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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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Growth phase dependent gene expression levels of individual cps gene clusters in L. plantarum WCFS1. Data is represented as average expression of cps genes of cluster 1 (circles), 2 (squares), 3 (upward pointing arrowheads) and 4(downward pointing arrowheads). Error bars represent the range of expression between the highest and lowest expressed gene in the different clusters.
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Figure 2: Growth phase dependent gene expression levels of individual cps gene clusters in L. plantarum WCFS1. Data is represented as average expression of cps genes of cluster 1 (circles), 2 (squares), 3 (upward pointing arrowheads) and 4(downward pointing arrowheads). Error bars represent the range of expression between the highest and lowest expressed gene in the different clusters.

Mentions: We previously generated global growth phase-dependent gene expression profiles of L. plantarum WCFS1 ranging from mid-logarithmic to late stationary growth phase (Daniela M. Remus, Fabrizia Fusetti, Jurgen Karczewski, Roger S. Bongers, Irene Konings, Bert Poolman, Maria L. Marco, Paul de Vos, Jerry M. Wells, Michiel Wels, Peter A. Bron and Michiel Kleerebezem, unpublished observations). Analysis of the cps cluster-related gene expression patterns revealed that the cps1A-I cluster-associated genes were highly expressed under laboratory conditions, whereas the genes of cps clusters 2A-K, 3A-I and 4A-J were expressed at much lower levels (Figure2).


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)

Growth phase dependent gene expression levels of individual cps gene clusters in L. plantarum WCFS1. Data is represented as average expression of cps genes of cluster 1 (circles), 2 (squares), 3 (upward pointing arrowheads) and 4(downward pointing arrowheads). Error bars represent the range of expression between the highest and lowest expressed gene in the different clusters.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Growth phase dependent gene expression levels of individual cps gene clusters in L. plantarum WCFS1. Data is represented as average expression of cps genes of cluster 1 (circles), 2 (squares), 3 (upward pointing arrowheads) and 4(downward pointing arrowheads). Error bars represent the range of expression between the highest and lowest expressed gene in the different clusters.
Mentions: We previously generated global growth phase-dependent gene expression profiles of L. plantarum WCFS1 ranging from mid-logarithmic to late stationary growth phase (Daniela M. Remus, Fabrizia Fusetti, Jurgen Karczewski, Roger S. Bongers, Irene Konings, Bert Poolman, Maria L. Marco, Paul de Vos, Jerry M. Wells, Michiel Wels, Peter A. Bron and Michiel Kleerebezem, unpublished observations). Analysis of the cps cluster-related gene expression patterns revealed that the cps1A-I cluster-associated genes were highly expressed under laboratory conditions, whereas the genes of cps clusters 2A-K, 3A-I and 4A-J were expressed at much lower levels (Figure2).

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