Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling
Authors: Remus Daniela M , van Kranenburg Richard , van Swam Iris I , Taverne Nico , Bongers Roger S , Wels Michiel , Wells Jerry M , Bron Peter A , Kleerebezem Michiel 
Published: November 2012

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

TheLactobacillus plantarumWCFS1 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-Jandcps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-Iandcps3A-J). We constructedL. plantarumWCFS1 gene deletion mutants that lack individual (Δcps1A-I,Δcps2A-J, Δcps3A-Jand Δcps4A-J) or combinations ofcpsclusters (Δcps1A-3Jand Δcps1A-3I, Δcps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. Thecpscluster deletions influenced the expression of variable gene sets in the individualcpscluster mutants, but also considerable numbers of up- and down-regulated genes were shared between mutants incpscluster 1 and 2, as well as between mutant incpsclusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness ofcps1A-Iandcps3A-J, all clusters are active and functional inL. plantarum. The Δcps1A-Istrain 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 ofcps2A-J,cps3A-Jorcps4A-Jproduced 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 thecpscluster 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 aL. plantarumwild-type derivative, TLR2 activation remained unaffected by the Δcps1A-Iand Δcps3A-Jmutants but appeared slightly increased after stimulation with the Δcps2A-Jand Δcps4A-Jmutants, while the Δcps1A-3Jand Δcps1A-3J,Δcps4A-Jmutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling.

Conclusions

Our study reveals that modulation of surface glycan characteristics inL. plantarumhighlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently completecpsclusters (cps2A-Jandcps4A-J) contributed individually to this shielding, the removal of allcpsclusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis inL. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.