BACKGROUND: Recent advances in sequencing technologies have enabled metagenomic analyses of many human body sites. Several studies have catalogued the composition of bacterial communities of the ...surface of human skin, mostly under static conditions in healthy volunteers. Skin injury will disturb the cutaneous homeostasis of the host tissue and its commensal microbiota, but the dynamics of this process have not been studied before. Here we analyzed the microbiota of the surface layer and the deeper layers of the stratum corneum of normal skin, and we investigated the dynamics of recolonization of skin microbiota following skin barrier disruption by tape stripping as a model of superficial injury. RESULTS: We observed gender differences in microbiota composition and showed that bacteria are not uniformly distributed in the stratum corneum. Phylogenetic distance analysis was employed to follow microbiota development during recolonization of injured skin. Surprisingly, the developing neo-microbiome at day 14 was more similar to that of the deeper stratum corneum layers than to the initial surface microbiome. In addition, we also observed variation in the host response towards superficial injury as assessed by the induction of antimicrobial protein expression in epidermal keratinocytes. CONCLUSIONS: We suggest that the microbiome of the deeper layers, rather than that of the superficial skin layer, may be regarded as the host indigenous microbiome. Characterization of the skin microbiome under dynamic conditions, and the ensuing response of the microbial community and host tissue, will shed further light on the complex interaction between resident bacteria and epidermis.
Lactobacilli are found in diverse environments and are widely applied as probiotic, health-promoting food supplements. Polysaccharides are ubiquitously present on the cell surface of lactobacilli and ...are considered to contribute to the species- and strain-specific probiotic effects that are typically observed. Two Lactobacillus plantarum strains, SF2A35B and Lp90, have an obvious ropy phenotype, implying high extracellular polysaccharide (EPS) production levels. In this work, we set out to identify the genes involved in EPS production in these L. plantarum strains and to demonstrate their role in EPS production by gene deletion analysis. A model L. plantarum strain, WCFS1, and its previously constructed derivative that produced reduced levels of EPS were included as reference strains. The constructed EPS-reduced derivatives were analyzed for the abundance and sugar compositions of their EPS, revealing cps2-like gene clusters in SF2A35B and Lp90 responsible for major EPS production. Moreover, these mutant strains were tested for phenotypic characteristics that are of relevance for their capacity to interact with the host epithelium in the intestinal tract, including bacterial surface properties as well as survival under the stress conditions encountered in the gastrointestinal tract (acid and bile stress). In addition, the Toll-like receptor 2 (TLR2) signaling and immunomodulatory capacities of the EPS-negative derivatives and their respective wild-type strains were compared, revealing strain-specific impacts of EPS on the immunomodulatory properties. Taken together, these experiments illustrate the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction.
This study evaluates the role of extracellular polysaccharides that are produced by different strains of Lactobacillus plantarum in the determination of the cell surface properties of these bacteria and their capacity to interact with their environment, including their signaling to human host cells. The results clearly show that the consequences of removal of these polysaccharides are very strain specific, illustrating the diverse and unpredictable roles of these polysaccharides in the environmental interactions of these bacterial strains. In the context of the use of lactobacilli as health-promoting probiotic organisms, this study exemplifies the importance of strain specificity.
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.
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.
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.
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.
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-κB 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-α after stimulation with the WTA mutants as compared to the wild-type.
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.
Bacterial lipoproteins are well-recognized microorganism-associated molecular patterns, which interact with Toll-like receptor (TLR) 2, an important pattern recognition receptor of the host innate ...immune system. Lipoproteins are conjugated with two- or three-acyl chains (di- or tri-acyl), which is essential for appropriate anchoring in the cell membrane as well as for the interaction with TLR2. Lipoproteins have mostly been studied in pathogens and have established roles in various biological processes, such as nutrient import, cell wall cross-linking and remodeling, and host-cell interaction. By contrast, information on the role of lipoproteins in the physiology and host interaction of probiotic bacteria is scarce. By deletion of
lgt
, encoding prolipoprotein diacylglyceryl transferase, responsible for lipidation of lipoprotein precursors, we investigated the roles of the collective group of lipoproteins in the physiology of the probiotic model strain
Lactobacillus plantarum
WCFS1 using proteomic analysis of secreted proteins. To investigate the consequences of the
lgt
mutation in host-cell interaction, the capacity of mutant and wild-type bacteria to stimulate TLR2 signaling and inflammatory responses was compared using (reporter-) cell-based models. These experiments exemplified the critical contribution of the acyl chains of lipoproteins in immunomodulation. To the best of our knowledge, this is the first study that investigated collective lipoprotein functions in a model strain for probiotic lactobacilli, and we show that the lipoproteins in
L. plantarum
WCFS1 are critical drivers of anti-inflammatory host responses toward this strain.
Although teichoic acids are major constituents of bacterial cell walls, little is known about the relationships between their spatial localization and their functional roles. Here, we used ...single-molecule atomic force microscopy (AFM) combined with fluorescence microscopy to image the distribution of wall teichoic acids (WTAs) in Lactobacillus plantarum, in relation with their physiological roles. Phenotype analysis of the wild-type strain and of mutant strains deficient for the synthesis of WTAs (ΔtagO) or cell wall polysaccharides (Δcps1-4) revealed that WTAs are required for proper cell elongation and cell division. Nanoscale imaging by AFM showed that strains expressing WTAs have a highly polarized surface morphology, the poles being much smoother than the side walls. AFM and fluorescence imaging with specific lectin probes demonstrated that the polarized surface structure correlates with a heterogeneous distribution of WTAs, the latter being absent from the surface of the poles. These observations indicate that the polarized distribution of WTAs in L. plantarum plays a key role in controlling cell morphogenesis (surface roughness, cell shape, elongation, and division).
Toll-like receptor (TLR) expression in patients with inflammatory bowel disease is increased when compared with healthy controls. However, the impact of TLR signaling during inflammatory bowel ...disease is not fully understood.
In this study, we used a murine model of acute phase inflammation in bone marrow chimeric mice to investigate in which cell type TLR2/4 signal induction is important in preventing intestinal inflammation and how intestinal dendritic cells are influenced. Mice were either fed with wild-type bacteria, able to initiate the TLR2/4 signaling cascade, or with mutant strains with impaired signal induction capacity.
The induction of the TLR2/4 signal cascade in epithelial cells resulted in inflammation in bone marrow chimeric mice, whereas induction in hematopoietic cells had an opposed function. Furthermore, feeding of wild-type bacteria prevented disease; however, differing signal induction of bacteria had no effect on lamina propria dendritic cell activation. In contrast, functional TLR2/4 signals resulted in increased frequencies of CD103-expressing lamina propria and mesenteric lymph node dendritic cells, which were able to ameliorate disease.
The TLR-mediated amelioration of disease, the increase in CD103-expressing cells, and the beneficial function of TLR signal induction in hematopoietic cells indicate that the increased expression of TLRs in patients with inflammatory bowel disease might result in counterregulation of the host and serve in preventing disease.
The use of superoxide dismutases (SODs) in inflammatory diseases is hampered by their short circulatory half-life. To determine whether a bacterial supply of SOD into the colon might improve an ...experimental colitis, the effects of oral treatment with live recombinant lactic acid bacteria producing different amounts of SOD and those of colonic infusion of SOD were compared. Wistar rats were fitted with a catheter in the proximal colon through which TNBS was administered to induce colitis. Animals received a continuous intracolonic infusion of bovine SOD (40 U per rat per day) for 4 days after TNBS or were treated orally with live recombinant Lactococcus lactis or Lactobacillus plantarum strains (10 colony-forming units (CFU)/d), producing or not producing SOD, for 4 days before and after TNBS. SOD activity of bacterial extracts was 0, 26, 74, and 624 units/10 CFU for L. plantarum, L. lactis, L. lactis SOD, and L. plantarum SOD, respectively. Four days after TNBS, macroscopic and microscopic damage, myeloperoxidase (MPO) activity, and nitrotyrosine immunostaining were evaluated. TNBS induced macroscopic and microscopic damages, an increase in MPO activity, and intense immunostaining for nitrotyrosine. Macroscopic damage and MPO activity were reduced by bovine SOD. These parameters and microscopic damages also were reduced by L. lactis, L. lactis SOD, and L. plantarum SOD, but not by L. plantarum. Nitrotyrosine immunostaining was attenuated after treatment with the 4 bacterial strains. Although not all of the anti-inflammatory effects could be attributed directly to SOD, our results suggest that SOD-producing lactic acid bacteria open a novel approach in inflammatory bowel disease treatment.
Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the ...more traditional genetic engineering approaches 'bottle-necks' are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected 'rate-limiting' steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.
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