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Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling.

Remus DM, van Kranenburg R, van Swam II, Taverne N, Bongers RS, Wels M, Wells JM, Bron PA, Kleerebezem M - Microb. Cell Fact. (2012)

Bottom Line: Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action.The Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production.In the quadruple mutant, the amount of surface polysaccharides was strongly reduced.

View Article: PubMed Central - HTML - PubMed

Affiliation: TI Food & Nutrition, Nieuwe Kanaal 9A, 6709 PA Wageningen, The Netherlands.

ABSTRACT

Background: Bacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well described. Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action.

Results: The Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production. Two of these clusters appear to encode all functions required for capsular polysaccharide formation (cps2A-J and cps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-I and cps3A-J). We constructed L. plantarum WCFS1 gene deletion mutants that lack individual (Δcps1A-I, Δcps2A-J, Δcps3A-J and Δcps4A-J) or combinations of cps clusters (Δcps1A-3J and Δcps1A-3I, Δcps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. The cps cluster deletions influenced the expression of variable gene sets in the individual cps cluster mutants, but also considerable numbers of up- and down-regulated genes were shared between mutants in cps cluster 1 and 2, as well as between mutant in cps clusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness of cps1A-I and cps3A-J, all clusters are active and functional in L. plantarum. The Δcps1A-I strain produced surface polysaccharides in equal amounts as compared to the wild-type strain, while the polysaccharides were characterized by a reduced molar mass and the lack of rhamnose. The mutants that lacked functional copies of cps2A-J, cps3A-J or cps4A-J produced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of the cps cluster mutations on toll-like receptor (TLR)-mediated human nuclear factor (NF)-κB activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to a L. plantarum wild-type derivative, TLR2 activation remained unaffected by the Δcps1A-I and Δcps3A-J mutants but appeared slightly increased after stimulation with the Δcps2A-J and Δcps4A-J mutants, while the Δcps1A-3J and Δcps1A-3J, Δcps4A-J mutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling.

Conclusions: Our study reveals that modulation of surface glycan characteristics in L. plantarum highlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently complete cps clusters (cps2A-J and cps4A-J) contributed individually to this shielding, the removal of all cps clusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis in L. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.

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Genetic organization of polysaccharide biosynthesis gene clusters in L. plantarum strains. (A) Genetic organization of the L. plantarum WCFS1 polysaccharide biosynthesis gene clusters 1, 2, and 3 (AL935263; lp_1176 through lp_1234) and comparison with the corresponding regions of L. plantarum strains ST-III (NC_014554; LPST_C0945 through LPST_C0997), JDM-1 (NC_012984; JDM1_1015 through JDM1_1041), and ATCC 14917 (ACGZ02000014; HMPREF0531_11685 through HMPREF0531_11729; ACGZ02000010; HMPREF0531_11316 through HMPREF0531_11319). Dark-grey colored connecting blocks indicate regions of high sequence conservation identified in the genomes of the indicated strains. The white area connecting msa homologues gene of ST-III and JDM-1, indicates that this gene is separated on two contigs in the draft genome sequence available for ATCC14917. (B) Genetic organization of the L. plantarum WCFS1 polysaccharide biosynthesis gene cluster 4. The corresponding regions are conserved in L. plantarum ST-III, ATCC 14917, and JDM1 sharing 99%, 99%, and 97% nucleotide identity with WCFS 1, respectively. Arrows indicate predicted promoters with e-value ≤ 10-5[27].
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Figure 1: Genetic organization of polysaccharide biosynthesis gene clusters in L. plantarum strains. (A) Genetic organization of the L. plantarum WCFS1 polysaccharide biosynthesis gene clusters 1, 2, and 3 (AL935263; lp_1176 through lp_1234) and comparison with the corresponding regions of L. plantarum strains ST-III (NC_014554; LPST_C0945 through LPST_C0997), JDM-1 (NC_012984; JDM1_1015 through JDM1_1041), and ATCC 14917 (ACGZ02000014; HMPREF0531_11685 through HMPREF0531_11729; ACGZ02000010; HMPREF0531_11316 through HMPREF0531_11319). Dark-grey colored connecting blocks indicate regions of high sequence conservation identified in the genomes of the indicated strains. The white area connecting msa homologues gene of ST-III and JDM-1, indicates that this gene is separated on two contigs in the draft genome sequence available for ATCC14917. (B) Genetic organization of the L. plantarum WCFS1 polysaccharide biosynthesis gene cluster 4. The corresponding regions are conserved in L. plantarum ST-III, ATCC 14917, and JDM1 sharing 99%, 99%, and 97% nucleotide identity with WCFS 1, respectively. Arrows indicate predicted promoters with e-value ≤ 10-5[27].

Mentions: The L. plantarum WCFS1 genome contains two regions with surface-associated polysaccharide biosynthesis genes that are designated cps genes (Figure1A and B). One region of 49 kb contains three gene clusters separated by transposase genes and has been identified as a genomic life-style island with high variability between L. plantarum strains[26]. Of these three gene clusters, cps1 and cps2 are identified only in strain WCFS1 and not in the other completely sequenced strains of the same species, while gene cluster cps3 is conserved in ST-III and ATCC 14917 but not in JDM1 (Figure1; Additional file1). A second region of 14 kb comprises the WCFS1 cps4 gene cluster and is conserved in other L. plantarum strains (Additional file1). Clusters 2 and 4 have a typical structure of a Wzy-dependent polymer gene cluster (Figure1; Additional file1). The first three genes (cps2ABC and cps4ABC) are homologous to the typical components of the tyrosine kinase phospho-regulatory circuit involved in control of capsule synthesis[14]. The fourth gene in both gene clusters is predicted to encode an UDP-N-acetylglucosamine 4-epimerase catalyzing the interconversion between UDP-N-acetyl-d-glucosamine and UDP-N-acetyl-d-galactosamine (cps2D, and cps4D). The fifth gene is predicted to encode the priming glycosyltransferase catalyzing the transfer of a sugar-1-phosphate from a UDP-sugar to the undecaprenyl-phosphate, the first step in the synthesis of the repeat unit (cps2E and cps4E). The cps clusters 2 and 4 contain three additional genes with high sequence similarity to glycosyltransferase genes (cps2FGJ and cps4FGI), indicating that the encoded polysaccharides would be made up of quatro-saccharide repeat units. The 3’ regions of both clusters encode homologues of the typical flippase (cps2I and cps4J) and polymerase (cps2H and cps4H) functions required for capsule synthesis[14].


Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling.

Remus DM, van Kranenburg R, van Swam II, Taverne N, Bongers RS, Wels M, Wells JM, Bron PA, Kleerebezem M - Microb. Cell Fact. (2012)

Genetic organization of polysaccharide biosynthesis gene clusters in L. plantarum strains. (A) Genetic organization of the L. plantarum WCFS1 polysaccharide biosynthesis gene clusters 1, 2, and 3 (AL935263; lp_1176 through lp_1234) and comparison with the corresponding regions of L. plantarum strains ST-III (NC_014554; LPST_C0945 through LPST_C0997), JDM-1 (NC_012984; JDM1_1015 through JDM1_1041), and ATCC 14917 (ACGZ02000014; HMPREF0531_11685 through HMPREF0531_11729; ACGZ02000010; HMPREF0531_11316 through HMPREF0531_11319). Dark-grey colored connecting blocks indicate regions of high sequence conservation identified in the genomes of the indicated strains. The white area connecting msa homologues gene of ST-III and JDM-1, indicates that this gene is separated on two contigs in the draft genome sequence available for ATCC14917. (B) Genetic organization of the L. plantarum WCFS1 polysaccharide biosynthesis gene cluster 4. The corresponding regions are conserved in L. plantarum ST-III, ATCC 14917, and JDM1 sharing 99%, 99%, and 97% nucleotide identity with WCFS 1, respectively. Arrows indicate predicted promoters with e-value ≤ 10-5[27].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539956&req=5

Figure 1: Genetic organization of polysaccharide biosynthesis gene clusters in L. plantarum strains. (A) Genetic organization of the L. plantarum WCFS1 polysaccharide biosynthesis gene clusters 1, 2, and 3 (AL935263; lp_1176 through lp_1234) and comparison with the corresponding regions of L. plantarum strains ST-III (NC_014554; LPST_C0945 through LPST_C0997), JDM-1 (NC_012984; JDM1_1015 through JDM1_1041), and ATCC 14917 (ACGZ02000014; HMPREF0531_11685 through HMPREF0531_11729; ACGZ02000010; HMPREF0531_11316 through HMPREF0531_11319). Dark-grey colored connecting blocks indicate regions of high sequence conservation identified in the genomes of the indicated strains. The white area connecting msa homologues gene of ST-III and JDM-1, indicates that this gene is separated on two contigs in the draft genome sequence available for ATCC14917. (B) Genetic organization of the L. plantarum WCFS1 polysaccharide biosynthesis gene cluster 4. The corresponding regions are conserved in L. plantarum ST-III, ATCC 14917, and JDM1 sharing 99%, 99%, and 97% nucleotide identity with WCFS 1, respectively. Arrows indicate predicted promoters with e-value ≤ 10-5[27].
Mentions: The L. plantarum WCFS1 genome contains two regions with surface-associated polysaccharide biosynthesis genes that are designated cps genes (Figure1A and B). One region of 49 kb contains three gene clusters separated by transposase genes and has been identified as a genomic life-style island with high variability between L. plantarum strains[26]. Of these three gene clusters, cps1 and cps2 are identified only in strain WCFS1 and not in the other completely sequenced strains of the same species, while gene cluster cps3 is conserved in ST-III and ATCC 14917 but not in JDM1 (Figure1; Additional file1). A second region of 14 kb comprises the WCFS1 cps4 gene cluster and is conserved in other L. plantarum strains (Additional file1). Clusters 2 and 4 have a typical structure of a Wzy-dependent polymer gene cluster (Figure1; Additional file1). The first three genes (cps2ABC and cps4ABC) are homologous to the typical components of the tyrosine kinase phospho-regulatory circuit involved in control of capsule synthesis[14]. The fourth gene in both gene clusters is predicted to encode an UDP-N-acetylglucosamine 4-epimerase catalyzing the interconversion between UDP-N-acetyl-d-glucosamine and UDP-N-acetyl-d-galactosamine (cps2D, and cps4D). The fifth gene is predicted to encode the priming glycosyltransferase catalyzing the transfer of a sugar-1-phosphate from a UDP-sugar to the undecaprenyl-phosphate, the first step in the synthesis of the repeat unit (cps2E and cps4E). The cps clusters 2 and 4 contain three additional genes with high sequence similarity to glycosyltransferase genes (cps2FGJ and cps4FGI), indicating that the encoded polysaccharides would be made up of quatro-saccharide repeat units. The 3’ regions of both clusters encode homologues of the typical flippase (cps2I and cps4J) and polymerase (cps2H and cps4H) functions required for capsule synthesis[14].

Bottom Line: Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action.The Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production.In the quadruple mutant, the amount of surface polysaccharides was strongly reduced.

View Article: PubMed Central - HTML - PubMed

Affiliation: TI Food & Nutrition, Nieuwe Kanaal 9A, 6709 PA Wageningen, The Netherlands.

ABSTRACT

Background: Bacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well described. Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action.

Results: The Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production. Two of these clusters appear to encode all functions required for capsular polysaccharide formation (cps2A-J and cps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-I and cps3A-J). We constructed L. plantarum WCFS1 gene deletion mutants that lack individual (Δcps1A-I, Δcps2A-J, Δcps3A-J and Δcps4A-J) or combinations of cps clusters (Δcps1A-3J and Δcps1A-3I, Δcps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. The cps cluster deletions influenced the expression of variable gene sets in the individual cps cluster mutants, but also considerable numbers of up- and down-regulated genes were shared between mutants in cps cluster 1 and 2, as well as between mutant in cps clusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness of cps1A-I and cps3A-J, all clusters are active and functional in L. plantarum. The Δcps1A-I strain produced surface polysaccharides in equal amounts as compared to the wild-type strain, while the polysaccharides were characterized by a reduced molar mass and the lack of rhamnose. The mutants that lacked functional copies of cps2A-J, cps3A-J or cps4A-J produced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of the cps cluster mutations on toll-like receptor (TLR)-mediated human nuclear factor (NF)-κB activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to a L. plantarum wild-type derivative, TLR2 activation remained unaffected by the Δcps1A-I and Δcps3A-J mutants but appeared slightly increased after stimulation with the Δcps2A-J and Δcps4A-J mutants, while the Δcps1A-3J and Δcps1A-3J, Δcps4A-J mutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling.

Conclusions: Our study reveals that modulation of surface glycan characteristics in L. plantarum highlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently complete cps clusters (cps2A-J and cps4A-J) contributed individually to this shielding, the removal of all cps clusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis in L. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.

Show MeSH
Related in: MedlinePlus