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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
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.
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.
). 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 (
). 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 (
). 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 (
). 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 (
). 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) (
). 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 (
). 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 (
). 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.
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.
The capsular EPS is thought to be involved in the functional properties of colonization and persistence of both commensal and pathogenic bacteria (
). 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 (
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 (
). 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 (
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 (
). 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 (
), 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 (
). 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 (
), 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 (
) 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 (
). 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 (
). 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 (
) 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
) 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 (
). 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.
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.
New perspectives for lactobacilli exopolysaccharides.