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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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Impact of cps cluster deletion on host cell signaling measured by a luminescence reporter in TLR2-expressing HEK-293 cells. Data is represented as average +/− the range between 2 independently grown bacterial cultures (15 CFU/HEK-293 cell) of the L. plantarum wild-type like derivative (NZ3400Cm) and cps deletion mutants (NZ3548cm/Δcps1A-I, NZ5333ACm/Δcps2A-J, NZ3549Cm/Δcps3A-J, NZ3534Cm/Δcps4A-J, NZ3550Cm/ Δcps1A-3J and NZ3680Cm/Δcps1A-3J, Δcps4A-J).
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Figure 3: Impact of cps cluster deletion on host cell signaling measured by a luminescence reporter in TLR2-expressing HEK-293 cells. Data is represented as average +/− the range between 2 independently grown bacterial cultures (15 CFU/HEK-293 cell) of the L. plantarum wild-type like derivative (NZ3400Cm) and cps deletion mutants (NZ3548cm/Δcps1A-I, NZ5333ACm/Δcps2A-J, NZ3549Cm/Δcps3A-J, NZ3534Cm/Δcps4A-J, NZ3550Cm/ Δcps1A-3J and NZ3680Cm/Δcps1A-3J, Δcps4A-J).

Mentions: The effects of cps cluster deletion on host cell signaling were examined in HEK-293 reporter cell lines stably expressing human Toll-like receptor (TLR)2, which harbor a reporter plasmid containing firefly luciferase under the control of the human nuclear factor (NF)-κB promoter. HEK-293 cells do not normally produce TLRs and have been previously shown to be unresponsive to several microbial-associated molecular patterns (MAMPs)[31,32] HEK-293 cells transiently transfected with only the NF-κB reporter plasmid (pNiFTY) were unresponsive to TLR2 agonists of L. plantarum demonstrating the requirement of human TLR2 in this signaling pathway (data not shown). An L plantarum wild-type like derivative (NZ3400Cm) activated TLR2-dependent NF-κB (Figure3). Overall, activation of NF-κB remained unaffected by Δcps1A-I and Δcps3A-J but was slightly increased after stimulation by the Δcps2A-J and Δcps4A-J mutants, which are the strains that displayed the largest reduction in the amount of surface polysaccharides produced. Notably, exposure of TLR2-expressing HEK-293 cells to Δcps1A-3J and Δcps1A-3J, Δcps4A-J elicited the strongest responses and clearly activated NF-κB. Although cps4A-J appears to be expressed at very low levels, its deletion in the Δcps1A-3J background led to a substantial increase of TLR2-mediated NF-κB activity of the resulting strain, demonstrating that deletion of all cps clusters and the concomitant severe reduction of surface polysaccharide production leads to enhanced release and/or exposure of TLR2-agonists. These results suggest a role of surface polysaccharides in the shielding of other L. plantarum cell envelope MAMPs such as lipoproteins and teichoic acids that could activate TLR signaling, as was previously proposed for Lactobacillus rhamnosus GG[21].


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)

Impact of cps cluster deletion on host cell signaling measured by a luminescence reporter in TLR2-expressing HEK-293 cells. Data is represented as average +/− the range between 2 independently grown bacterial cultures (15 CFU/HEK-293 cell) of the L. plantarum wild-type like derivative (NZ3400Cm) and cps deletion mutants (NZ3548cm/Δcps1A-I, NZ5333ACm/Δcps2A-J, NZ3549Cm/Δcps3A-J, NZ3534Cm/Δcps4A-J, NZ3550Cm/ Δcps1A-3J and NZ3680Cm/Δcps1A-3J, Δcps4A-J).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Impact of cps cluster deletion on host cell signaling measured by a luminescence reporter in TLR2-expressing HEK-293 cells. Data is represented as average +/− the range between 2 independently grown bacterial cultures (15 CFU/HEK-293 cell) of the L. plantarum wild-type like derivative (NZ3400Cm) and cps deletion mutants (NZ3548cm/Δcps1A-I, NZ5333ACm/Δcps2A-J, NZ3549Cm/Δcps3A-J, NZ3534Cm/Δcps4A-J, NZ3550Cm/ Δcps1A-3J and NZ3680Cm/Δcps1A-3J, Δcps4A-J).
Mentions: The effects of cps cluster deletion on host cell signaling were examined in HEK-293 reporter cell lines stably expressing human Toll-like receptor (TLR)2, which harbor a reporter plasmid containing firefly luciferase under the control of the human nuclear factor (NF)-κB promoter. HEK-293 cells do not normally produce TLRs and have been previously shown to be unresponsive to several microbial-associated molecular patterns (MAMPs)[31,32] HEK-293 cells transiently transfected with only the NF-κB reporter plasmid (pNiFTY) were unresponsive to TLR2 agonists of L. plantarum demonstrating the requirement of human TLR2 in this signaling pathway (data not shown). An L plantarum wild-type like derivative (NZ3400Cm) activated TLR2-dependent NF-κB (Figure3). Overall, activation of NF-κB remained unaffected by Δcps1A-I and Δcps3A-J but was slightly increased after stimulation by the Δcps2A-J and Δcps4A-J mutants, which are the strains that displayed the largest reduction in the amount of surface polysaccharides produced. Notably, exposure of TLR2-expressing HEK-293 cells to Δcps1A-3J and Δcps1A-3J, Δcps4A-J elicited the strongest responses and clearly activated NF-κB. Although cps4A-J appears to be expressed at very low levels, its deletion in the Δcps1A-3J background led to a substantial increase of TLR2-mediated NF-κB activity of the resulting strain, demonstrating that deletion of all cps clusters and the concomitant severe reduction of surface polysaccharide production leads to enhanced release and/or exposure of TLR2-agonists. These results suggest a role of surface polysaccharides in the shielding of other L. plantarum cell envelope MAMPs such as lipoproteins and teichoic acids that could activate TLR signaling, as was previously proposed for Lactobacillus rhamnosus GG[21].

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