Strain-Specific Features of Extracellular Polysaccharides and Their Impact on Lactobacillus plantarum-Host Interactions.

UNLABELLED: 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. IMPORTANCE: 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.

GtfA and GtfB are both required for protein O-glycosylation in Lactobacillus plantarum.

Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively.

The quest for probiotic effector molecules--unraveling strain specificity at the molecular level.

Pharmaceutical agents are widely applied for the treatment of gastrointestinal (and systemic) disorders and their role as modulators of host cell responses is relatively well characterized. By contrast, we are only beginning to understand the molecular mechanisms by which health-promoting, probiotic bacteria act as host cell modulators. The last decade has seen a rapid development of the genomics field for the widely applied probiotic genus Lactobacillus, and nowadays dozens of full genome sequences are available, as well as sophisticated post genomic and genetic engineering tools. This development has enabled comparative (functional) genomics approaches to identify the bacterial effector molecules involved in molecular communication with the host system that may underlie the probiotic effects observed. These efforts can also be complemented with dedicated mutagenesis approaches to eliminate or alter these effector molecules, followed by assessment of the host interaction consequences thereof, allowing the elucidation of the molecular mechanisms involved in probiotic health effects. Many of these approaches have pinpointed that the Lactobacillus cell envelope contains several effector molecules that are pivotal in the direct signaling capacity of these bacteria that underlies their immunomodulatory effects, including lipoteichoic acid, peptidoglycan, and (glyco)proteins. Moreover, the cell envelope contains several compounds such as wall teichoic acid and capsular polysaccharides that may not be involved in direct signaling to the host cell, but still affect signaling through shielding of other bacterial effector molecules. Initial structural studies revealed subtle strain- and species-specific biochemical differences in the canonical cell envelope compounds that are involved in these host interactions. These biochemical variations include the degree and positioning of d-alanyl and glycosyl substitution in lipoteichoic acids, and acetylation of peptidoglycan. Furthermore, specific peptides derived from peptidoglycan and envelope associated (glyco)proteins were recently identified as potent immunomodulators. The latter findings are exciting in the light of the possibility of more pharmacological application of these bioactive probiotic molecules, and especially cost-effective production and targeted delivery of bioactive peptides seems to emerge as a feasible strategy to harness this knowledge.

Lipoproteins Contribute to the Anti-inflammatory Capacity of Lactobacillus plantarum WCFS1.

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.

Draft Genome Sequence of Lactobacillus plantarum SF2A35B.

The lactic acid bacterium Lactobacillus plantarum is intensively studied as a model probiotic species. Here, we present the draft genome sequence of the exopolysaccharide-producing strain SF2A35B.

The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids) and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

Modulation of Lactobacillus plantarum gastrointestinal robustness by fermentation conditions enables identification of bacterial robustness markers.

BACKGROUND: Lactic acid bacteria (LAB) are applied worldwide in the production of a variety of fermented food products. Additionally, specific Lactobacillus species are nowadays recognized for their health-promoting effects on the consumer. To optimally exert such beneficial effects, it is considered of great importance that these probiotic bacteria reach their target sites in the gut alive. METHODOLOGY/PRINCIPAL FINDINGS: In the accompanying manuscript by Bron et al. the probiotic model organism Lactobacillus plantarum WCFS1 was cultured under different fermentation conditions, which was complemented by the determination of the corresponding molecular responses by full-genome transcriptome analyses. Here, the gastrointestinal (GI) survival of the cultures produced was assessed in an in vitro assay. Variations in fermentation conditions led to dramatic differences in GI-tract survival (up to 7-log) and high robustness could be associated with low salt and low pH during the fermentations. Moreover, random forest correlation analyses allowed the identification of specific transcripts associated with robustness. Subsequently, the corresponding genes were targeted by genetic engineering, aiming to enhance robustness, which could be achieved for 3 of the genes that negatively correlated with robustness and where deletion derivatives displayed enhanced survival compared to the parental strain. Specifically, a role in GI-tract survival could be confirmed for the lp_1669-encoded AraC-family transcription regulator, involved in capsular polysaccharide remodeling, the penicillin-binding protein Pbp2A involved in peptidoglycan biosynthesis, and the Na(+)/H(+) antiporter NapA3. Moreover, additional physiological analysis established a role for Pbp2A and NapA3 in bile salt and salt tolerance, respectively. CONCLUSION: Transcriptome trait matching enabled the identification of biomarkers for bacterial (gut-)robustness, which is important for our molecular understanding of GI-tract survival and could facilitate the design of culture conditions aimed to enhance probiotic culture robustness.