Molecular analysis of candidate probiotic effector molecules of Lactobacillus plantarum

Probiotics are health-promoting microorganisms that exert their beneficial effects in several ways. While it is known that probiotic bacteria interact with cells of the host gastrointestinal tractand modulate cell-signaling responses by which they might promote health, the underlying molecular mechanism and probiotic “effector” molecules that are responsible for these effects remain largely unexplored. In this thesis, effector molecules of the lactic acid bacterium Lactobacillus plantarum were discovered that interact with diverse host cell types and manipulate cell-associated signaling pathways. This work represents crucial steps to better understand the exact mode of probiotic action, which is a prerequisite for their controlled, safe, purpose-directed, and person-specific applications in the context of health improvement and disease prevention.</p

An intimate tete-a-tete - How probiotic lactobacilli communicate with the host

Pharmaceutical agents are routinely used in the treatment of gastrointestinal disorders and their role as modulators of host cell responses is well characterized. In contrast, the understanding of the molecular mechanisms, which determine the role of probiotics, i.e. health-promoting bacteria, as host cell modulators is still in its infancy. Both in vitro and in vivo studies are just starting to reveal the capability of probiotic lactobacilli to modulate host cell-signaling networks and the associated influences on downstream regulatory pathways, including modulation of mucosal cytokine profiles that dictate host immune functions. The communication between probiotic lactobacilli and intestinal host cells is multifactorial and involves an integrative repertoire of receptors on the host side that recognize multiple effector molecules on the bacterial side, of which most have been found to be cell wall- or cell surface-associated compounds and proteins. This review describes the discovery of these bacterial effector molecules and their role in strain- and species-specific modulation of host signaling pathways. Unraveling the mechanisms responsible for probiotic-host interactions will progress this research field towards molecular science and will provide markers for probiotic product quality control as well as host-response efficacy. These developments can ultimately lead to a more dedicated, personalized application of probiotics with strong molecular and scientific support for health promotion

Lactobacillus plantarum possesses the capability for wall teichoic acid backbone alditol switching

Contains fulltext : 108484.pdf (publisher's version ) (Open Access)ABSTRACT: BACKGROUND: Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background. RESULTS: Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-kappaB reporter cells expressing Toll like receptor (TLR)-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-alpha after stimulation with the WTA mutants as compared to the wild-type. CONCLUSIONS: The study presented here correlates structural differences in WTA to their functional characteristics, thereby providing important information aiding to improve our understanding of molecular host-microbe interactions and probiotic functionality

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

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 (¿cps1A-I, ¿cps2A-J, ¿cps3A-J and ¿cps4A-J) or combinations of cps clusters (¿cps1A-3J and ¿cps1A-3I, ¿cps4A-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 ¿cps1A-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)-¿B 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 ¿cps1A-I and ¿cps3A-J mutants but appeared slightly increased after stimulation with the ¿cps2A-J and ¿cps4A-J mutants, while the ¿cps1A-3J and ¿cps1A-3J, ¿cps4A-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