      Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).
      ,
      • Whitfield 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.
      ) (Fig. 4). A comparison of the 13C NMR spectra from O2a and O2afg polysaccharides showed that the latter contained six additional signals, including a signal for an additional anomeric carbon at δ 101.6 ppm (Fig. 4, O2afg, G1). The assignment of 1H and 13C signals was performed based on 2D COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments (Fig. S1 and Table 1). NMR data demonstrated that the O2afg polysaccharide was composed of an O2a backbone in which the Galp residue was substituted with an α-(1→4)-linked Galp side group. 1H and 13C NMR chemical shifts were in good agreement with those previously reported for the O2afg structure (
      • Kelly 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.
      ,
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ). Comparison of the HSQC spectra of the O2a and the O2afg polysaccharides revealed a series of small signals that could originate from unmodified repeat units. Based on integral intensities of Galf H-2 signals (δH 4.33 and 4.39 in modified and non-modified repeat units, respectively), more than 90% of the backbone Galp residues were substituted with α-d-Galp. These data demonstrated that the OPS expressed from DH5α [rfb2a, gmlABC2afg] was identical to the O2afg antigen originally reported for CWK55 (
      • Kelly 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.
      ) and confirmed the involvement of gmlABC2afg reported previously (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ). Furthermore, the recapitulation of this structure in E. coli defined the required minimal gene complement for authentic O2afg biosynthesis. This provided an essential foundation for subsequent work.
      Figure thumbnail gr4
      Figure 413C NMR spectra of the OPSs isolated from recombinant E. coli K-12 strains expressing the rfb2a locus alone (O2a) and in combination with the gmlABC2afg genes from K. pneumoniae CWK55 (O2afg) and gmlABD2aeh from CWK53 (O2aeh). Signals were assigned based on 1H NMR and 2D COSY, TOCSY, ROESY, 1H-13C HSQC, and HMBC experiments (for HSQC and HMBC spectra, see ). The alphanumeric designations above the NMR peaks refer to carbons in the sugar residues labeled on the repeat-unit structure at the right of each spectrum. The 13C NMR glycosylation effects on G C-1 (+8.1 ppm in O2afg and +3.2 ppm in O2aeh) are in good agreement with the reported values of +8.0 ppm and +4.0 ppm for the α-(1→4) and α-(1→2) linkages, respectively (
      • Lipkind G.M.
      • Shashkov A.S.
      • Knirel Y.A.
      • Vinogradov E.V.
      • Kochetkov N.K.
      A computer-assisted structural analysis of regular polysaccharides on the basis of 13C-n.m.r. data.
      ). Downfield displacement of the signals for P C-4 in the O2afg spectrum and P C-2 in the O2aeh spectrum (by 9.0 and 2.5 ppm, respectively, compared with their position in →3)-substituted P′) confirmed the positions of side-chain galactose. A small portion of the repeating units were unmodified in both the O2afg (∼6%) and the O2aeh (∼15%) polysaccharides. The rfb2a genes were expressed from pWQ288. The gmlABC2afg and gmlABD2aeh gene clusters were contained on pWQ393 and pWQ394, respectively.
      Table 11H and 13C NMR data (δ, ppm)
      a The signals for N-acetyl group are at δH 2.06 and δC 23.5 (CH3) and 176.0 (CO).
      Some Klebsiella isolates with the O2afg genes also contain the unlinked wbbY gene responsible for producing the O1 antigen (
      • Hsieh P.-F. F.
      • Wu M.-C. C.
      • Yang F.-L. L.
      • Chen C.-T. T.
      • Lou T.-C. C.
      • Chen Y.-Y. Y.
      • Wu S.-H. H.
      • Sheu J.-C. C.
      • Wang J.-T. T.
      d-Galactan II is an immunodominant antigen in O1 lipopolysaccharide and affects virulence in Klebsiella pneumoniae: implication in vaccine design.
      ), indicating that the O1 polysaccharide (Fig. 1) can be added to the non-reducing end of either modified or unmodified O2a antigen (
      • Stojkovic 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.
      ). The O1 antigen is not produced by CWK55 (
      • Whitfield C.
      • Perry M.B.
      • MacLean L.L.
      • Yu S.H.
      Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).
      ). The wbbY gene is present near the xynB locus in the O2afg reference genome of K. pneumoniae NTUH-K2044 (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ,
      • Stojkovic 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.
      ) but was absent from this region of the CWK55 genome (data not shown). Furthermore, a BLASTP search failed to identify a predicted WbbY homolog from the total CWK55 genomic data.

      A gml locus unlinked to the rfb region is required for biosynthesis of the O2aeh O antigen

      The O antigen of K. pneumoniae O2aeh differs from the O2afg polymer in the linkage ((1→2) versus (1→4)) of its α-d-Galp side group (
      • Kelly 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.
      ) (Fig. 1). Although not discussed in the original report (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ), analysis of the gmlABC2afg gene products indicates that they are derived from a three-component cassette, resembling other systems that direct the periplasmic modification of glycan backbones in a variety of contexts, including the addition of glucosyl side groups to OPSs of Salmonella and Shigella (reviewed in Ref.
      • Mann E.
      • Whitfield C.
      A widespread three-component mechanism for the periplasmic modification of bacterial glycoconjugates.
      ). In these well-documented glucosylation systems, products of the gtrABC genes modify nascent OPS in the periplasm. GtrB proteins (GmlB homolog) are related to eukaryotic dolichol-phosphate mannose synthase enzymes participating in protein glycosylation (
      • Loibl M.
      • Strahl S.
      Protein O-mannosylation: what we have learned from baker's yeast.
      ) and synthesize a Und-P–glucose intermediate from the UDP-glucose donor at the cytoplasmic face of the inner membrane (
      • Ardiccioni C.
      • Clarke O.B.
      • Tomasek D.
      • Issa H.A.
      • von Alpen D.C.
      • Pond H.L.
      • Banerjee S.
      • Rajashankar K.R.
      • Liu Q.
      • Guan Z.
      • Li C.
      • Kloss B.
      • Bruni R.
      • Kloppmann E.
      • Rost B.
      • et al.
      Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis.
      ). The use of a monophosphoryl intermediate presumably prevents crosstalk between the side-group modification reactions and OPS polymerization, which utilizes Und-PP. Indirect evidence implicates GtrA (GmlA homolog) as the component required to translocate the lipid-linked sugar to the periplasmic face. Predicted structures of GtrA proteins (and the homologous GmlA) are similar to the multi-antimicrobial extrusion (MATE) family of proteins, which includes the ArnEF heterodimer, a Und-P–Ara4N flippase involved in modification of LPS lipid A (
      • Yan A.
      • Guan Z.
      • Raetz C.R.H.
      An undecaprenyl phosphate-aminoarabinose flippase required for polymyxin resistance in Escherichia coli.
      ). GtrC (GmlC homolog) is a GT-C family glucosyltransferase and adds the side-group glucose to the nascent OPS before ligation to the lipid A-core molecule (
      • Nair A.
      • Korres H.
      • Verma N.K.
      Topological characterisation and identification of critical domains within glucosyltransferase IV (GtrIV) of Shigella flexneri.
      ).
      It was predicted that biosynthesis of the O2aeh OPS occurred through a pathway analogous to O2afg, requiring the activities of the GmlABC2afg homologs. Whole-genome shotgun sequencing was performed on K. pneumoniae CWK53, and the nucleotide sequence between orf7 and hisI revealed a single open reading frame (Fig. 2; orf8). A query of the NCBI Conserved Domain Database (
      • Marchler-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.
      ) revealed that the orf8 gene product contained a domain with similarity to the acyltransferase-3 superfamily. Members of this superfamily include OafA and the Oac proteins involved in O-acetylation of OPSs in Salmonella and Shigella, respectively (
      • Slauch J.M.
      • Lee A.A.
      • Mahan M.J.
      • Mekalanos J.J.
      Molecular characterization of the oafA locus responsible for acetylation of Salmonella typhimurium O-antigen: OafA is a member of a family of integral membrane trans-acylases.
      • Sun Q.
      • Knirel Y.A.
      • Wang J.
      • Luo X.
      • Senchenkova S.N.
      • Lan R.
      • Shashkov A.S.
      • Xu J.
      Serotype-converting bacteriophage SfII encodes an acyltransferase protein that mediates 6-O-acetylation of GlcNAc in Shigella flexneri O-antigens, conferring on the host a novel O-antigen epitope.
      ,
      • Clark C.A.
      • Beltrame J.
      • Manning P.A.
      The oac gene encoding a lipopolysaccharide O-antigen acetylase maps adjacent to the integrase-encoding gene on the genome of Shigella flexneri bacteriophage Sf6.
      • Verma N.K.
      • Brandt J.M.
      • Verma D.J.
      • Lindberg A.A.
      Molecular characterization of the O-acetyl transferase gene of converting bacteriophage SF6 that adds group antigen 6 to Shigella flexneri.
      ). The CKW53 O antigen is O-acetylated non-stoichiometrically (
      • Kelly 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.
      ), and orf8 provides a candidate for the required enzyme. Using BLASTP, a database of predicted polypeptides derived from the CWK53 genomic sequence was queried with the K. pneumoniae CWK55 GmlB amino acid sequence, and a candidate gml locus was identified at a position between proA and a tRNA-Thr coding region on the CWK53 genome (Fig. 2). The predicted amino acid sequences for the CWK55 and CWK53 GmlA and GmlB proteins shared identities of 78% (over 115 amino acids) and 79% (over 308 amino acids), respectively (Fig. S2). The product of the third gene (which we name gmlD) in the O2aeh locus shows no primary structure homology with GmlC2afg. This is expected from the Gtr glucosylation loci, where the initial two enzymes in the pathway provide conserved reactions and the GtrC proteins are serotype-specific glucosyltransferases with differing sequences (
      • Mann E.
      • Whitfield C.
      A widespread three-component mechanism for the periplasmic modification of bacterial glycoconjugates.
      ). The carbohydrate structure of the K. pneumoniae O2ae OPS is identical to O2aeh (
      • Kelly 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.
      ). Analysis of a whole-genome shotgun sequence of the O2ae prototype, CWK52, identified the gmlABD2ae genes at the same genomic locus as in CWK53. The respective GmlA and GmlB amino acid sequences were identical in the two strains, and the GmlD homologs shared 99% identity. The O1 antigen is not expressed in either CWK52 or CWK53 (
      • Kelly 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.
      ,
      • Whitfield 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.
      ), and the wbbY locus was absent in the genomes of these strains.
      The amplified gmlABD2aeh genes were cloned to generate pWQ394. Like the corresponding experiments with O2afg LPS, the ladder in the silver-stained LPS profile from DH5α [rfb2a, gmlABD2aeh] exhibited a band shift, compared with DH5α [rfb2a] LPS (Fig. 3D). This LPS only reacted with antibodies raised against O2aeh LPS from the reference strain (Fig. 3, E and F). These data suggested that the gmlABD2aeh gene cluster from CWK53 was indeed responsible for serotype conversion of the O2a OPS to the O2aeh serotype, and this was confirmed by structural analysis.
      1H and 13C NMR spectroscopy of the OPS fraction purified from DH5α [rfb2a, gmlABD2aeh] was used to confirm the presence of the α-(1→2)–linked Galp side group (Fig. 4). 2D COSY and TOCSY spectra of the recombinant O2aeh polysaccharide revealed spin systems for three sugar residues (designated F, P, and G), all having a galacto configuration. Correlations of the anomeric protons H-1 with H-2, H-3, and H-4 were traced for each spin system, and the remaining H-5 and H-6 were assigned based 1H,1H ROESY and 1H,13C HMBC data. The assignment of 13C chemical shifts was performed using 1H,13C HSQC and HMBC experiments (Fig. S3 and Table 1). The position of F C-1 at δC 110.4, F C-2-C-4 at the region δC 82–86 ppm, and a strong H-1/C-4 correlation observed in the HMBC spectrum are characteristic of β-furanose residues (
      • Bock K.
      • Pedersen C.
      Carbon-13 nuclear magnetic resonance spectroscopy of monosaccharides.
      ). Based on 13C chemical shifts, residues P and G are pyranoses. The α-anomeric configuration of P was established by the small 3J1,2 coupling constant of 3.6 Hz. The signal for G H-1 was unresolved with ν½ < 5 Hz. The α-anomeric configuration of G was inferred from its C-5 chemical shift at δ 72.3 and confirmed by H-1/H-2 correlation in the ROESY spectrum. Interresidue correlations between anomeric carbons and protons at the linkage carbons G C-1/P H-2, P C-1/F H-3, and F C-1/P H-3 observed in the HMBC spectrum (Fig. S3) revealed the positions of substitution and the sequence of the residues in the repeat unit. Hence, the O2aeh OPS obtained from the recombinant strain has a backbone containing [→3)-β-d-Galf-(1→3)-α-d-Galp-(1→] disaccharide repeat units, whose Galp residues are substituted with α-Galp at position 2, identical to the carbohydrate structure of the authentic product (
      • Kelly 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.
      ).

      Production of the K. pneumoniae O2c antigen is thermoregulated, and the biosynthesis genes are unlinked to the rfb2a locus

      The K. pneumoniae O2c antigen is a polymer of alternating β-d-Galf and β-d-GlcpNAc residues co-expressed on the cell surface with the O2a OPS (Fig. 1) (
      • Whitfield C.
      • Perry M.B.
      • MacLean L.L.
      • Yu S.H.
      Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).
      ). NMR data predicted a tandem arrangement of the O2a and O2c antigens in a format resembling the O1 OPS. Using a PCR-based approach, Fang et al (
      • Fang C.-T.
      • Shih Y.-J.
      • Cheong C.-M.
      • Yi W.-C.
      Rapid and accurate determination of lipopolysaccharide O-antigen types in Klebsiella pneumoniae with a novel PCR-based O-genotyping method.
      ) demonstrated that the K. pneumoniae O1 and O2ac serotype strains could be distinguished by the differences in their respective wbbY alleles. In O2ac isolates, wbbY is truncated relative to the O1 allele, and their DNA sequences diverge toward the 3′ end (
      • Fang C.-T.
      • Shih Y.-J.
      • Cheong C.-M.
      • Yi W.-C.
      Rapid and accurate determination of lipopolysaccharide O-antigen types in Klebsiella pneumoniae with a novel PCR-based O-genotyping method.
      ), but the functional implications of this variation are unclear. The O1 polysaccharide was not identified in the Klebsiella O2ac prototype strain 5053 (
      • Whitfield C.
      • Perry M.B.
      • MacLean L.L.
      • Yu S.H.
      Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).
      ), so the impact of “wbbY2ac” from 5053 on OPS biosynthesis was assessed by introducing a plasmid (pWQ398) containing “wbbY2ac” into E. coli DH5α [rfb2a]. The LPS from E. coli DH5α [rfb2a, wbbY2ac] was not recognized by either an O1-specific mAb or O2c-specific antiserum (data not shown), and it was concluded that the truncated O2c “wbbY” allele does not encode a functional protein.
      To identify the genes required for biosynthesis of the O2c antigen, whole-genome shotgun sequence from K. pneumoniae 5053 was examined for candidate glycosyltransferases that were not ascribed to known glycan biosynthesis pathways. A cluster of three open reading frames (designated wbmV, wbmW, and wbmX), each encoding a putative glycosyltransferase, was identified as part of a 12,706-bp contig (Fig. 2). The gene cluster was cloned from 5053 DNA to produce the recombinant plasmid pWQ395. DH5α [rfb2a, wbmVWX] produced LPS composed of both the O2a antigen and an additional OPS recognized by antiserum specific for the O2c antigen (Fig. 5). All three genes in the cluster are essential for the production of the 2c antigen (Fig. 6). Interestingly, expression of the O2c antigen in both the wildtype Klebsiella 5053 and the E. coli-based recombinant was low at 37 °C but enhanced significantly by growth at 30 °C (Fig. 5), indicating that synthesis of this glycan is temperature-regulated. In the wildtype Klebsiella 5053, the increase in 2c antigen was accompanied by a marked reduction in the amount of O2a antigen. These LPS phenotypes were more striking in K. pneumoniae 5053, presumably because the elevated gene copy in the recombinant strain masked some of the regulatory effects. Temperature regulation of the O2a or O1 antigens was not observed in K. pneumoniae O1 strains (data not shown).
      Figure thumbnail gr5
      Figure 5A gene cluster unlinked to the rfb region is involved in thermoregulated biosynthesis of the O2c antigen. A, silver-stained SDS-PAGE of LPS from whole-cell lysates of the wild-type K. pneumoniae 5053 and E. coli DH5α harboring recombinant plasmids containing genes required for the biosynthesis of the O2a and the O2c OPS. B and C, corresponding immunoblots probed with antisera specific for the O2c and O2a OPS, respectively. Cultures were grown at 30 or 37 °C. The rfb2a genes were expressed from pWQ288. The wbmVWX genes were provided by pWQ395.
      Figure thumbnail gr6
      Figure 6Production of the O2c antigen requires three genes (wbmVWX). A, to determine whether all three genes in the wbmVWX are necessary for the expression of the O2c antigen, a series of constructs were made that each eliminate one of the genes. B, silver-stained SDS-PAGE of LPS from whole-cell lysates of E. coli DH5α harboring recombinant plasmids containing genes required for the biosynthesis of the O2a OPS and derivatives of the O2c wbm gene cluster. C and D, corresponding immunoblots probed with antisera specific for the O2c and O2a OPS, respectively. Cultures were grown at 30 °C. The rfb2a genes were expressed from pWQ288. Plasmids pWQ395, pWQ895, pWQ896, and pWQ897 expressed the gene combinations wbmVWX, wbmVX, wbmVW, and wbmWX, respectively.
      The chemical structure of the OPS produced by DH5α [rfb2a, wbmVWX] was confirmed by NMR spectroscopy. The complete assignment of 1H and 13C resonances (Table 1) was performed by a combination of 1D and 2D experiments, including 2D COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC (Figs. S4 and S5). The 1H and 13C chemical shifts are in good agreement with those reported previously for the O2ac polysaccharide (
      • Whitfield C.
      • Perry M.B.
      • MacLean L.L.
      • Yu S.H.
      Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).
      ). Based on the integral intensities in the 1H NMR spectrum, the ratio between the O2a and O2c polymers is ∼1:1.4. In the original report establishing the chemical structure of the O2ac OPS from K. pneumoniae 5053, periodate oxidation studies suggested that the O2a and O2c antigens represented two distinct glycans (each attached to independent LPS molecules) on the bacterial cell surface (
      • Whitfield C.
      • Perry M.B.
      • MacLean L.L.
      • Yu S.H.
      Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c).
      ). However, subsequent NMR data were consistent with a direct linkage between a terminal β-d-Galf residue in the O2a polysaccharide and a GlcpNAc residue from the O2c antigen (
      • Vinogradov E.
      • Frirdich E.
      • MacLean L.L.
      • Perry M.B.
      • Petersen B.O.
      • Duus J.Ø.
      • Whitfield C.
      Structures of lipopolysaccharides from Klebsiella pneumoniae: eluicidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains.
      ) in a format resembling the O1 OPS (Fig. 1). Attempts to determine the type of linkage (if present) between them by interpreting the minor signals in the NMR spectra here were unsuccessful, due to very low intensity and significant overlap with the signals of the internal repeat units. An in vivo approach was therefore used to unequivocally establish the structural format, by examining whether assembly of the O2c antigen was dependent on concurrent biosynthesis of the O2a polymer. E. coli K-12 strain CWG286 was transformed with plasmid pWQ395 (wbmVWX) as well as one of a series of plasmids containing rfb2a loci with single mutations in each gene. CWG286 has a deletion spanning the rfbK-12 region (
      • Köplin 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.
      ), ruling out any contribution to OPS biosynthesis from the E. coli K-12 pathway. Biosynthesis of the O2c OPS was clearly dependent on all genes in the rfb2a cluster (Fig. 7). A dependence on glf to provide the UDP-Galf sugar donor was expected, but dependence on the O2a glycosyltransferases (wbbMNO) and transporter (wzm, wzt) implicated nascent O2a antigen as an acceptor for polymerization, supporting the conclusion that the O2c antigen is indeed added to the non-reducing terminus of O2a OPS chains.
      Figure thumbnail gr7
      Figure 7Biosynthesis of the K. pneumoniae O2c antigen is dependent on a functional rfb2a gene cluster. E. coli CWG286 was transformed with pWQ395 (wbmVWX) together with a series of plasmids containing single mutations in each gene in the rfb2a cluster. LPS in whole-cell lysates was separated by SDS-PAGE and visualized by silver-staining (A) and immunoblotting with antisera specific for O2a (B) and the O2c antigen (C). Und-PP–linked OPS accumulates intracellularly in ABC-transporter (wzm wzt) mutants. This material is poorly stained with silver but detectable by immunoblotting (
      • Kos V.
      • Cuthbertson L.
      • Whitfield C.
      The Klebsiella pneumoniae O2a antigen defines a second mechanism for O antigen ATP-binding cassette transporters.
      ). Individual rfb2a mutations were provided by plasmids pWQ517 (ΔwbbM), pWQ549 (wbbN*; frameshift mutation), pWQ516 (ΔwbbO), pWQ289 (Δwzm Δwzt), and pWQ633 (Δglf).

      Discussion

      Published serological re-evaluations of the Klebsiella O-serotype reference strains led collectively to a consensus of 11 O serotypes (O1, O2a, O2ac, O2aeh(O9), O3, O4, O5, O7, O8, O11, and O12) (
      • Trautmann 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.
      ,
      • Hansen D.S.
      • Mestre F.
      • Alberti S.
      • Hernández-Allés S.
      • Alvarez D.
      • Doménech-Sánchez A.
      • Gil J.
      • Merino S.
      • Tomás J.M.
      • Benedí V.J.
      Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries.
      ). The carbohydrate structures of O1 and O8 are identical (
      • Kelly 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.
      ), and the structure of O11 remains unknown. However, considering the chemical structures of the known OPS molecules, as well as the biosynthesis genes unique to the serotypes and the distribution of these structures in clinical isolates, the number of distinct O-antigen types can minimally be expanded to include O2afg, which is linked to the ST258 clone (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ). Here we described the molecular basis of additional O2 subgroups, focusing only on the carbohydrate components of the structures. Partial O-acetylation can generate additional epitopes that contribute additional O2 subfactors (
      • Kelly 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.
      ,
      • MacLean L.L.
      • Whitfield C.
      • Perry M.B.
      Characterization of the polysaccharide antigen of Klebsiella pneumoniae O:9 lipopolysaccharide.
      ) and provide the only difference between O1 and O8 (
      • Kelly 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.
      ). However, these modifications do not prevent recognition by antibodies generated against the non-acetylated versions of these polysaccharides (
      • Trautmann 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.
      ). Without exhaustive cross-absorption of polyconal sera or more precise monoclonal antibodies, the acetylated/non-acetylated forms will not be distinguished in conventional serological tests.
      Serological typing methods do not easily distinguish all of the galactose-containing O antigens. A PCR-based genotyping method was developed to differentiate the major Klebsiella O groups based on variation among the wzm–wzt alleles within the rfb region (
      • Fang C.-T.
      • Shih Y.-J.
      • Cheong C.-M.
      • Yi W.-C.
      Rapid and accurate determination of lipopolysaccharide O-antigen types in Klebsiella pneumoniae with a novel PCR-based O-genotyping method.
      ), but the shared rfb region present in all O1 and “O2” strains precluded subclassification within this serogroup. This method did discriminate between the O1 and O8 serotypes, which seems surprising, given the conserved function of these transporters. However, this is explained by the phylogenetic separation of O8 isolates from O1 and “O2” and consistent with rfb DNA hybridization analyses (
      • Kelly 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.
      ). Fang et al. (
      • Fang C.-T.
      • Shih Y.-J.
      • Cheong C.-M.
      • Yi W.-C.
      Rapid and accurate determination of lipopolysaccharide O-antigen types in Klebsiella pneumoniae with a novel PCR-based O-genotyping method.
      ) distinguished serotype O1 and O2ac strains by differences in the wbbY alleles, which are truncated in O2ac isolates and shown here to generate a non-functional enzyme. With data reported here, O-genotyping could now be refined with a positive PCR by utilizing oligonucleotides specific for the O2c biosynthesis gene(s). As a diagnostic tool, genetic classification of Klebsiella O types should be considered complementary to serological typing, as strains testing positive for a particular OPS biosynthesis locus may harbor mutations preventing expression of the antigen on the cell surface (
      • Hansen D.S.
      • Mestre F.
      • Alberti S.
      • Hernández-Allés S.
      • Alvarez D.
      • Doménech-Sánchez A.
      • Gil J.
      • Merino S.
      • Tomás J.M.
      • Benedí V.J.
      Klebsiella pneumoniae lipopolysaccharide O typing: revision of prototype strains and O-group distribution among clinical isolates from different sources and countries.
      ,
      • Fang C.-T.
      • Shih Y.-J.
      • Cheong C.-M.
      • Yi W.-C.
      Rapid and accurate determination of lipopolysaccharide O-antigen types in Klebsiella pneumoniae with a novel PCR-based O-genotyping method.
      ).
      The gmlABC2afg and gmlABD2aeh loci are responsible for modifying the O2a-antigen backbone with α-(1→4) and α-(1→2)-linked galactose residues, respectively. The sequence differences in the GmlC2afg and GmlD2aeh orthologues make these genes useful markers in a genotyping scheme for distinguishing these O-serotypes. The O2aeh OPS is structurally similar to that of O2ae (which is identical to O9). These polymers are acetylated, and the only detectable difference is the frequency of both acetylation and modification by the α-(1→-2)-d-Galp side group (
      • Kelly 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.
      ). The physical separation of polymerization (cytoplasm) and side-chain addition (periplasm) creates an opportunity to have populations of LPS species with varying amounts of modification or potentially subdomains with an individual chain that varies in the extent of modification. The gmlABD sequences of O2aeh and O2ae are nearly identical, so the variable stoichiometry of Galp substitution is most likely due to differences in transcription/translation of one or more biosynthetic genes or some (unknown) instability that leads to loss of the locus in a portion of the population. Serotype O2afg has been associated with the drug-resistant strain ST258 (
      • Szijártó V.
      • Guachalla L.M.
      • Hartl K.
      • Varga C.
      • Banerjee P.
      • Stojkovic K.
      • Kaszowska M.
      • Nagy E.
      • Lukasiewicz J.
      • Nagy G.
      Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.
      ) and shown to be frequently distributed in a globally representative collection of K. pneumoniae isolates (
      • Follador R.
      • Heinz E.
      • Wyres K.L.
      • Ellington M.J.
      • Kowarik M.
      • Holt K.E.
      • Thomson N.R.
      The diversity of Klebsiella pneumoniae surface polysaccharides.
      ). In this collection of 573 whole-genome sequenced isolates, 387 isolates (67%) contain the rfb2a locus. We re-examined this collection using the new genetic insight. Of these, 164 isolates (42%) were identified as containing the gmlABC2afg cluster, showing the seroepidemiological importance of the O2afg antigen (Fig. S6). Surprisingly, no gmlABD2aeh cluster was detected in any of these isolates, leaving the natural distribution of serotype O2aeh uncertain.
      The majority of three-component glycan modification systems described so far in Gram-negative bacteria modify OPS produced by Wzy-dependent biosynthesis pathways, where Und-PP-linked oligosaccharide repeat units are translocated to the periplasm for polymerization (
      • Mann E.
      • Whitfield C.
      A widespread three-component mechanism for the periplasmic modification of bacterial glycoconjugates.
      ). The Klebsiella O antigens are synthesized by the ABC-transporter pathway, in which the entire OPS is polymerized on Und-PP in the cytoplasm, before export to the periplasm. In a hybrid ABC transporter–dependent pathway reconstituted in E. coli K-12, a host Gtr periplasmic glucosylation system modifies the K. pneumoniae O12 OPS (
      • Mann E.
      • Ovchinnikova O.G.
      • King J.D.
      • Whitfield C.
      Bacteriophage-mediated glucosylation can modify lipopolysaccharide O-antigens synthesized by an ATP-binding cassette (ABC) transporter-dependent assembly mechanism.
      ), illustrating that the polymerization strategy poses no barrier for these types of modification. The Klebsiella O2afg and O2aeh strains are the only examples in which Gtr-like modifications have been unequivocally demonstrated for native ABC transporter–dependent OPS biosynthesis pathways. In E. coli, Shigella, and Salmonella, the gtrABC genes are often associated with lysogenic bacteriophages, and the associated OPS side-group modifications may function to prevent superinfection by bacteriophage that recognize the unmodified OPS as a binding receptor (
      • Mann E.
      • Ovchinnikova O.G.
      • King J.D.
      • Whitfield C.
      Bacteriophage-mediated glucosylation can modify lipopolysaccharide O-antigens synthesized by an ATP-binding cassette (ABC) transporter-dependent assembly mechanism.
      ). Altering the OPS may also promote bacteriophage reproduction by affecting immune evasion and enhanced survival of the host bacteria (
      • Davies M.R.
      • Broadbent S.E.
      • Harris S.R.
      • Thomson N.R.
      • van der Woude M.W.
      Horizontally acquired glycosyltransferase operons drive Salmonellae lipopolysaccharide diversity.
      ,
      • Kintz E.
      • Heiss C.
      • Black I.
      • Donohue N.
      • Brown N.
      • Davies M.R.
      • Azadi P.
      • Baker S.
      • Kaye P.M.
      • van der Woude M.
      Salmonella enterica serovar Typhi lipopolysaccharide O-antigen modification impact on serum resistance and antibody recognition.
      ). In one study, 20 of 22 Salmonella enterica subspecies enterica genomes contained between two and four gtr loci (
      • Davies M.R.
      • Broadbent S.E.
      • Harris S.R.
      • Thomson N.R.
      • van der Woude M.W.
      Horizontally acquired glycosyltransferase operons drive Salmonellae lipopolysaccharide diversity.
      ), each conferring a distinct OPS modification. These gtr loci are located on prophages or within bacteriophage-derived regions, giving rise to a mobile trait that can diversify by recombination. The gmlABD loci in K. pneumoniae O2aeh and O2ea are located next to a tRNA-Thr gene, whereas the gmlABC2afg locus is situated between the rfb2a cluster and hisI. There is no evidence of homology to bacteriophage DNA within or near either of the gml gene clusters.
      The K. pneumoniae O2ac genes wbmVWX are required for the biosynthesis of the O2c OPS composed of alternating d-Galf and d-GlcpNAc added to the end of the rfb2a-encoded O2a antigen. The OPS structure (Fig. 1) predicts three required glycosyltransferases creating three linkage types, two for the disaccharide O2c-antigen repeat unit itself and one for the transition between the two glycan domains (i.e. between O2a and O2c). In K. pneumoniae 5053, this gene cluster was flanked by putative insertion elements, suggesting that this region was acquired by a transposition event. Horizontal acquisition of the locus is supported by the relatively low G+C content (35%) compared with 56% G+C for the entire genomic sequence (data not shown). In Klebsiella, the O2c antigen is thought to be extended on a Galf residue from the O2a polysaccharide (
      • Vinogradov E.
      • Frirdich E.
      • MacLean L.L.
      • Perry M.B.
      • Petersen B.O.
      • Duus J.Ø.
      • Whitfield C.
      Structures of lipopolysaccharides from Klebsiella pneumoniae: eluicidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains.
      ). It is conceivable that the O2c antigen could also be assembled on glycans with different repeat unit structures, providing the appropriate terminal Galf is available to provide an acceptor. We re-examined the collection of 573 Klebsiella genomes and identified 14 isolates where the full wbmVWX locus was present and two others with a truncated cluster lacking wbmX (Fig. S6). Three of these 14 isolates also possess the gml2afg locus, potentially giving rise to more serotype complexity. Furthermore, 10 of the 16 isolates are from the pre-antibiotic collection, consisting of isolates collected before 1947. Interestingly, those strains with the full wbmVWX locus lack wbbY, whereas those possessing only wbmV and wbmW possess an intact wbbY and would probably serotype as O1.
      The O2c antigen provides the only example of thermoregulated OPS in K. pneumoniae. The details of the thermoregulation have not been investigated and are not central to the objectives of the current study. Unfortunately, we do not have other authenticated O2ac isolates to confirm that this property is conserved in all representatives of this serotype. Temperature regulation has been described for the biosynthesis of a few other OPSs in other bacteria. The Yersinia enterocolitica O:8 OPS is expressed at 22–25 °C but transcriptionally down-regulated at 37 °C (
      • Bengoechea J.A.
      • Zhang L.
      • Toivanen P.
      • Skurnik M.
      Regulatory network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica includes cell envelope-dependent signals.
      ). Its expression is critical for virulence of Y. enterocolitica (
      • Zhang L.
      • Radziejewska-Lebrecht J.
      • Krajewska-Pietrasik D.
      • Toivanen P.
      • Skurnik M.
      Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O:8.
      ,
      • Young G.M.
      • Miller V.L.
      Identification of novel chromosomal loci affecting Yersinia enterocolitica pathogenesis.
      • Darwin A.J.
      • Miller V.L.
      Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis.
      ), but it has been proposed that after colonization, down-regulation of OPS is necessary for exposure or function of other virulence factors (
      • Bengoechea J.A.
      • Zhang L.
      • Toivanen P.
      • Skurnik M.
      Regulatory network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica includes cell envelope-dependent signals.
      ,
      • Straley S.C.
      • Perry R.D.
      Environmental modulation of gene expression and pathogenesis in Yersinia.
      ). Why such a process would be required in O2ac and not in other serotypes is unclear. It is conceivable that the O2c antigen plays no active role in pathogenesis, perhaps explaining its limited distribution.
      With these studies, the molecular genetic basis for the known serological complexity in serotypes O1 and O2 is now resolved. Establishing the essential genetic complement for the biosynthesis of these OPS structures affords the opportunity to synthesize precise glycans for therapeutic applications in defined recombinant E. coli backgrounds. However, it has also provided the tools necessary to rule out isolates of the O2ac and O2aeh serotypes as major components in collections of clinical isolates.

      Experimental procedures

      Bacterial strains and growth conditions

      The bacterial strains and plasmids used in this study are listed in Table 2. Cultures were grown either in lysogeny broth (LB) (
      ) or on LB agar, and the antibiotics, ampicillin (100 μg/ml) or chloramphenicol (34 μg/ml), were added when required. For growth of CWG286, overnight starter cultures contained 0.4% (w/v) d-glucose, and these were subcultured into LB containing 0.1% (w/v) d-galactose.
      Table 2Bacterial strains and plasmids
      Strain or plasmidDescriptionReference
      Bacterial strains
          E. coli DH5αK-12 Fϕ80lacZΔM15 Δ(lacZYA-argF) U169 deoR recA1 endA1 hsdR17 (rk, mk+) gal phoA supE44 thi gyrA96 relA1Ref.
      • Bethesda Research Laboratories
      BRL pUC host: E. coli DH5αTM competent cells.
          E. coli CWG286K-12 lacZ trp Δ(sbc-rfb) upp rel rpsL galE::Tn10Ref.
      • Köplin 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.
          K. pneumoniae CWK2Serotype O1:K. Acapsular mutant of strain 889/50 (O1:K20)Ref.
      • McCallum K.L.
      • Schoenhals G.
      • Laakso D.
      • Clarke B.
      • Whitfield C.
      A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.
          K. pneumoniae CWK37Derivative of CWK2 expressing only the O2a antigenRef.
      • McCallum K.L.
      • Schoenhals G.
      • Laakso D.
      • Clarke B.
      • Whitfield C.
      A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.
          K. pneumoniae CWK52Serotype O2ae:K. Acapsular mutant of strain 7444 (O2ae:K35)Ref.
      • Kelly 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.
          K. pneumoniae CWK53Serotype O2aeh:K. Acapsular mutant of strain 2212/52 (O2aeh:K59)Ref.
      • Kelly 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.
          K. pneumoniae CWK55Serotype O2afg:K. Acapsular mutant of strain 6613 (O2afg:K27)Ref.
      • Kelly 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.
          K. pneumoniae 5053Serotype O2ac:KRef.
      • Ørskov I.
      O antigens in the Klebsiella group.
      Plasmids
          pWQ288pACYC184 derivative containing the rfb2a gene cluster (wzm, wzt, wbbM, glf, wbbN, wbbO)Ref.
      • Kos V.
      • Cuthbertson L.
      • Whitfield C.
      The Klebsiella pneumoniae O2a antigen defines a second mechanism for O antigen ATP-binding cassette transporters.
          pWQ289pWQ288 derivative with a deletion in wzm and wztRef.
      • Kos V.
      • Cuthbertson L.
      • Whitfield C.
      The Klebsiella pneumoniae O2a antigen defines a second mechanism for O antigen ATP-binding cassette transporters.
          pWQ392pBRC322 derivative containing the wbbOhisI region from CWK55This work
          pWQ393pBR322 derivative containing the gmlABC2afg genes from CWK55This work
          pWQ394pBR322 derivative containing the gmlABD2aeh genes from CWK53This work
          pWQ395pBR322 derivative containing wbmVWX, from 5053This work
          pWQ398pBR322 derivative containing wbbYZ from 5053This work
          pWQ516pWQ288 derivative with a deletion in wbbORef.
      • Kos V.
      • Cuthbertson L.
      • Whitfield C.
      The Klebsiella pneumoniae O2a antigen defines a second mechanism for O antigen ATP-binding cassette transporters.
          pWQ517pWQ288 derivative with a deletion in wbbMRef.
      • Kos V.
      • Cuthbertson L.
      • Whitfield C.
      The Klebsiella pneumoniae O2a antigen defines a second mechanism for O antigen ATP-binding cassette transporters.
          pWQ549pWQ288 derivative with a frame-shift mutation in wbbN (encodes the first 79 amino acids)Ref.
      • Kos V.
      • Cuthbertson L.
      • Whitfield C.
      The Klebsiella pneumoniae O2a antigen defines a second mechanism for O antigen ATP-binding cassette transporters.
          pWQ633pWQ288 derivative with a deletion in glfUnpublished
          pWQ895pWQ395 derivative with a λ-red–mediated deletion of wbmWThis work
          pWQ896pBR322 derivative containing wbmVW from 5053This work
          pWQ897pBR322 derivative containing wbmWX from 5053This work

      Construction of recombinant plasmids

      KOD Hot Start DNA polymerase (Novagen) was used to amplify DNA fragments by PCR. Oligonucleotide primers (Sigma) along with their relevant characteristics are listed in Table S1. PCR products were purified from reactions using the PureLink PCR purification kit (Invitrogen). Plasmid and genomic DNA were purified with the PureLink plasmid miniprep kit and the PureLink genomic minikit, respectively (Invitrogen). Recombinant plasmids used in this study were constructed by cloning PCR fragments into the vector pBR322 by Gibson Assembly (New England Biolabs). Briefly, pBR322 was digested with the restriction endonucleases BamHI and SalI (New England Biolabs), and inserts were incorporated downstream of the tetracycline promoter by homologous recombination, mediated by primer sequences homologous to DNA flanking the restriction sites in pBR322. BamHI and SalI sites were retained in the recombinant plasmids. When cloning the convergently transcribed K. pneumoniae 5053 wbmVWX genes to produce pWQ395, potential promoter and/or regulatory elements were accommodated by cloning a DNA fragment that included non-coding sequences between the putative gene cluster and transposase genes flanking the locus (Fig. 2). Plasmid pWQ895 was constructed by replacing the wbmW gene with the kanamycin resistance gene from pKD4 by λ-red–mediated recombination (
      • Datsenko K.A.
      • Wanner B.L.
      One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.
      ,
      • Datta S.
      • Costantino N.
      • Court D.L.
      A set of recombineering plasmids for Gram-negative bacteria.
      ). Plasmid constructs were assessed by restriction endonuclease digestion and confirmed by DNA sequencing performed by the Advanced Analysis Centre, Genomics Facility, University of Guelph.

      Genomic DNA sequencing

      Whole-genome shotgun sequence data (Illumina paired end reads; ∼100× coverage) for K. pneumoniae 5053 and CWK2 were obtained from the Advanced Analysis Centre, University of Guelph, using an Illumina Miseq platform. Libraries were prepared with the Nextera XT kit (Illumina) to give ∼675-bp inserts. De novo assembly was performed using SPAdes (
      • Bankevich A.
      • Nurk S.
      • Antipov D.
      • Gurevich A.A.
      • Dvorkin M.
      • Kulikov A.S.
      • Lesin V.M.
      • Nikolenko S.I.
      • Pham S.
      • Prjibelski A.D.
      • Pyshkin A.V.
      • Sirotkin A.V.
      • Vyahhi N.
      • Tesler G.
      • Alekseyev M.A.
      • Pevzner P.A.
      SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing.
      ), and the contigs were ordered with MAUVE (
      • Darling A.C.E.
      • Mau B.
      • Blattner F.R.
      • Perna N.T.
      Mauve: multiple alignment of conserved genomic sequence with rearrangements.
      ), using the K. pneumoniae NTUH-K2044 genomic sequence (accession number AP006725) as a reference. Annotation of predicted protein sequences was performed using the RAST server (
      • Aziz R.K.
      • Bartels D.
      • Best A.A.
      • DeJongh M.
      • Disz T.
      • Edwards R.A.
      • Formsma K.
      • Gerdes S.
      • Glass E.M.
      • Kubal M.
      • Meyer F.
      • Olsen G.J.
      • Olson R.
      • Osterman A.L.
      • Overbeek R.A.
      • et al.
      The RAST server: rapid annotations using subsystems technology.
      ). Genomic sequencing of CWK52, CWK53, and CWK55 was performed on the Solexa paired-end sequencing platform (Illumina). Genomic DNA was prepared using the Illumina sample preparation kit to give paired libraries with 500-bp inserts. De novo assembly of sequencing reads (∼100× coverage) was performed with Velvet (
      • Zerbino D.R.
      • Birney E.
      Velvet: algorithms for de novo short read assembly using de Bruijn graphs.
      ). The sequences of wbbYZ (from CWK2; GenBankTM accession number MG458672), gmlABC2afg(MG458669), gmlABD2aeh (MG458670), gmlABD2ae (MG458671),rfb2aeh (MG280710), and wbmVWX (MG602074) were deposited at NCBI.

      Bioinformatics analyses

      BLAST analyses (
      • Altschul S.F.
      • Gish W.
      • Miller W.
      • Myers E.W.
      • Lipman D.J.
      Basic local alignment search tool.
      ) of nucleotide and amino acid sequences were performed using the National Center for Biotechnology Information (NCBI) server (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and the standalone BLAST software. Protein motifs were searched using the Conserved Domain Database server (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (
      • Marchler-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.
      ) and the Pfam protein motif database (http://xfam.org).
      Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
      Insertion elements were identified using ISFinder (https://www-is.biotoul.fr)5 (
      • Siguier P.
      • Perochon J.
      • Lestrade L.
      • Mahillon J.
      • Chandler M.
      ISfinder: the reference centre for bacterial insertion sequences.
      ). The WbbY amino acid sequence used for BLASTP queries was from K. pneumoniae NTUH-K2044 (GenBankTM accession number KJ451390). The origin of the globally representative collection of 573 K. pneumoniae isolates has been described in detail elsewhere (
      • Follador R.
      • Heinz E.
      • Wyres K.L.
      • Ellington M.J.
      • Kowarik M.
      • Holt K.E.
      • Thomson N.R.
      The diversity of Klebsiella pneumoniae surface polysaccharides.
      ). In short, four different collections have been analyzed: a global data set consisting of isolates from six different countries (
      • Holt K.E.
      • Wertheim H.
      • Zadoks R.N.
      • Baker S.
      • Whitehouse C.A.
      • Dance D.
      • Jenney A.
      • Connor T.R.
      • Hsu L.Y.
      • Severin J.
      • Brisse S.
      • Cao H.
      • Wilksch J.
      • Gorrie C.
      • Schultz M.B.
      • et al.
      Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health.
      ), a UK hospital data set collected over a period of 7 years, a Nepal hospital data set from a single outbreak (
      • Chung The H.
      • Karkey A.
      • Pham Thanh D.
      • Boinett C.J.
      • Cain A.K.
      • Ellington M.
      • Baker K.S.
      • Dongol S.
      • Thompson C.
      • Harris S.R.
      • Jombart T.
      • Le Thi Phuong T.
      • Tran Do Hoang N.
      • Ha Thanh T.
      • Shretha S.
      • et al.
      A high-resolution genomic analysis of multidrug-resistant hospital outbreaks of Klebsiella pneumoniae.
      ), and a pre-antibiotic data set from strains isolated before the widespread use of antibiotics (
      • Wand M.E.
      • Baker K.S.
      • Benthall G.
      • McGregor H.
      • McCowen J.W.I.
      • Deheer-Graham A.
      • Sutton J.M.
      Characterization of pre-antibiotic era Klebsiella pneumoniae isolates with respect to antibiotic/disinfectant susceptibility and virulence in Galleria mellonella.
      ). The presence/absence analysis of the genetic elements rfb2a, wbbYZ, gmlABC2afg, gmlABD2aeh, and wbmVWX has been performed as described previously (
      • Follador R.
      • Heinz E.
      • Wyres K.L.
      • Ellington M.J.
      • Kowarik M.
      • Holt K.E.
      • Thomson N.R.
      The diversity of Klebsiella pneumoniae surface polysaccharides.
      ).

      Antiserum preparation

      Rabbit anti-O2a serum and monoclonal antibody O1–52.6 (anti-O1) were reported previously (
      • McCallum K.L.
      • Schoenhals G.
      • Laakso D.
      • Clarke B.
      • Whitfield C.
      A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.
      ). Antisera against the O2afg (CWK55), the O2aeh (CWK53), and the O2ac (5053) antigens were prepared by immunizing New Zealand White rabbits. Formalin-killed cells were suspended in 0.85% (w/v) NaCl at ∼108 cfu/ml and mixed 1:1 with Freund’s incomplete adjuvant (Sigma). Cells (0.5 ml) were injected intramuscularly every 2 weeks for 6 weeks, after which blood was collected and the sera were stored at −80 °C. Antibodies cross-reacting with the O2a antigen were removed by adsorption with whole cells of CWK37 for anti-O2afg and anti-O2aeh and with DH5α [pWQ288] for anti-O2ac.

      SDS-PAGE and immunoblotting

      LPS samples for SDS-PAGE were prepared by proteinase K treatment of whole-cell lysates (
      • Hitchcock P.J.
      • Brown T.M.
      Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.
      ) from 1 A600 nm unit of bacterial culture. Samples (5 μl) were separated on 12% acrylamide gels in Tris-glycine buffer (
      • Laemmli U.K.
      Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
      ), and LPS was visualized by silver staining (
      • Tsai C.M.
      • Frasch C.E.
      A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels.
      ). For immunoblotting, LPS was transferred to nitrocellulose membrane (Protran, GE Healthcare) in 25 mm Tris, 150 mm glycine, 20% (v/v) methanol at 200 mA for 45 min, and the membranes were incubated in 5% (w/v) skim milk powder (BD Difco) in TBST (10 mm Tris-Cl, pH 7.5, 150 mm NaCl, 0.005% (v/v) Tween 20). The immunoblots were probed with antisera diluted as follows in 5% (w/v) skim milk (in TBST): anti-O2a, anti-O2afg, anti-O2ac (1:1000), and anti-O2aeh (1:3000). Goat anti-rabbit alkaline phosphatase (Cedar Lane) was used as a secondary antibody, and detection was performed using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche Applied Science).

      Isolation of O-polysaccharides

      Overnight cultures (10 liters) of E. coli DH5α [pWQ288] (rfb2a), DH5α [pWQ288, pWQ393] (rfb2a, gmlABC2afg), DH5α [pWQ288, pWQ394] (rfb2a, gmlABD2aeh), and DH5α [pWQ288, pWQ395] (rfb2a, wbmVWX) were grown with shaking at 200 rpm. All cultures were grown at 37 °C except for DH5α [pWQ288, pWQ395], which was grown at 30 °C. Cells were harvested by centrifugation at 5,000 × g, washed with distilled water, and lyophilized. LPS was isolated by hot phenol-water extraction (
      • Westphal O.
      • Jann K.
      Bacterial lipopolysaccharides: extraction with phenol water and further application of procedure.
      ). Briefly, 4–5 g of dry cells were extracted with 300 ml of 45% (v/v) aqueous phenol at 70 °C with constant stirring. After cooling, the phenol and water phases were separated by centrifugation, and the phenol phase was re-extracted with an equal volume of preheated water. The pooled water phase was dialyzed against tap water to remove the phenol and then concentrated with a rotary evaporator. The crude LPS solution was adjusted to pH 2 with cold aqueous CCl3COOH. Precipitated proteins and nucleic acids were removed by centrifugation at 12,000 × g, and the supernatant was dialyzed against distilled water and lyophilized. LPS isolated from E. coli DH5α [pWQ288, pWQ395] was subjected to an additional ultracentrifugation step at 105,000 × g for 16 h at 4 °C. Purified LPS samples (150 mg) were hydrolyzed with 2% (v/v) acetic acid at 100 °C until precipitation was observed (2–4 h). The lipid precipitate was removed by centrifugation at 13,000 × g, and the carbohydrate-containing supernatant was fractionated on a Sephadex G-50 superfine column (2.5 cm × 75 cm) in 50 mm pyridinium acetate buffer (pH 4.5) at a flow rate of 0.6 ml min−1. Elution was monitored with a Smartline 2300 refractive index detector (Knauer).

      Nuclear magnetic resonance spectroscopy

      NMR studies were performed at the University of Guelph Advanced Analysis Centre. Polysaccharide samples were deuterium-exchanged by lyophilizing twice from 99.9% D2O and then analyzed as solutions in 99.96% D2O. NMR spectra were recorded at 50 °C (O2a, O2afg, and O2aeh polysaccharides) and 30 °C (O2ac polysaccharide) on a Bruker AvanceII 600-MHz spectrometer equipped with a cryoprobe. The Bruker TopSpin 3.2 program was used to acquire and process the NMR data. Mixing times of 100 and 200 ms were used in TOCSY and ROESY experiments, respectively. The HMBC experiment was optimized for the JH,C coupling constant of 8 Hz. The chemical shifts are referenced to 3-trimethylsilylpropanoate-2,2,3,3-d4H 0, δC–1.6) added as an internal standard.

      Author contributions

      B. R. C., O. G. O., T. L. L., and C. W. conceptualization; B. R. C., O. G. O., S. D. K., M. L. W., J. E. B., B. L., L. W., X. G., and R. F. data curation; B. R. C., O. G. O., S. D. K., B. L., R. F., T. L. L., and C. W. formal analysis; B. R. C., O. G. O., S. D. K., M. L. W., J. E. B., B. L., and R. F. investigation; B. R. C., O. G. O., T. L. L., and C. W. writing-original draft; B. R. C., O. G. O., R. F., T. L. L., and C. W. writing-review and editing; L. W., X. G., and R. F. methodology; T. L. L. and C. W. funding acquisition; C. W. supervision.

      Note added in proof

      While this paper was in review Pennini et al. (
      • Pennini M.E.
      • De Marco A.
      • Pelletier M.
      • Bonnell J.
      • Cvitkovic R.
      • Beltramello M.
      • Cameroni E.
      • Bianchi S.
      • Zatta F.
      • Zhao X.
      • Camara M.M.
      • DiGiandomenico A.
      • Semenova E.
      • Lanzavecchia A.
      • Warrener P.
      • Suzich J.
      • Wang Q.
      • Corti D.
      • Stover C.K.
      Immune stealth-drive driven O2 serotype prevalence and potential for therapeutic antibodies against multidrug resistant Klebsiella pneumoniae.
      ) published further data on the clinical distribution of O2 serotype Klebsiella pneumoniae, with a focus on multidrug-resistant isolates, and showed the protective ability of anti-O2 monoclonal antibodies in a mouse infection model.

      Supplementary Material

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