Host-interaction effector molecules of Lactobacillus plantarum WCFS1

Abstract Lactobacillus plantarum is found in various environmental habitats, including fermentation products and the mammalian gastrointestinal tract, and specific strains are marketed as probiotics, which are defined as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’. Throughout the studies of the mechanisms underlying probiotic activity, it became apparent that the probiotic effects are often species and/or strain specific. This situation has led more researchers to focus on the molecular characteristics of probiotic strains intending to link specific molecular structures to specific probiotic functions, and thereby deduce the mechanisms of molecular communication of probiotics. This thesis focuses on potential cell envelope effector molecules involved in interaction with the mammalian host cells, including lipoteichoic acid (LTA), lipo- and glyco-proteins, and extracellular polysaccharides (EPS), of L. plantarum WCFS1, a model strain for probiotic lactobacilli with a well-annotated genome sequences and sophisticated genetic engineering tools. First, existing research regarding the potential roles in probiotic functionality of Lactobacillus surface molecules in terms of their biosynthesis pathways and structure variations as well as interaction with host Pattern Recognition Receptors (PRRs) and immunomodulatory properties of these molecules are summarized and compared to provide an overview of the state-of-the-art in probiotic effector molecule research. Subsequently, specific molecules that reside in the cell envelope of L. plantarum WCFS1 were study for their role in bacterial physiology, as well as their role as ligands in Toll-like receptor (TLR) 2 signaling and immunomodulatory properties using human-cell co-incubation models. Our results showed that the deficiency of LTA had a drastic impact on cell division, cell morphology and growth in L. plantarum WCFS1, while LTA-deficient cells also elicited more pro-inflammatory responses in PBMCs rather than the expected loss of pro-inflammatory capacity as was observed with similar mutants of Lactobacillus acidophilus NCFM. Further studies on the signaling capacity of the purified LTA from L. plantarum WCFS1 revealed that these molecules are poor TLR2 activators, which is in clear contrast to the highly potent TLR2 stimulatory capacity of LTA obtained from Bacillus subtilis, implying that structural differences of the LTA produced by different bacteria are prominent determinants of their TLR2 signaling capacity and immunomodulatory properties. Lipoproteins of L. plantarum WCFS1 were studied using a derivative strain that is deficient in prolipoprotein diacylglyceryltransferase (Lgt), which transfers acyl chain moieties onto lipoproteins. The lipid moiety was shown to be important for proper anchoring of lipoproteins and TLR1/2 signaling capacity, but did not affect TLR2/6 signaling, suggesting that lipoproteins of L. plantarum WCFS1 are predominantly (if not exclusively) triacylated. The Lgt deficient strain elicited more pro-inflammatory responses in PBMCs as compared to the wild type, indicating that the native lipoproteins could play a role in dampening inflammation upon host-probiotic interaction. In addition, we explored the protein glycosylation machinery in L. plantarum WCFS1, responsible for the glycosylation of the major autolysin (Acm2) of this bacterium, which was previously shown to be O-glycosylated with N-acetylhexosamine conjugates. Using sequence similarity searches in combination with a lectin-based glycan detection and mass spectrometry analysis, two glycosyl-transferases, GtfA and GtfB (formerly annotated as TagE5 and TagE6, respectively), were shown to be required for the glycosylation of Acm2 and other unidentified L. plantarum WCFS1 glycosylated proteins. These results provide the first example of a general protein-glycosylation machinery in a Lactobacillus species. Finally, extracellular polysaccharides (EPS) in L. plantarum were studied in two strains that produce large amounts of EPS: L. plantarum SF2A35B and Lp90, in comparison to the lowly producing model strain WCFS1. Based on genome sequence comparison, both of the high producer strains were found to possess strain-specific and unique polysaccharide gene clusters. These gene clusters were deleted and the mutants were shown to have lost the capacity to produce large amounts of EPS, and were studied in relation to their properties in host-bacteria interaction. The results illustrate strain-specific and variable impacts of the removal of the EPS in the background of individual L. plantarum strains, supporting the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction. Overall, this thesis showed that surface molecules not only play important roles in bacterial physiology, but also in the interaction with the host mucosa through pattern recognition receptors expressed by the host cells. With the growing amount of evidence of structural variations in surface molecules, which are influenced by genetic background, physiological status, environmental factors, and other biological processes, these molecules form a unique signature associated with each strain that as a consequence elicits a strain-specific response when interacting with host cells.