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Structure and Biosynthesis of Two Exopolysaccharides Produced by Lactobacillus johnsonii FI9785*

  • Author Footnotes
    1 Supported by the Turkish Ministry of Education.
    Enes Dertli
    Footnotes
    1 Supported by the Turkish Ministry of Education.
    Affiliations
    From the Gut Health and Food Safety Programme, Institute of Food Research, Colney, Norwich NR4 7UA, United Kingdom,

    the Department of Food Engineering, Faculty of Engineering, Bayburt University, Bayburt 69000, Turkey, and
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  • Ian J. Colquhoun
    Affiliations
    the Analytical Sciences Unit, Institute of Food Research,
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  • A. Patrick Gunning
    Affiliations
    the Food and Health Programme, Institute of Food Research, and
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  • Roy J. Bongaerts
    Affiliations
    the Analytical Sciences Unit, Institute of Food Research,
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  • Gwénaëlle Le Gall
    Affiliations
    the Analytical Sciences Unit, Institute of Food Research,
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  • Author Footnotes
    2 Recipient of instrument funding from BBSRC Grant BB/C510924 and a contribution from Varian Inc. (Agilent Technologies).
    Boyan B. Bonev
    Footnotes
    2 Recipient of instrument funding from BBSRC Grant BB/C510924 and a contribution from Varian Inc. (Agilent Technologies).
    Affiliations
    the School of Biomedical Sciences, University of Nottingham, Nottingham NG7 2UH, United Kingdom
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  • Melinda J. Mayer
    Correspondence
    To whom correspondence should be addressed: Gut Health and Food Safety Programme, Institute of Food Research, Colney, Norwich, NR4 7UA, United Kingdom. Tel.: 44-1603-255284; Fax: 44-1603-507723
    Affiliations
    From the Gut Health and Food Safety Programme, Institute of Food Research, Colney, Norwich NR4 7UA, United Kingdom,
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  • Arjan Narbad
    Affiliations
    From the Gut Health and Food Safety Programme, Institute of Food Research, Colney, Norwich NR4 7UA, United Kingdom,
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  • Author Footnotes
    2 Recipient of instrument funding from BBSRC Grant BB/C510924 and a contribution from Varian Inc. (Agilent Technologies).
    * This work was supported in part by Biotechnology and Biological Science Research Council (BBSRC) Strategic Core Grants BB/J004529/1 and BB/J004545/1 and the European Union FP7 TORNADO program.
    1 Supported by the Turkish Ministry of Education.
Open AccessPublished:September 09, 2013DOI:https://doi.org/10.1074/jbc.M113.507418
      Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1–3)-β-Glcp-(1–5)-β-Galf-(1–6)-α-Glcp-(1–4)-β-Galp-(1–4)-β-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.
      Background: Bacterial cell surface polysaccharides are important in pathogenesis, cell adhesion, and protection against harsh environments.
      Results: Two novel exopolysaccharide (EPS) structures were identified in Lactobacillus johnsonii.
      Conclusion: The eps cluster is essential for production of both EPS, but epsE is required only for the heteropolymer.
      Significance: This study will guide functional analysis of EPS in survival and colonization of gut commensals.

      Introduction

      Exopolysaccharides (EPS)
      The abbreviations used are: EPS
      exopolysaccharide(s)
      AFM
      atomic force microscopy
      TEM
      transmission electron microscopy
      pN
      piconewtons
      TOCSY
      total correlation spectroscopy
      ROESY
      rotating frame NOE spectroscopy
      HMBC
      heteronuclear multiple-bond correlation spectroscopy
      HSQC
      heteronuclear single quantum correlation
      MAS
      magic angle spinning.
      encapsulate some bacteria, either remaining bound to the cell or being released into the environment (
      • Badel S.
      • Bernardi T.
      • Michaud P.
      New perspectives for lactobacilli exopolysaccharides.
      ,
      • De Vuyst L.
      • Degeest B.
      Heteropolysaccharides from lactic acid bacteria.
      ). They have been shown to be important for the genus Lactobacillus for their putative roles in colonization, adhesion, stress resistance, host-bacteria interactions, and also immunomodulation, which are all important properties related to their probiotic functions (
      • Delcour J.
      • Ferain T.
      • Deghorain M.
      • Palumbo E.
      • Hols P.
      The biosynthesis and functionality of the cell-wall of lactic acid bacteria.
      ). EPS are also of considerable interest to the food industry, due to their rheological properties and GRAS (generally regarded as safe) status (
      • Badel S.
      • Bernardi T.
      • Michaud P.
      New perspectives for lactobacilli exopolysaccharides.
      ). The structure of bacterial EPS has a wide diversity among different species due to the different sugar monomers and glycosidic linkages present in their repeating units. Those containing only one type of sugar molecule are described as homopolysaccharides, whereas heteropolysaccharides are composed of different sugar monomers (
      • De Vuyst L.
      • Degeest B.
      Heteropolysaccharides from lactic acid bacteria.
      ,
      • Delcour J.
      • Ferain T.
      • Deghorain M.
      • Palumbo E.
      • Hols P.
      The biosynthesis and functionality of the cell-wall of lactic acid bacteria.
      ). The structural differences of the capsular EPS influence their functional characteristics in relation to colonization and regulation of host response (
      • Delcour J.
      • Ferain T.
      • Deghorain M.
      • Palumbo E.
      • Hols P.
      The biosynthesis and functionality of the cell-wall of lactic acid bacteria.
      ,
      • Hidalgo-Cantabrana C.
      • López P.
      • Gueimonde M.
      • los Reyes-Gavilán C.
      • Suárez A.
      • Margolles A.
      • Ruas-Madiedo P.
      Immune modulation capability of exopolysaccharides synthesised by lactic acid bacteria and Bifidobacteria.
      ,
      • Lin M.H.
      • Yang Y.L.
      • Chen Y.P.
      • Hua K.F.
      • Lu C.P.
      • Sheu F.
      • Lin G.H.
      • Tsay S.S.
      • Liang S.M.
      • Wu S.H.
      A novel exopolysaccharide from the biofilm of Thermus aquaticus YT-1 induces the immune response through Toll-like receptor 2.
      ). Therefore, identification of the primary structure of capsular polysaccharides produced by members of the genus Lactobacillus may provide valuable information on the functional properties of EPS.
      Lactobacillus johnsonii FI9785 is a poultry-derived isolate that is being investigated as a potential probiotic that may be given to poultry for use as a competitive exclusion agent to control Clostridium perfringens (
      • La Ragione R.M.
      • Narbad A.
      • Gasson M.J.
      • Woodward M.J.
      In vivo characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry.
      ). C. perfringens is a cause of human food poisoning, but some strains are also responsible for necrotic enteritis in poultry, causing problems of animal welfare as well as huge economic losses to the poultry industry worldwide. L. johnsonii FI9785 has been shown to adhere well to tissue culture and chick gut explant tissues, out-competing pathogenic bacteria in challenge models. However, the mode of action by which L. johnsonii FI9785 achieves this protective effect is unknown.
      L. johnsonii 142 and L. johnsonii NCC533 have also been shown to produce capsular EPS, and deletion of the eps cluster in the strain NCC533 resulted in an acapsular phenotype and affected residence time in the murine gut (
      • Górska S.
      • Jachymek W.
      • Rybka J.
      • Strus M.
      • Heczko P.B.
      • Gamian A.
      Structural and immunochemical studies of neutral exopolysaccharide produced by Lactobacillus johnsonii 142.
      ,
      • Denou E.
      • Pridmore R.D.
      • Berger B.
      • Panoff J.M.
      • Arigoni F.
      • Brüssow H.
      Identification of genes associated with the long-gut-persistence phenotype of the probiotic Lactobacillus johnsonii strain NCC533 using a combination of genomics and transcriptome analysis.
      ). Little is known about the function of the capsular EPS and the mechanism of the biosynthesis for the genus Lactobacillus. Previously, the genome of L. johnsonii FI9785 was shown to include a 14.9-kb region that harbors 14 putative genes that may be responsible for the EPS biosynthesis in this strain (Fig. 1) (
      • Horn N.
      • Wegmann U.
      • Dertli E.
      • Mulholland F.
      • Collins S.R.
      • Waldron K.W.
      • Bongaerts R.J.
      • Mayer M.J.
      • Narbad A.
      Spontaneous mutations reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.
      ). The predicted roles of these genes include regulation of sugar biosynthesis, chain length determination, biosynthesis of the repeating unit, polymerization, and export. This cluster has six putative genes encoding glycosyltransferases, which transfer a sugar moiety to the activated acceptor molecule (
      • De Vuyst L.
      • Degeest B.
      Heteropolysaccharides from lactic acid bacteria.
      ,
      • Jolly L.
      • Newell J.
      • Porcelli I.
      • Vincent S.J.
      • Stingele F.
      Lactobacillus helveticus glycosyltransferases. From genes to carbohydrate synthesis.
      ). On the basis of homology to conserved domains, the product of the first glycosyltransferase gene, epsE, was predicted to initiate the capsular EPS biosynthesis by adding the first sugar to the undecaprenylphosphate, whereas another gene in this cluster, epsC, was predicted to encode a tyrosine-protein kinase involved in regulation of capsular EPS biosynthesis (Fig. 1). Changes in the eps cluster resulted in alterations in the accumulation level of EPS in derivatives of L. johnsonii FI9785; a ΔepsE deletion mutant was still able to produce EPS but in lower quantities, whereas an increase in EPS production was observed for a spontaneous epsCD88N mutant (
      • Horn N.
      • Wegmann U.
      • Dertli E.
      • Mulholland F.
      • Collins S.R.
      • Waldron K.W.
      • Bongaerts R.J.
      • Mayer M.J.
      • Narbad A.
      Spontaneous mutations reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.
      ). In order to understand the changes in EPS production after these mutations, knowledge of the primary structure of the EPS produced by the wild type and derivative strains is a prerequisite.
      Figure thumbnail gr1
      FIGURE 1Molecular organization of the eps cluster of L. johnsonii FI9785. The cluster has 14 genes that are predicted to encode a transcriptional regulator (epsA), a polymerization and chain length determination protein (epsB), a tyrosine-protein kinase (epsC), a protein-tyrosine phosphate phosphohydrolase (epsD), the priming glycosyltransferase UDP-phosphate galactosephosphotransferase (epsE) and five glycosyltransferases (1178–1174), an oligosaccharide repeat unit polymerase (1173), a mutase (glf), an oligosaccharide translocase (epsU), and an EPS biosynthesis protein (1170) (
      • Horn N.
      • Wegmann U.
      • Dertli E.
      • Mulholland F.
      • Collins S.R.
      • Waldron K.W.
      • Bongaerts R.J.
      • Mayer M.J.
      • Narbad A.
      Spontaneous mutations reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.
      ).
      In the present study, we identified the structure of two different capsular EPS produced by L. johnsonii FI9785. We also investigated strains with mutations in specific genes of the eps cluster to examine effects on the structure and biosynthesis of these EPS polymers as well as on the cell surface structure of L. johnsonii FI9785. Moreover, we confirmed the localization of specific sugar residues in situ. These characterizations may help us to identify the importance of the structure of the capsular EPS to the bacterial cell surface, which may have an impact on colonization and pathogen exclusion by commensal resident gut bacteria.

      DISCUSSION

      The capsular EPS is thought to be involved in the functional properties of colonization and persistence of both commensal and pathogenic bacteria (
      • Deutsch S.M.
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      • Bouchoux A.
      • Guyomarc'h F.
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      • Falentin H.
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      Contribution of surface β-glucan polysaccharide to physicochemical and immunomodulatory properties of Propionibacterium freudenreichii.
      ,
      • Alp G.
      • Aslim B.
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      The role of hemagglutination and effect of exopolysaccharide production on bifidobacteria adhesion to Caco-2 cells in vitro.
      ). In pathogens, the production of a capsule can be a major virulence factor, yet many of the biosynthetic mechanisms for EPS production are similar between pathogens and commensals. There are few reports on the structure determination and identification of biosynthetic mechanisms of capsular EPS produced by commensal gut bacteria, such as L. johnsonii FI9785. In this study, we determined the structure of two different EPS produced in situ by this bacterium. We assessed the effects on EPS resulting from the deletion of the epsE gene (predicted to encode a UDP-phosphate galactose phosphotransferase that initiates EPS biosynthesis), a spontaneous mutation in the epsC gene (epsCD88N) (described as a putative tyrosine protein kinase) that has a role in the regulation of EPS biosynthesis, and a mutation where the entire eps gene cluster had been removed (
      • Horn N.
      • Wegmann U.
      • Dertli E.
      • Mulholland F.
      • Collins S.R.
      • Waldron K.W.
      • Bongaerts R.J.
      • Mayer M.J.
      • Narbad A.
      Spontaneous mutations reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.
      ).
      It was interesting to find that L. johnsonii FI9785 was capable of producing two different types of capsular EPS: EPS-1 and EPS-2. EPS-1 is a novel dextran with the unusual feature that every α-(1,6)-linked Glcp backbone residue was substituted at O2 with a terminal α-Glcp unit. EPS-2 is a heteropolysaccharide that has a unique hexasaccharide repeating unit composed of four glucose and two galactose residues. To our knowledge, the structures of the two exopolysaccharides are unique among EPS produced by any bacteria. The production of α-glucan with different linkages is quite common for the genus Lactobacillus, and glucosyltransferases encoded by genes designated as gtf are commonly responsible for the production of these dextran-type exopolysaccharides (
      • Kralj S.
      • van Geel-Schutten G.H.
      • Dondorff M.M.
      • Kirsanovs S.
      • van der Maarel M.J.
      • Dijkhuizen L.
      Glucan synthesis in the genus Lactobacillus. Isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains.
      ,
      • Kralj S.
      • van Geel-Schutten G.H.
      • Rahaoui H.
      • Leer R.J.
      • Faber E.J.
      • van der Maarel M.J.
      • Dijkhuizen L.
      Molecular characterization of a novel glucosyltransferase from Lactobacillus reuteri strain 121 synthesizing a unique, highly branched glucan with α-(1→4) and α-(1→6) glucosidic bonds.
      ,
      • van Leeuwen S.S.
      • Kralj S.
      • van Geel-Schutten I.H.
      • Gerwig G.J.
      • Dijkhuizen L.
      • Kamerling J.P.
      Structural analysis of the α-d-glucan (EPS180) produced by the Lactobacillus reuteri strain 180 glucansucrase GTF180 enzyme.
      ,
      • van Hijum S.A.
      • Kralj S.
      • Ozimek L.K.
      • Dijkhuizen L.
      • van Geel-Schutten I.G.
      Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria.
      ). The L. johnsonii FI9785 genome does not contain any annotated genes with clear homology to glucansucrases. The production of more than one EPS has also been demonstrated in other lactic acid bacteria; Lactobacillus plantarum EP56 expressed two heteropolysaccharides, one cell-bound and one released (
      • Tallon R.
      • Bressollier P.
      • Urdaci M.C.
      Isolation and characterization of two exopolysaccharides produced by Lactobacillus plantarum EP56.
      ), whereas the two EPS produced by Leuconostoc pseudomesenteroides R2 were both linear dextrans with different characteristics (
      • Paulo E.M.
      • Boffo E.F.
      • Branco A.
      • Valente A.M.
      • Melo I.S.
      • Ferreira A.G.
      • Roque M.R.
      • Assis S.A.
      Production, extraction and characterization of exopolysaccharides produced by the native Leuconostoc pseudomesenteroides R2 strain.
      ).
      EPS phosphorylation has been shown to affect interactions with the host; phosphate groups associated with EPS from Lactobacillus delbrueckii subsp. bulgaricus have been shown to be required for lymphocyte activation (
      • Kitazawa H.
      • Harata T.
      • Uemura J.
      • Saito T.
      • Kaneko T.
      • Itoh T.
      Phosphate group requirement for mitogenic activation of lymphocytes by an extracellular phosphopolysaccharide from Lactobacillus delbrueckii ssp. bulgaricus.
      ), whereas artificial phosphorylation of a dextran from Leuconostoc mesenteroides increased its immunostimulatory potential (
      • Sato T.
      • Nishimura-Uemura J.
      • Shimosato T.
      • Kawai Y.
      • Kitazawa H.
      • Saito T.
      Dextran from Leuconostoc mesenteroides augments immunostimulatory effects by the introduction of phosphate groups.
      ). EPS-1 was found to be partly substituted with the 1-phosphoglycerol moiety. Such substitution increases the net charge of the EPS, which could play an important role as determinant of interactions between cells, with host surfaces and with ions and peptides in the environment (
      • Tallon R.
      • Bressollier P.
      • Urdaci M.C.
      Isolation and characterization of two exopolysaccharides produced by Lactobacillus plantarum EP56.
      ,
      • Looijesteijn P.J.
      • Trapet L.
      • de Vries E.
      • Abee T.
      • Hugenholtz J.
      Physiological function of exopolysaccharides produced by Lactococcus lactis.
      ), as well as modulating EPS packing and permeability. Different degrees of phosphorylation and unique phosphorylation patterns may influence the observed differences in cellular adhesion between the wild type and the ΔepsE mutant. We found evidence for partial acetylation of both EPS-1 (at multiple sites) and EPS-2 (at a single site), although we did not establish the precise location of the substituents. O-Acetylation of bacterial EPS is frequently reported in both lactic acid bacteria (
      • Faber E.J.
      • van den Haak M.J.
      • Kamerling J.P.
      • Vliegenthart J.F.
      Structure of the exopolysaccharide produced by Streptococcus thermophilus S3.
      ,
      • Robijn G.W.
      • van den Berg D.J.
      • Haas H.
      • Kamerling J.P.
      • Vliegenthart J.F.
      Determination of the structure of the exopolysaccharide produced by Lactobacillus sake 0–1.
      ,
      • Rodríguez-Carvajal M.A.
      • Sánchez J.I.
      • Campelo A.B.
      • Martínez B.
      • Rodríguez A.
      • Gil-Serrano A.M.
      Structure of the high-molecular weight exopolysaccharide isolated from Lactobacillus pentosus LPS26.
      ,
      • Staaf M.
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      Structural elucidation of the viscous exopolysaccharide produced by Lactobacillus helveticus Lb161.
      ) and others, including Klebsiella aerogenes, E. coli O8:K27, and the plant pathogen Pseudomonas flavescens (
      • Cescutti P.
      • Toffanin R.
      • Fett W.F.
      • Osman S.F.
      • Pollesello P.
      • Paoletti S.
      Structural investigation of the exopolysaccharide produced by Pseudomonas flavescens strain B62. Degradation by a fungal cellulase and isolation of the oligosaccharide repeating unit.
      ,
      • Sutherland I.W.
      • Jann K.
      • Jann B.
      The isolation of O-acetylated fragments from the K antigen of Escherichia coli 08:K27 (A):H by the action of phage-induced enzymes from Klebsiella aerogenes.
      ,
      • Sutherland I.W.
      • Wilkinson J.F.
      The exopolysaccharide of Klebsiella aerogens A3 (S1) (type 54). The isolation of O-acetylated octasaccharide, tetrasaccharide and trisaccharide.
      ). Acetylation can alter the physical properties of the EPS, giving, for example, increased viscosity in solution. In the context of the gut environment, we speculate that acetylation provides protection of the EPS from many types of hydrolases produced by gut bacteria.
      AFM was used to investigate cell surface differences using a d-galactose-specific lectin-functionalized tip. The adhesion maps obtained for the wild type (which produces EPS-1 and EPS-2) and the ΔepsE mutant (which only produces EPS-1) reveal a clear difference in the frequency and magnitude of adhesive events captured, showing higher adhesion in the wild type, agreeing with the loss of a galactose-rich EPS in this mutant. In addition to detecting and spatially locating the galactose-bearing EPS-2 on the wild type sample, further analysis of the force spectra yielded information about the physical properties of the polysaccharide. Force spectra obtained on the wild type sample fitted the wormlike chain model (
      • Kratky O.
      • Porod G.
      Röntgenuntersuchung gelöster Fadenmoleküle.
      ,
      • Flory P.
      ), indicating that EPS-2 adopts a semiflexible random coil conformation. The fact that this information can be obtained in situ without the need to isolate the polysaccharide illustrates the power of AFM to measure important intrinsic properties of bacterial cell surfaces (
      • Dupres V.
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      • Cuenot S.
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      Nanoscale mapping and functional analysis of individual adhesins on living bacteria.
      ).
      Recently, Fanning et al. (
      • Fanning S.
      • Hall L.J.
      • Cronin M.
      • Zomer A.
      • MacSharry J.
      • Goulding D.
      • Motherway M.O.
      • Shanahan F.
      • Nally K.
      • Dougan G.
      • van Sinderen D.
      Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.
      ) showed that the putative priming glycosyltransferase Bbr_0430 was essential for the biosynthesis of EPS in Bifidobacterium breve UCC2003. In contrast, we found that the ΔepsE mutant was still producing EPS-1; this suggested that the production of EPS-1 could be independent from the eps gene cluster of L. johnsonii FI9785. But deleting this entire eps cluster from the genome of L. johnsonii FI9785 resulted in the loss of both EPS-1 and EPS-2 production, suggesting that at least one of the genes in this cluster is required for the production of EPS-1. These results are consistent with previous reports where the deletion of the eps gene cluster in L. johnsonii NCC533 resulted in an acapsular strain (
      • Denou E.
      • Pridmore R.D.
      • Berger B.
      • Panoff J.M.
      • Arigoni F.
      • Brüssow H.
      Identification of genes associated with the long-gut-persistence phenotype of the probiotic Lactobacillus johnsonii strain NCC533 using a combination of genomics and transcriptome analysis.
      ). The eps gene cluster of L. johnsonii FI9785 has a genetic organization similar to those of identified gene clusters for the biosynthesis of capsular or extracellular heteropolysaccharides (
      • Fanning S.
      • Hall L.J.
      • Cronin M.
      • Zomer A.
      • MacSharry J.
      • Goulding D.
      • Motherway M.O.
      • Shanahan F.
      • Nally K.
      • Dougan G.
      • van Sinderen D.
      Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.
      ,
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      • Murphy L.
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      • Samuel G.
      • Skovsted I.C.
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      • Barrell B.
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      • Spratt B.G.
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      ,
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      • Dufrêne Y.
      • Vanderleyden J.
      • De Keersmaecker S.C.
      Identification of a gene cluster for the biosynthesis of a long, galactose-rich exopolysaccharide in Lactobacillus rhamnosus GG and functional analysis of the priming glycosyltransferase.
      ). We suggest that this gene cluster, which harbors six putative glycosyltransferase genes, might be responsible for the biosynthesis of heteropolysaccharide EPS-2; in addition, one of these glycosyltransferases may have a bifunctional role to produce the homopolymer EPS-1 (
      • Luzhetskyy A.
      • Bechthold A.
      Features and applications of bacterial glycosyltransferases. Current state and prospects.
      ). Alternatively, a novel gene from the genome of L. johnsonii FI9785 may be involved in EPS-1 production in conjunction with a gene(s) in the eps cluster. Potentially, the six monosaccharide units in the heteropolysaccharide EPS-2 might be added by each glycosyltransferase to form the long-chain capsular EPS-2 initiated by the priming glycosyltransferase epsE. Another gene supporting the role of the eps cluster in EPS-2 production is the glf gene, which putatively encodes the UDP-galactopyranose mutase (
      • Horn N.
      • Wegmann U.
      • Dertli E.
      • Mulholland F.
      • Collins S.R.
      • Waldron K.W.
      • Bongaerts R.J.
      • Mayer M.J.
      • Narbad A.
      Spontaneous mutations reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.
      ). This has been predicted to convert UDP-galactopyranose to UDP-galactofuranose in Lactobacillus rhamnosus GG (
      • Lebeer S.
      • Verhoeven T.L.
      • Francius G.
      • Schoofs G.
      • Lambrichts I.
      • Dufrêne Y.
      • Vanderleyden J.
      • De Keersmaecker S.C.
      Identification of a gene cluster for the biosynthesis of a long, galactose-rich exopolysaccharide in Lactobacillus rhamnosus GG and functional analysis of the priming glycosyltransferase.
      ) and may be responsible for the presence of the galactofuranose residue in the repeating unit structure of EPS-2.
      Based on our findings, we propose that EpsE is the first glycosyltransferase responsible for attachment of the first sugar monomer to a lipid carrier because the ΔepsE mutant was not able to produce EPS-2. The role of this glycosyltransferase has been demonstrated in both Gram-positive and Gram-negative bacteria (
      • Bentley S.D.
      • Aanensen D.M.
      • Mavroidi A.
      • Saunders D.
      • Rabbinowitsch E.
      • Collins M.
      • Donohoe K.
      • Harris D.
      • Murphy L.
      • Quail M.A.
      • Samuel G.
      • Skovsted I.C.
      • Kaltoft M.S.
      • Barrell B.
      • Reeves P.R.
      • Parkhill J.
      • Spratt B.G.
      Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes.
      ,
      • Lebeer S.
      • Verhoeven T.L.
      • Francius G.
      • Schoofs G.
      • Lambrichts I.
      • Dufrêne Y.
      • Vanderleyden J.
      • De Keersmaecker S.C.
      Identification of a gene cluster for the biosynthesis of a long, galactose-rich exopolysaccharide in Lactobacillus rhamnosus GG and functional analysis of the priming glycosyltransferase.
      ,
      • Cieslewicz M.J.
      • Kasper D.L.
      • Wang Y.
      • Wessels M.R.
      Functional analysis in type Ia group B Streptococcus of a cluster of genes involved in extracellular polysaccharide production by diverse species of streptococci.
      ,
      • Minic Z.
      • Marie C.
      • Delorme C.
      • Faurie J.M.
      • Mercier G.
      • Ehrlich D.
      • Renault P.
      Control of EpsE, the phosphoglycosyltransferase initiating exopolysaccharide synthesis in Streptococcus thermophilus, by EpsD tyrosine kinase.
      ,
      • van Kranenburg R.
      • Marugg J.D.
      • van Swam I.I.
      • Willem N.J.
      • de Vos W.M.
      Molecular characterization of the plasmid-encoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis.
      ). Previously, it was shown that the inactivation of the priming glycosyltransferase of L. rhamnosus GG resulted in the absence of the galactose-rich EPS layer on the cell surface, whereas a glucose-rich polysaccharide was still detectable attached to the cell surface (
      • Lebeer S.
      • Verhoeven T.L.
      • Francius G.
      • Schoofs G.
      • Lambrichts I.
      • Dufrêne Y.
      • Vanderleyden J.
      • De Keersmaecker S.C.
      Identification of a gene cluster for the biosynthesis of a long, galactose-rich exopolysaccharide in Lactobacillus rhamnosus GG and functional analysis of the priming glycosyltransferase.
      ). Similarly, it was shown that deletion of the cpsIaE gene, which initiates the polysaccharide biosynthesis in streptococci, resulted in a non-capsular phenotype (
      • Cieslewicz M.J.
      • Kasper D.L.
      • Wang Y.
      • Wessels M.R.
      Functional analysis in type Ia group B Streptococcus of a cluster of genes involved in extracellular polysaccharide production by diverse species of streptococci.
      ). In the current study, we showed that after inactivation of the epsE gene, a second capsular EPS that was formed by glucose monomers only was still detectable in L. johnsonii FI9785. These results demonstrate the essential role of the epsE gene in EPS-2 accumulation on the cell surface of lactobacilli, and further work to investigate the L. johnsonii FI9785 EpsE protein may confirm its proposed role as the priming glycosyltransferase and identify the first monosaccharide of the chain.
      Our previous work on the epsCD88N mutant showed that there was an increase in the production of EPS in this strain (
      • Horn N.
      • Wegmann U.
      • Dertli E.
      • Mulholland F.
      • Collins S.R.
      • Waldron K.W.
      • Bongaerts R.J.
      • Mayer M.J.
      • Narbad A.
      Spontaneous mutations reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.
      ). This mutant could produce both EPS-1 and EPS-2, and the alteration of EPS accumulation level was not related to structural changes in the EPS. The increase in EPS content was possibly due to the production of a higher level of EPS-2 than the wild type, related to the putative role of EpsC in the regulation of EPS-2 biosynthesis (
      • Cieslewicz M.J.
      • Kasper D.L.
      • Wang Y.
      • Wessels M.R.
      Functional analysis in type Ia group B Streptococcus of a cluster of genes involved in extracellular polysaccharide production by diverse species of streptococci.
      ,
      • Morona J.K.
      • Morona R.
      • Miller D.C.
      • Paton J.C.
      Mutational analysis of the carboxy-terminal (YGX)4 repeat domain of CpsD, an autophosphorylating tyrosine kinase required for capsule biosynthesis in Streptococcus pneumoniae.
      ). The characterization of the role of capsular EPS and investigation of the potential genes for EPS-1 biosynthesis is currently in progress.
      The structure of capsular EPS has been shown to have an impact on the immunomodulation, biofilm formation, and colonization properties of producing bacteria (
      • Hidalgo-Cantabrana C.
      • López P.
      • Gueimonde M.
      • los Reyes-Gavilán C.
      • Suárez A.
      • Margolles A.
      • Ruas-Madiedo P.
      Immune modulation capability of exopolysaccharides synthesised by lactic acid bacteria and Bifidobacteria.
      ,
      • Fanning S.
      • Hall L.J.
      • Cronin M.
      • Zomer A.
      • MacSharry J.
      • Goulding D.
      • Motherway M.O.
      • Shanahan F.
      • Nally K.
      • Dougan G.
      • van Sinderen D.
      Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.
      ,
      • Lebeer S.
      • Verhoeven T.L.
      • Perea Vélez M.
      • Vanderleyden J.
      • De Keersmaecker S.C.
      Impact of environmental and genetic factors on biofilm formation by the probiotic strain Lactobacillus rhamnosus GG.
      ,
      • Sims I.M.
      • Frese S.A.
      • Walter J.
      • Loach D.
      • Wilson M.
      • Appleyard K.
      • Eason J.
      • Livingston M.
      • Baird M.
      • Cook G.
      • Tannock G.W.
      Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100–23.
      ). In terms of the lifestyle of the poultry gastrointestinal tract-derived commensal L. johnsonii FI9785, these two EPS could have a protective effect, improving the survival of the bacteria in the external environment and during transit through the gut. Previously, we have reported that differences in the cell surface-associated EPS caused by mutations in the eps cluster affect the adhesion and aggregation properties of L. johnsonii FI9785 (
      • Horn N.
      • Wegmann U.
      • Dertli E.
      • Mulholland F.
      • Collins S.R.
      • Waldron K.W.
      • Bongaerts R.J.
      • Mayer M.J.
      • Narbad A.
      Spontaneous mutations reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.
      ). Both of these characteristics can have an impact upon intra- and interspecies interactions as well as interactions with the host gastrointestinal tract. Here we have detected the cell surface changes after mutations in the eps gene cluster using anti-L. johnsonii FI9785 antibody responses. Górska and co-workers (
      • Górska S.
      • Jachymek W.
      • Rybka J.
      • Strus M.
      • Heczko P.B.
      • Gamian A.
      Structural and immunochemical studies of neutral exopolysaccharide produced by Lactobacillus johnsonii 142.
      ) found that the heteropolysaccharide from L. johnsonii 142, isolated from the murine gut, reacted to a whole cell antibody. Interestingly, the ΔepsE mutant, which could only produce the α-glucan as a capsular EPS, showed a higher antibody response to the L. johnsonii antibody than the wild type, and this increase was intensified in the acapsular Δeps_cluster mutant, whereas strains producing higher levels of EPS did not show an increased response. The inability to produce EPS-2 or the EPS-1/EPS-2 mixture as a capsular material at the cell surface may have resulted in the exposure and presentation of cell surface epitopes like surface proteins for antibody binding in Δeps_cluster and ΔepsE mutants. Another explanation for increased antibody response in ΔepsE might be that glucose-containing epitopes could be more antigenic than galactose-containing epitopes, as noted previously (
      • Iwamori M.
      • Shibagaki T.
      • Nakata Y.
      • Adachi S.
      • Nomura T.
      Distribution of receptor glycolipids for Lactobacilli in murine digestive tract and production of antibodies cross-reactive with them by immunization of rabbits with lactobacilli.
      ). Deletion of a gene producing a levan EPS from Lactobacillus reuteri prevented the induction of regulatory T cells caused by colonization with the wild type strain (
      • Sims I.M.
      • Frese S.A.
      • Walter J.
      • Loach D.
      • Wilson M.
      • Appleyard K.
      • Eason J.
      • Livingston M.
      • Baird M.
      • Cook G.
      • Tannock G.W.
      Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100–23.
      ), whereas EPS-deficient strains of B. breve elicited a stronger immune response than the wild type (
      • Fanning S.
      • Hall L.J.
      • Cronin M.
      • Zomer A.
      • MacSharry J.
      • Goulding D.
      • Motherway M.O.
      • Shanahan F.
      • Nally K.
      • Dougan G.
      • van Sinderen D.
      Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.
      ). EPS layers in these two examples were shown to have a positive effect on persistence and colonization during in vivo studies (
      • Fanning S.
      • Hall L.J.
      • Cronin M.
      • Zomer A.
      • MacSharry J.
      • Goulding D.
      • Motherway M.O.
      • Shanahan F.
      • Nally K.
      • Dougan G.
      • van Sinderen D.
      Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.
      ,
      • Sims I.M.
      • Frese S.A.
      • Walter J.
      • Loach D.
      • Wilson M.
      • Appleyard K.
      • Eason J.
      • Livingston M.
      • Baird M.
      • Cook G.
      • Tannock G.W.
      Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100–23.
      ). Our findings suggest that the gastrointestinal colonization and recognition of the wild type L. johnsonii FI9785, the Δeps_cluster and the ΔepsE strains by the immune system would be different because of the described structural differences and imply a further biological role for the EPS in protecting the bacteria against an immune response.
      In conclusion, this study has revealed simultaneous synthesis of two novel polysaccharide structures by L. johnsonii FI9785. Synthesis of both polymers is dependent on the identified eps gene cluster; however, the precise regulation of the biosynthesis of individual EPS has yet to be identified. Further structural functional characterization using the isolated mutants will allow us to elucidate the physiological importance of these cell surface structures in bacterial survival, host colonization, and pathogen exclusion.

      Acknowledgments

      We thank Kathryn Cross (Imaging Platform, Institute of Food Research) for TEM analysis and Dr. E. Maguin (Institut National de la Recherche Agronomique) for provision of the pG+host vector.

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